The menstrual cycle influences HIV infection-risk in women, although the timing and underlying mechanism are unclear. Here we investigated the contribution of the menstrual cycle to HIV susceptibility through evaluating immune behavior with infection-risk over time.n = 10) were prospectively collected over the course of a menstrual cycle to profile T cell populations. Immune properties from PBMC and vaginal lavage samples were measured by flow cytometry. Plasma progesterone was measured by enzyme immunoassay. The oscillation frequency of progesterone concentration and CCR5 expression on CD4 T cells was calculated using the Lomb-Scargle periodogram. SHIV infection was monitored in plasma by RT-PCR. Immune measures were compared using generalized estimating equations (GEE).Blood and vaginal lavage samples were collected from 18 pig-tailed macaques to evaluate immune changes over reproductive cycles, and from 5 additional animals undergoing repeated vaginal exposures to simian HIV (SHIV). Peripheral blood mononuclear cell (PBMC) samples from healthy women (T cell response with corresponding CCR5+ memory CD4 T cell (HIV target cell) infiltration into the vaginal lumen at the late luteal phase. Power spectral analysis identified CCR5 oscillation frequencies synchronized with reproductive cycles. In a repetitive low-dose vaginal challenge model, productive SHIV163P3 infection only occurred during intervals of mounting type-1 T cell responses (n = 5/5). Finally, we identify similar type-1 inflammatory T cell responses over the menstrual cycle are occurring in healthy women.Macaques cycle-phases were associated with fluctuations in systemic immune properties and a type-1 inflammatory T cell recruitment and HIV susceptibility in the female reproductive tract.These data demonstrate that periodic shifts in the immune landscape under menstrual cycle regulation drives bystander CCR5+ CD4 This study was supported by the U.S. Centers for Disease Control and Prevention, Atlanta, GA 30329 and NIH grants to Emory University .The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention or the Department of Health and Human Services. T cells (HIV target cells) to mucosal barriers are considered HIV-risk factors, when and how the immune system is regulated by the menstrual cycle to influence infection in the FRT remains undefined.Understanding the contribution of the menstrual cycle to STI/HIV infection from sexual contact may help prevent new infections, and inform upon other potential risk factors in women such as hormonal contraception. Exposure to HIV in the female reproductive tract (FRT) during fertility windows has been previously proposed to increase infection susceptibility through heightened immune tolerance. Although proinflammatory responses and the proximity of CCR5+ CD4 T cell expression of proinflammatory properties and the HIV co-receptor CCR5 within the late luteal phase. Furthermore, in macaques we find that the sinusoidal fluctuations of CCR5+ memory CD4 T cells in circulation corresponds with infiltration of CCR5+ CD4 T cells into the vaginal mucosa primarily within the late luteal phase of the cycle, when SHIV inoculation also led to the establishment of infection from weekly repetitive low-dose vaginal challenge.We identify in humans and pig-tailed macaques that the menstrual cycle regulates a type-1 specific adaptive immune response, identified in part by increased CD4 This study finds evidence that over the menstrual cycle HIV-risk from sexual contact is increased by the FRT tissue remodeling functions that occur during the late luteal phase of the menstrual cycle.Alt-text: Unlabelled box1Despite the discovery and implementation of antiretroviral drugs to prevent and treat Human Immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS)-related illness are still the leading cause of death globally in women aged 15–49 . In 2017T cells, at mucosal tissue barrier sites of exposure is an indicator of susceptibility to HIV or simian immunodeficiency virus (SIV) in both human and non-human primates (NHP) T cells can also indicate the likelihood of establishing an infection. In some studies, HIV acquisition was found to correlate with the frequency of activation-associated properties and CCR5 from CD4 T cells in blood [T cells identify increased HIV infection risk.Although we have yet to understand the precise mechanisms by which HIV infection is established following mucosal exposure, the immune system is a principal component of the process. The localization of the main target of HIV infection, CCR5 expressing CD4 in blood . Thus, cIt has long been recognized that preexisting inflammation, such as occurs in response to certain sexually transmitted infections (STIs) or bacterial vaginosis, increases the odds of HIV or SIV acquisition vaginally . Such inAlthough the process of menstruation is not a result of any infection it comprises elements that are consistent with an inflammatory response. In menstruating mammals such as humans and some species of NHP, the unique function of spontaneous decidualization by endometrial stromal cells which are hypothesized to limit harmful levels of invasion by a potential embryo during implantation also demand extensive tissue remodeling of the uterine lining in order to maintain reproductive cycles Chlamydia trachomatis, Candida albicans and Neisseria Gonorrhoeae infections [. However progesterone has an inhibitory effect on HIV infectivity [ex-vivo FRT cross-sectional infection studies provide conflicting evidence as to whether HIV replication is more likely to occur at either the follicular or luteal phase [Although an overwhelming majority of women of reproductive age globally including sub-Saharan Africa experience periodic menstruation fections . Howeverectivity ,35 and eal phase . In macaal phase ,26, yet Macaca nemestrina) model as well as samples from healthy women to study immune properties with HIV-risk factors over time. Using this approach we identified systemic type-1 inflammatory shifts with CCR5 CD4 T cell infiltration into the vaginal mucosa most often occurring at the late luteal phase when progesterone production is decreased. Furthermore, we mapped cycle-associated variations in HIV risk factors by constructing periodograms of CD4 T cell expression of CCR5 over consecutive reproductive cycles to find novel sinusoidal components with frequencies coinciding with progesterone oscillations. We also document that productive SHIV infection in pig-tailed macaques correlates most strongly with rising CCR5 expression frequency, activation-associated properties, and proinflammatory responses by CD4 T cells. By monitoring healthy typical-cycling women over the course of a menstrual cycle we found that in addition to a type-1 inflammatory bias during the late luteal phase, CD4 T cell populations were more likely to express chemokine receptors that play a vital role in T cell trafficking into type-1 inflammatory signals. Our results demonstrate that menstrual cycle regulation of the immune system control inflammatory-driven trafficking properties by HIV target cells and can dictate infection opportunities in the female reproductive tract (FRT).To evaluate HIV susceptibility throughout the menstrual cycle we used the pig-tailed macaque . Macaques were housed at the CDC under the full care of veterinarians in accordance with the Guide for the Care and Use of Laboratory Animals . All efforts were made to minimize suffering, improve housing conditions, and provide enrichment opportunities. Procedures were performed under anesthesia using standard doses of ketamine hydrochloride.2.250) of an R5-tropic SHIV162P3 isolate (obtained from the NIH AIDS Reagent Program) was added into the vaginal vault once a week for up to 15 weeks by non-traumatic inoculation. Challenges stopped once animal was determined SHIV RNA positive The repeated low dose exposures model was used to measure susceptibility from vaginal challenge 2.3Weekly plasma samples were analyzed for progesterone concentrations at the University of Wisconsin National Primate Center using an enzyme immunoassay  2.4This study was approved by the Emory University Institutional Review Board, the U.S. Centers for Disease Control and Prevention Institutional Review Board, and the Grady Research Oversight Committee. All participants provided informed consent.2.5T cell measurements from blood were acquired from healthy female volunteers in Atlanta, Georgia, participating in a prospective study assessing pharmacokinetics of the antiretroviral drug maraviroc as a candidate HIV prevention drug . Cellular data used in our analysis were generated from the control arm specimens of the study (not administered drug). Enrollment criteria were that participants were women were aged older than 18 years, HIV-negative, exhibiting normal menses (22–35 days cycle for at least 3 cycles), not using any systemic hormonal contraception or hormonal intrauterine device for at least 6 months prior and were not pregnant or breastfeeding for at least 3 months prior, had no signs or symptoms of vaginal infection at time of screening, had no history of cervical surgeries or procedures, and had no evidence of cardiac or liver disease. The age range of participants were 19–44 years old. Weekly blood specimens were collected at each visit, processed, and analyzed as described below. Progesterone and estradiol were measured from blood plasma using the Milliplex multi-species hormone magnetic bead assay  and read on a Bio-Plex® 200 System (Bio-Rad).2.6T cells subtracted from those values measured with MIP-1β incubation.To measure CCR5 receptor occupancy, PBMC were incubated in the presence or absence of the chemokine MIP-1β  which binds to CCR5 and inhibits antibody binding and detection of cell surface CCR5 otherwise measured without MIP-1β incubation, as previously described 2.7Blood was collected in BD Vacutainer® CPT™ cell preparation tubes (CPT) . Plasma and PBMC were separated by centrifugation using standard procedures and resuspended in saline solution for characterization. Vaginal lavage was performed as previously described ,46 speci2.8Soluble cytokines were measured from blood plasma using a bead-based immunoassay kit as indicated by the manufacturer . Samples were run on an LSRII flow cytometer. Data was acquired using FACS DIVA software (BD Biosciences) and analyzed using Biolegend® legendplex software.2.9f is frequency, T is time, and A, B, and C are fitting parameters determined by minimizing the sum of squared residuals. The fit is performed at many closely spaced frequencies over the range of interest. The Lomb-Scargle power  is computed at each frequency asThe Lomb-Scargle Periodogram was calculated for progesterone concentrations and CCR5 expression frequency over time using previously established algorithms ,48. The 2.10Cellular immune markers and levels of soluble inflammatory cytokines concentrations by cycle phase were fit using generalized estimating equations (GEE) with an exchangeable working correlation structure to compare repeated measures with continuous variables as previously described 2.11This study was supported by the U.S. Centers for Disease Control and Prevention, Atlanta, GA 30329 and NIH grants to Emory University . The Funders of this study had no role in study design, data collection, data analysis, interpretation, or writing of the manuscript.33.1T cell properties from the peripheral blood mononuclear cells (PBMC) of uninfected cycling pig-tailed macaques with progesterone (P4) concentrations in blood plasma. From CD4 T cells we monitored CCR5, and PD-1 frequency which are molecules associated with immune activated states [T cells (regulatory T cells: Treg) exhibiting immunosuppressive function T cell measurements into follicular (lowest P4 levels immediately preceding increases), luteal (rising or peak P4 levels), and late luteal (declining P4 levels post peak) phases of the menstrual cycle  as well as the levels of soluble inflammatory cytokines. From PBMC populations  were significantly decreased. Although this cDC population are a small proportion of circulating PBMCs and most likely immature, numerical changes in this population have been observed in response to tissue recruitment Because ulations a, b we fB cell and monocyte populations , inflammatory (CD14negCD16+) or intermediate (CD14+CD16+) phenotype Immune phenotyping of properties expressed by innate cell populations showed consistent trends with ulations b. From Ble, CD40 ,59 was rDue to the type-1 bias we detected from the cellular immune populations we further measured soluble proinflammatory properties over the menstrual cycle, by measuring key cytokines involved in type-1 inflammatory responses  c. Indivi3.3T cells into tissue sites experiencing type-1 inflammation including the FRT [T cells corresponded with migratory changes in local T cell properties at the site of HIV exposure in the FRT. To test this, we performed FRT barrier sampling in parallel to blood collection from pig-tailed macaques over a period of 24 weeks  within the epithelial layer of cutaneous tissue identified that CD69, a molecule which functions to inhibit cellular re-entry into circulation through antagonizing lymphatic and blood trafficking signals EΒ7 integrin epithelial cell adhesion molecule) was unlikely to be expressed on T cells that had recently immigrated into the epidermal layer [T cells and compared values by cycle phase  were most abundant at the late luteal phase compared with the follicular or luteal phase which predominantly yielded CD4 T cells expressing TRM phenotypes. Next, we examined HIV target cell populations by measuring CCR5 on FRT CD4 T cells  while also monitoring in parallel, circulating immune properties and P4 kinetics in order to identify immune trends at the time of infection , we found that the TOI consistently occurred during a state of increasing CCR5, PD-1, and TNFα expression from CD4 T cells while the immunosuppressive marker, FoxP3 was decreasing. To further understand this pattern we combined T cell susceptibility characteristics  into a composite analysis .To determine whether cycle-based variations in circulating HIV risk factors occur in humans we measured CD4 araviroc , and weraraviroc a. We meatibility ,71 as weBy comparing the change in HIV-risk properties over the course of a menstrual cycle, we observed mounting expression in all markers at the late luteal phase  with challenges that did not result in a productive infection, we found no overall difference in the frequency of immune properties at the late luteal phase or other cycle phases save an increased mean expression of CCR5. However, the frequency range of all T cell activation through antigen-specific recognition indicates little to no change within the target T cell population that might increase HIV infection risk in the FRT. However, accumulating work on “bystander” T cell activation demonstrates that type-1 inflammatory signals are sufficient to stimulate certain effector responses from T cells in the absence of TCR signaling, in particular non-cognate CD4 T cell trafficking into inflamed tissues sites [T cells found that recently immigrated T cells within some epithelial barrier sites do not express tissue retention properties such as CD69 and maintain the ability to respond to migratory cues that would facilitate reentry from the tissues back into the circulation through afferent lymphatic trafficking [As the menstrual cycle is not the result of an infection, it is reasonable to suppose that a lack of archetypal CD4 es sites ,76. Furtfficking . It is tT cell immune properties might better inform upon detectable and potentially uniform markers of increased HIV risk.In regard to other HIV risk factors in women such as progestin-based hormonal contraception  and STI'To conclude, our study provides valuable new insights into the role of the immune response in vaginal HIV transmission under menstrual cycle regulation. We define for the first time, to our knowledge, periodic shifts in type-1 inflammatory behaviors that are associated with increased susceptibility to HIV infection through target cell recruitment into the site of pathogen exposure.We posit that novel HIV prevention strategies that reduce local inflammation events in the FRT, such as occurs under menstrual cycle regulation, may help prevent HIV transmission events in women ASK performed experiments, analyzed, and interpreted data. FH performed statistical and modeling analysis. JB and EWB designed, analyzed, and interpreted periodogram data. ASK, IM, and JGGL designed the NHP study. AS, ASK, and IO designed the human study. SS performed and analyzed cytokine testing. JB, AS, FH, SS, EWB, IO, and IM provided critical advice and helped with the interpretation and discussion of the results. ASK and JGGL wrote the manuscript, with contributions from all authors.Access to data generated and analyzed in this study will be provided upon reasonable request to the corresponding author.The authors declare no competing interests.


These findings could not be reproduced using MSCs alone. These results demonstrate cECFC treatment exerts beneficial effects on multiple cellular components in the FGR brain and may act as a neuroprotectant.The foetal brain is particularly vulnerable to the detrimental effects of foetal growth restriction (FGR) with subsequent abnormal neurodevelopment being common. There are no current treatments to protect the FGR newborn from lifelong neurological disorders. This study examines whether pure foetal mesenchymal stromal cells (MSC) and endothelial colony-forming cells (ECFC) from the human term placenta are neuroprotective through modulating neuroinflammation and supporting the brain vasculature. We determined that one dose of combined MSC-ECFCs (cECFC; 10 FGR is often caused by placental insufficiency, resulting in an inadequate supply of oxygen and nutrients to the developing foetus in utero4. The foetal brain is particularly vulnerable to FGR conditions and subsequent abnormal neurodevelopment is common5. Adverse neurodevelopmental outcomes include learning and behavioural disorders and cerebral palsy, which have lifelong medical and financial consequences10. Due to medical advances, more FGR babies now survive, although many remain at risk of these neurodevelopmental disabilities. No treatments currently exist to protect the FGR newborn brain.Foetal growth restriction (FGR) occurs in around 3–15% of pregnancies with even greater rates in developing countries16. Recent animal studies have shown that inflammation plays a key role in these alterations19. High concentrations of pro-inflammatory cytokines have been reported in the blood of preterm FGR infants two weeks after birth20. Increases in pro-inflammatory cytokines are correlated with adverse neurological outcomes at 2 years of age in preterm small for gestational age newborns21. An increase in inflammatory mediators in the blood may affect the brain because of the disruption to the structural and functional integrity of the neurovascular unit (NVU) in the FGR newborn24 and toxic mediators may pass freely in and out of the brain. Therapeutic targeting of inflammatory pathways and the NVU hold promise as neuroprotectants in FGR newborns25.Both neuronal and white matter alterations are observed in both the human FGR infant and animal models of FGR27. As the NVU is developed in the newborn, maintaining the structural and functional integrity of the NVU is likely an essential element in neuroprotection in the newborn.The NVU separates the brain from the blood circulation and is composed of vascular endothelial cells, glial cells (astrocytes and microglia), neurons, pericytes and the basement membrane. The cells of the NVU share close and complex interactions that are crucial in maintaining blood–brain barrier (BBB) integrity and cerebral homoeostasis, ensuring healthy brain development. Disruption to the NVU plays an essential role in progression of numerous central nervous system (CNS) pathologies allowing toxic substances, such as pro-inflammatory cytokines to infiltrate the brain, which can exacerbate neuroinflammation and injure neurons and white matter28. They reside throughout the vasculature, have self-renewal as well as a capacity to engraft forming both de novo neo-vessels as well as chimeric vessels within ischaemic sites of the host, facilitating reperfusion and initiating tissue regeneration30. They have been isolated from numerous sources, classically the umbilical cord blood (UCB) and the term placenta32. The vasculogenic capacity of ECFCs is greatly enhanced upon co-incubation or co-delivery with mesenchymal stromal cells (MSCs), a process known as priming33. Moreover, engraftment of ECFCs if combined with MSCs (cECFC) can bypass the host immune system34. Recent evidence suggests that cECFC outperformed MSCs alone in reperfusing ischaemic limbs through neo-vascularisation29. Additionally, this combination of cECFCs did not require additional immunosuppressive therapy in immunocompetent animals29 highlighting their potential use as an allogeneic off-the-shelf cell therapy.Endothelial colony forming cells (ECFCs) are the vascular precursors, devoid of hematopoietic and myeloid contamination35. We hypothesised that the cECFC treatment provides neuroprotection in the FGR newborn. Using our established preclinical piglet model of FGR37, we assessed whether one dose on the first day of life in a term FGR piglet would regenerate damaged vasculature, restore the NVU, reduce brain inflammation and improve adverse neuronal and white matter changes present in the FGR newborn piglet brain.In the present study, we obtained pure foetal MSC and ECFC from the healthy human term placentap < 0.0001; brain p < 0.05; Table p = 0.735), brain weight (p = 0.999) or liver weight (p = 0.721) between NG piglet groups  compared with normally grown (NG) piglets  in the FGR group  stem cells (either cECFC or MSC) versus Sham. Core body temperature was regularly monitored following stem cell administration, with altered body temperature being a hallmark of immunological or inflammatory response. No differences in body temperature (°C) were evident between any of the five experimental groups . Normal temperature for a piglet is approximately 38.5 °CBiodistribution of human stem cells was detected in the piglet brain at P4 based on labelling with a human-specific Lamin A/C antibody. Cells positive for Lamin A/C were found to engraft into the vasculature of microvessels as well as sporadic instances of parenchymal labelling in FGR + cECFC brain Fig. . No Lamic.f. 4.45 ± 0.22%, p = 0.009; Fig. p = 0.048; Fig. 39, closely reflected the observations of Col IV labelling with significantly less labelling in FGR brains (2.21 ± 0.12%) compared with NG (3.08 ± 0.19%) , and significantly recovered in FGR following cECFC treatment  with significant restoration upon cECFC treatment . Although we could not find any evidence of cells in the NG + cECFC piglet brain comparison of NG and NG + cECFC of vessel length and branching showed no differences suggesting cECFC administration does not promote unwarranted angiogenic effects , which contributes to ~50% of the basement membrane of the NVU. All groups displayed robust labelling of Col IV across the length of the vasculature, however analysis of area labelled showed a significant reduction in the FGR brain compared with NG  and was significantly reduced upon cECFC treatment  and IgG (~155 kDa) extravasation to assess whether cECFC-treated animals demonstrated improvement to BBB permeability. FGR brains displayed predominantly perivascular labelling of albumin Fig.  with no Similarly, we examined permeability of the BBB with the endogenous immunogobulin IgG which is a larger molecular weight protein. Labelling of IgG showed sporadic extravasation into the parenchyma of FGR brains , with juxtavascular astrocytes in these regions displaying activated morphology and reduced end-feet contact with the vessel Fig. ’. NG and+ cells and reduced S100β-GFAP positive cells along the vasculature compared with NG animals  and GFAP to investigate altered astrocyte interaction with microvessels. In NG animals, we observed an abundance of S100β-positive juxtavascular astrocytes and co-localisation with GFAP with consistent contact of end-feet with the vasculature Fig. . FGR dem41. We examined if the administration of cECFC modulates the pro-inflammatory environment of the FGR brain. Labelling with the microglia marker Iba-1 revealed microglia in the parietal cortex of NG brain that displayed characteristic ramified (resting) morphology, with long fine process extensions and even distribution across the cortex  and activated microglia (p < 0.0001) in FGR when compared with NG  and activated microglia (p < 0.0001) compared with FGR brain  with significantly lower numbers of total microglia  arrays of chemokines and cytokines, we report altered expression of both pro- and anti-inflammatory genes in the FGR cortex compared with NG brains Fig. . CriticaL10 Fig. . Anti-inL10 Fig. . Our fin17. FGR cortex demonstrated regions sparse in neurons, as labelled with the neuronal nuclei marker (NeuN) and the structural neuronal marker (MAP2)  and MAP2-positive labelling resembled that observed in NG animals  Fig. . In contP2) Fig. . Quantif37. We therefore proceeded to determine whether administration of cECFC could also modulate apoptotic activity in the FGR brain.We have previously demonstrated an increase in cellular apoptosis in the FGR brain and the ability of the anti-inflammatory ibuprofen to ameliorate this increasep < 0.001; Fig. p < 0.001; Fig. p < 0.001; Fig. p = 0.002; Fig. p < 0.0001) in FGR compared with only 29% in NG animals, suggesting that neuronal cells have been flagged to undergo apoptosis  animals (p = 0.201).FGR brain showed a significantly higher number of cells positive for cleaved caspase-3 (Casp3) compared with NG brain , neurofilament (NF), and the pan-oligodendrocyte marker (Olig2) in white matter. NG brains demonstrated well organised and consistent labelling for each of these markers along lengths of the white matter tracts, with strong MBP and NF co-localisation  follow both umbilical cord blood (UCB) and ECFC treatment at 7 days and 12 weeks after treatment in neonatal hypoxic-ischaemic encephalopathy (HIE) rat model46. The authors also showed an increase in cerebral blood flow at 12 weeks for both treatments demonstrating evident functional improvement to the cerebral vasculature.MSC only treatment had no significant effect on improving the vasculature in the FGR brain. However, cECFC treatment not only increased vessel density it also improved vascular length in the FGR brain. cECFC treatment did not show a significant difference in vessel branching suggesting cECFC treatment does not promote unwarranted angiogenesis. These findings support the potential of cECFCs to improve cerebrovascularisation in the FGR neonate. On the contrary to our findings of reduced endothelial cells in FGR brain, an increase in endothelial cells (GLUT-1) was reported in both FGR and FGR UCB treated lambs in comparison to normally grown lambs24.There was evidence of BBB disruption in the piglet FGR brain, with endogenous proteins albumin located in the perivascular space and extravasation of IgG into the brain parenchyma. Juxtavascular astrocytes displayed activated morphology including hypertropic end-feet, and in some instances we found absence of vessel–glial contact. cECFC treatment improved BBB integrity with low-intensity albumin labelling restricted to the vessel lumen and juxtavascular astrocytes reverting to a ramified astrocyte morphology. Even though cECFC treatment did not significantly reduce the percentage of vessels with perivascular albumin labelling it did significantly reduce albumin labelled area compared with FGR. In a lamb FGR model, albumin was shown to enter the tissue parenchyma which we did not observe in our current study, however, the authors also showed a reduction in albumin extravasation following UCB treatment supporting the effect of stem cells on BBB integrity24. Following cECFC treatment, the FGR piglets displayed similar characteristics to NG animals with an abundance of juxtavascular astrocytes in consistent contact along the vasculature. This combination of ECFCs and MSCs may be working together to stabilise the NVU and by reducing the pro-inflammatory environment which in turn results in juxtavascular astrocytes returning to their normal function at the NVU. In the FGR lamb, while UCB treatment did not alter astrogliosis, the authors observed an increased association of smooth muscle proteins of the basal lamina with pericytes in the NVU24.Impaired astrocyte end–feet interaction with neurovasculature may be associated with altered NVU integrity in the FGR brain48. We and others have reported that early and persistent inflammation in the brain is associated with glial activation in the FGR neonate37. In the current study we have demonstrated an overt glial response in the FGR piglet brain after birth with an increase in activated microglia and astrocytes in both the grey and white matter. Microglia responded to both MSC only treatment and cECFC treatment with a decrease in both the number and activation of microglia throughout the brain parenchyma. However, cECFC treatment was more efficacious than treatment with MSC alone at minimising astrocyte activation in the FGR piglet brain. The MSC alone treatment results are corroborated by an FGR lamb study24. The authors demonstrated when allogeneic UCB mononuclear cells (25 million/kg) was administered 1 h after birth, activated microglial cells were reduced but not astrocytes in the white matter 24 h post-treatment24. An FGR rat study also showed that umbilical cord-derived mesenchymal stromal cells (UC-MSC) (1 × 105) did not affect the number of astrocytes 7 days after treatment49. Astrocytes have many functions in the brain and are located in the brain parenchyma as well as at the NVU. The positive response of glia in the tissue parenchyma we observe following cECFC treatment may be due to restoration of cerebrovascularisation and NVU integrity. As the combination of ECFCs and MSCs are stabilising the NVU and increasing the anti-inflammatory state (discussed below), these interrupt the perpetual inflammatory environment in the brain. However, one study showed a similar inflammatory response between UBC and ECFC treatment in a neonatal HIE rat model46. This study administered stem cells intraperitoneally 48 h after insult—105 ECFC or 107 UCB. At 7 days post treatment, they demonstrated a reduction in neuroinflammation (iNOS) after both treatments, but no change in astrocyte numbers. However, at 12 weeks a significant reduction in GFAP-positive cells was reported in both groups demonstrating a delayed decrease in astrogliosis. Demonstrating long-term studies are necessary to determine the longevity of treatment effects.Clinical and animal studies provide evidence that inflammation is perpetuated long after birth, providing the opportunity of targeting inflammation soon after birth which may be critical to reducing detrimental inflammatory events and subsequent injury to the FGR brain47. The current study concurs with previous reports showing an increase in pro-inflammatory cytokines in the FGR brain and an altered anti-inflammatory profile37. We have previously demonstrated anti-inflammatory treatment (ibuprofen) in the FGR newborn piglet reduces levels of pro-inflammatory cytokines17, however in the current study cECFC treatment exerted a greater effect on increasing anti-inflammatory cytokines. The prominent increases in anti-inflammatory cytokines may be due to reprograming of the microglia into an anti-inflammatory state. Recent in vitro studies suggest that a key therapeutic function of MSCs is their ability to reprogramme brain microglia into an anti-inflammatory state characterised by increased phagocytic activity and upregulated expression of anti-inflammatory mediators, which in turn contributes to reduced inflammation and promotes tissue repair50. These findings are corroborated in an FGR rat study examining UC-MSC treatment49. The authors demonstrate no significant effect of treatment on pro-inflammatory microglia however anti-inflammatory microglia were significantly increased in the treatment group compared with sham, demonstrating an anti-inflammatory effect of stem cell treatment in the FGR rat. An FGR lamb study reported a reduction in the pro-inflammatory cytokine TNFα following UCB treatment, similar to the response we demonstrated following cECFC treatment. However, they report no effect of UCB on any other pro-inflammatory cytokines measured24. This finding is further supported by recent study which has shown the ability of ECFCs in suppressing T-cell proliferation and programming toward less pro-inflammatory phenotypes. The study demonstrated the significance of the TNFα/TNFR2 pathway in mediating this response51. Recent study has shown that priming of ECFCs with TNFα (1 ng/mL) prior to administration was able to further enhance the immunosuppressive phenotype, potentially resulting in prolonged presence in vivo to enhance regeneration52. We demonstrated that cECFCs have a combined effect of reducing pro-inflammatory cytokines while concurrently increasing anti-inflammatory cytokines. MSCs and ECFCs target different cell populations to maintain a healthy brain environment, and therefore this multicellular effect may be the key to modulating the inflammatory state in the brain.Increased glial cell activation is associated with a pro-inflammatory environment in the FGR brain53. Ongoing neuronal and white matter impairment is also evident in the FGR piglet brain after birth at a microscopic level36. In the current study, we saw regions sparse in neurons  and disruption to mature myelin and oligodendrocytes throughout the white matter in the FGR brain. Inflammation is a key driver of neuronal and white matter injury and when postnatally targeted with an anti-inflammatory intervention as we have done here with cECFCs and previously with the anti-inflammatory ibuprofen, a corresponding reduction in neuronal and white matter disruption is evident17.Clinical imaging studies describe persistent grey and white matter disruption in the human FGR neonate that are associated with developmental disabilities at 1 year of age37. cECFC treatment reduced neuronal cells that labelled for both caspase-3 and caspase-9. The modulation of the inflammatory response following stem cell administration likely contributed, either directly or indirectly, to the reduced apoptotic activity in the FGR brain, An HI rat study reported similar reduction in apoptotic cells (TUNEL) and concurrent recovery to neuronal cell counts 7 days after treatment with either UCB or ECFCs46. This recovery to neurons could be due to reversal of apoptosis or reinstatement of normal development.The loss in neurons observed in the FGR brain may be attributed to pro-apoptotic events as a result of the pro-inflammatory environment. We reported a significant increase in the early apoptotic initiator, caspase-9 in neurons in the FGR brain. We have previously demonstrated an increase in cellular apoptosis and the ability of the anti-inflammatory ibuprofen to ameliorate this increase36, but also disruption to oligodendrocytes as observed in other animal models of FGR55. We demonstrate partial loss of myelination along the axon in the FGR brain with increased apoptotic oligodendrocytes. White matter tracts in the FGR brain displayed more dispersion and varied orientation suggesting that loss of oligodendrocytes in FGR results in decreased and disorganised myelination of white matter tracts which likely contributes to the long-term white matter alterations reported in the FGR brain56. Administration of cECFC treatment promoted oligodendrocyte survival, maintaining axonal myelination and organisation of the white matter tracts. Only one other study has examined white matter response to stem cell treatment in the FGR newborn. This study in the FGR rat showed no difference in white matter volume following UC-MSC treatment49. However, this FGR model may not be appropriate to study FGR with no detectable white matter injury. White matter injury is a hallmark feature of FGR57 as such there should be noticeable white matter changes in any FGR animal model. In the current study, we demonstrate recovery to both grey and white matter cellular impairment within the parietal cortex following cECFC treatment in the FGR piglet brain.We show not only disruption to myelination as previously demonstrated58. A similar response is observed in the FGR piglet where significant brain injury is observed at 104 days gestation  but not prior36, demonstrating this brain injury commences during the third trimester. As this injury occurs later in gestation and a large percentage of FGR fetuses are not detected until the time of birth, stem cell treatment around the time of birth is a feasible and appropriate option to protect the FGR newborn from sustained brain injury.The developmental progression of FGR brain injury likely occurs after 27 weeks gestation as reported in a human studyThe use of postmortem histological examination allows for in-depth analysis of key structural changes contributing to neuropathology, however, there are limitations with the range of phenomena one can observe. In the present study, we utilised several markers to interrogate interactions between cell types following cECFC treatment in the FGR brain, however, these do not allow mechanistic insight of cell-cell communication. This limitation is highlighted by our inability to separate vascular and neuronal effects of the cECFC treatment. It is unknown whether the benefits we observed following treatment are a direct consequence of increased vessel density and reduced BBB permeability or whether the cECFCs directly communicate with other cells such as astrocytes or microglia. Further in vitro studies would address this limitation. We observed no sex-related differences in any of the analyses undertaken. However, another limitation of small animal numbers in the current study make it difficult to fully discern sex differences.62. However, with the addition of ECFCs to aid in vascular regeneration, this enhanced stem cell preparation has the potential to significantly improve brain outcomes for FGR neonates. Previous studies have shown cECFCs are not rejected in immunocompetent animal studies29 demonstrating important findings where these cells can be used in allogeneic situations. The use of healthy placentas and in an off-the-shelf allogenic scenario would ensure continued availability and provide a more appropriate solution than using autologous cells from a pathologic FGR placenta63.MSC therapy alone has variable neuroprotective effects through modulation of inflammation in small animal models of cerebral ischaemiaWe regard cECFC treatment as a promising multicellular approach to protecting the FGR newborn brain. This combination of fetally derived stem cells improved the brain microenvironment only 3 days after treatment in the FGR newborn piglet. Future preclinical studies to confirm long-term efficacy and safety are essential in the success of human clinical trials for stem cell treatment in the FGR newborn.Approval for this study was granted by The University of Queensland Animal Ethics Committee (UQCCR/420/17) and was carried out with respect to the National Health and Medical Research Council guidelines  and ARRIVE guidelines. Animals care was in accordance with institutional guidelines.n = 36; <18 h) were collected from the UQ Gatton Piggery monitored and cared for at the Herston Medical Research Centre until day of euthanasia on postnatal day 4 (P4). Litter matched pairs were obtained from multiple sows (n = 14 litters). Piglets were divided into five experimental groups with pigs randomly assigned to treatment groups: NG (n = 8), FGR (n = 8), FGR + cECFC (n = 8); FGR + MSC (n = 5), and NG + cECFC (n = 7); with equal males and females in each group. FGR piglets were defined by birth bodyweight (<10th percentile on the day of birth) and confirmed through brain to liver weight ratio (B:L) ≥ 1 assessed post-mortem at P465.Newborn Large White piglets . Brains were transcardially flushed with phosphate-buffered saline, and brain tissue was collected, weighed, hemisected and coronally sliced. The right hemisphere sections were immersion fixed in 4% paraformaldehyde as previously described35. The isolated placental CD34+ single-cell suspension was incubated with human CD34-phycoerythrin (PE) , human CD45-FITC  and human CD31-V450 BD Biosciences: dilution 1:30) for 20 min at 4 °C. Cells were flow sorted using FACSAria Fusion (Becton Dickinson). Cell doublets were removed and 7-amino-actinomycin D  was used to exclude dead cells. Fluorescence minus one (FMO) control was used in gating the population of interest. To remove any remaining contaminating CD45+ cells from the hematopoietic lineage, only CD45-CD34+Hi population was gated and further sorted according to their level of CD31 expression. The population of interest, the CD31- and CD31Int populations were sorted directly into 100% foetal bovine serum  before being plated for cell culture.Primary conjugated antibodies were used for fluorescence-activated cell sorting (FACS). Placental tissues were processed, and single-cell suspension was prepared as reported previously32. ECFC and MSC preparations were sourced from three different donors. Human term placenta were obtained with written informed consent from healthy women undergoing caesarean deliveries at term (38–39 weeks of gestation) at the Royal Brisbane and Women’s Hospital, as approved by both the University of Queensland and the Royal Brisbane and Women’s Hospital human research ethics committees (RBWH HREC/09/QRBW/14 & UQ 2009000508). This approval allows for the isolation and use of any stem cell populations obtained from the placental tissue.Human placental foetal ECFC and MSC were isolated using our previously published protocol35  (Lonza) with 10% of FBS. The isolated cells demonstrated similar functional profiles. The foetal ECFC and MSC were characterised via flow cytometry using a suite of cell surface markers to ensure their correct phenotype. The cell surface markers include: CD144, CD31, CD34, CD105, CD44, CD146, HLA-ABC, HLA-DR, HLA-DR/DP/DQ, CD45, CD29, CD73, CD90 and expression was as expected and previously observedets Fig. . In a fuWe used magnetic resonance imaging (MRI) techniques to determine whether we could detect in vivo brain alterations in the FGR piglet at postnatal day 4. Piglets were anesthetised with isoflurane (1–3%) mixed with oxygen (2 L/min) for the duration of the scanning protocol. Each piglet was placed in a 300 mm bore 7T ClinScan MR scanner , running Siemens VB17. A 150 mm ID MRI rf coil was used to acquire the dynamic images of sagittal, coronal and axial slices. Images were obtained at TR/TE 8500/60 ms, 1.6 mm slices thickness, 64 × 64 acquisition matrix. Region of interest over the parietal cortex was defined on the T2 map and applied to the ADC map and raw values extracted.3 voxel using single-voxel spectroscopy with the following parameters: TR = 6000 ms, TE = 60 ms and 128 averages. Metabolite spectra were exported, processed, and analysed using the Advanced Method for Accurate, Robust and Efficient Spectral Fitting tool within jMURI and manually phased67. Before quantification, apodisation (10 Hz) was applied to the N-acetyl aspartate (NAA), creatine (Cr) and choline (Cho) peaks with Lorentzian peaks. Peak areas of metabolites were estimated using Lorentzian profiles and a priori knowledge with soft constraints . NAA was set as a reference at 2 ppm chemical shift. Peak area ratios were calculated for NAA/Lac, NAA/Cho, NAA/Cr, Lac/Cr, Lac/Cho, Lac/NAA and Cho/Cr.1H-MR spectroscopy were obtained using the 7T Bruker/Siemens whole-body. A single spectrum was acquired the frontoparietal region of the brain from a 10 mmThere were no overt differences in brain structure as assessed with T2 and ADC analysis between FGR and NG animals. There were no significant MRS alterations in metabolite levels between NG and FGR. Administration of cECFCs did not result in any noticeable changes when compared with NG and FGR . For vessel structure and glial interaction analyses brain sections of 12 µm thickness were used. Sections were affixed to Menzel Superfrost Plus adhesive slides and dried overnight at 37 °C. All sections were dewaxed and rehydrated using standard protocols followed by heat-induced epitope retrieval with 10 mM citrate buffer (pH 6) or TRIS-EDTA buffer (pH 9) at 90 °C for 20 min before cooling to room temperature (RT). Sections were blocked with 5% donkey serum in PBS with 0.5% Triton-X 100 for 1 h at RT. Primary antibodies  were embedded in paraffin and sectioned at 6 µm .Acquisition of immunolabelled sections was performed using a Zeiss Axio Microscope fitted with an Axiocam503 camera and ZEN 2012 software, using either an EC Plan-Neofluar 20x/0.50 M27 (FWD = 2.0 mm) or EC Plan-Neofluar 40x/0.75 M27 (FWD = 0.71 mm) objective. Four pictomicrographs  of the parietal cortex and white matter regions were captured for analysis from each section. For all markers, replicates were conducted with sections separated by at least 70 µm. All imaging and analyses were conducted under blind conditions . Microglia were manually counted and categorised with respect to morphology as per previous studies37. Total RNA from frozen parietal cortex samples was isolated using RNeasy Tissue Mini Kit (Qiagen). Yield and quality were determined using a NanoDrop spectrophotometer (ND-1000 system). Reverse transcription kits  were used for cDNA synthesis. cDNA was pooled for each experimental group giving equal concentrations from every animal in the pooled sample. Pooled cDNA was combined with RT2 SYBR Green qPCR Mastermix (Qiagen) and loaded into the Pig Inflammatory Cytokines & Receptors RT2 ProfilerTM PCR Array . The qPCR reactions were performed using a Qiagen Rotor-Gene Q real-time cycler. The amplified transcripts were quantified with the comparative CT method using actin, gamma 1 (ACTG1) mRNA expression levels for normalisation. The same CT threshold value was used across all arrays to allow comparison between runs. Each experimental group was run across two arrays with samples randomly assigned to each array.qPCR was conducted as previously reportedp < 0.05. Indicated sample sizes (n) represent biological replicates including individual samples and have been listed in the figure legends where appropriate.Unless otherwise specified, Two-way ANOVA with the Tukey post-hoc analyses was used to determine differences between NG and FGR animals under non-treated and stem cell treated conditions . Results are expressed as mean ± SEM with statistical significance accepted at Further information on research design is available in the Supplementary InformationReporting Summary


Colonic enteroendocrine cells (EECs) store and release potent anorectic hormones that are key regulators of satiety. EECs express multiple nutrient sensing receptors, particularly for medium-chain fatty acids (MCFAs): GPR84 and FFAR4. Here we show a non-surgical approach with targeted colonic delivery of MCFA, which induces EEC and neuronal activation leading to anorectic effects.A randomised, double-blind, placebo-controlled, cross-over study was performed in obese adults given combined GPR84 and FFAR4 agonists in colonic release capsules before meals. We measured serum hormones, energy intake and appetite perception. Cell type, activation by agonists and hormone/serotonin release were determined in human colonic explants. Mouse colonic afferent nerve responses to nutrients/mediators were recorded electrophysiologically.Subjects receiving GPR84 and FFAR4 agonists had reduced overall calorific intake and increased postprandial levels of PYY versus placebo. Receptors including GPR84 and FFAR4 were coexpressed on human colonic EEC. Activation of GPR84 exclusively induced intracellular pERK, whereas FFAR4 selectively activated pCaMKII. Coactivation of GPR84 and FFAR4 induced both phosphoproteins, and superadditive release of GLP-1 and PYY. Nutrients and hormones convergently activated murine colonic afterent nerves via GLP-1, Y2 and 5-HT3 receptors.Colonic GPR84 and FFAR4 agonists reduce energy intake and increase postprandial PYY in obese adults. Human colonic EECs coexpress these receptors, which activate cells via parallel intracellular pathways and synergistically evoke hormone release. Further synergism occurs in sensory nerve responses to MCFA and EEC mediators. Thus, synergistic activation of colonic endocrine cells via nutrient receptors is an important target for metabolic regulation.NCT04292236. The most effective current treatment for obesity and type 2 diabetes is gastric bypass surgery, but it is restricted in availability, irreversible, costly and may cause harm. Shunting of undigested food (including medium chain fatty acid (MCFA)) to the distal gut triggers hormone release by activating enteroendocrine cell (EEC) via specific nutrient sensing receptors.Boosting colonic levels of the bacterial product propionate increases hormone release and reduces energy intake in humans, further implicating the colon as a site for metabolic regulation.Administering a colonic release formulation of MCFA  before meals reduced food intake and boosted plasma PYY in obese adults without adverse effects.MCFA cooperatively stimulate nutrient receptors, which synergise through different intracellular pathways in EEC to maximise hormone release. This action on colonic EEC leads in turn to synergistic activation of afferent nerves projecting to the central nervous system (CNS).The stimuli used in this study were safe and unrestricted for human use; furthermore, side effects were absent.Later phase clinical trials in obesity could begin soon after refinement of capsules. We also now understand the biology of an important pathway for regulation of energy intake.Gastric bypass surgery, Roux-en-Y gastric bypass (RYGB) in particular, is currently the treatment of choice for obesity and associated type 2 diabetes.Energy intake, appetite and satiety are controlled by several sensory, mechanical and psychological factors with both short-term and long-term effects. EECs are one important molecular regulator of appetite as they store and release orexigenic  and anorexigenic hormones /transmitters (5-HT) that balance feelings of hunger and satiety.EECs, expressed from stomach to rectum, are traditionally classified according to hormone/peptide content, although emerging evidence suggests hormonal expression shifts during stages of cell maturation.et alThe development of new effective medical treatments for obesity will require delineation of molecular mechanisms that underpin the normal physiological processes of nutrient-mediated appetite regulation. Sumithran The aim of this study was to determine how dual nutrient stimulation may impact EEC activity using in vivo, ex vivo and clinical trial methodologies. The current study, a proof-of-concept trial in obese volunteers, demonstrates that coadministration of colonic GPR84 and FFAR4 agonists decreases food intake and increases PYY release in active versus placebo. We show that the underlying cellular mechanism is the expression of multiple nutrient sensing receptors on EECs that when stimulated concurrently, synergistically boosts cell activation and anorectic hormone release. Using mouse neuronal recordings, we demonstrate that peripheral afferent neuronal activity is subsequently increased in response to nutrient content and EEC activity.www.clinicaltrials.org.All subjects provided informed, written consent to participate. It was also registered with Volunteers for the clinical trial were recruited from the general public after newspaper coverage of the study. Patients undergoing resection were asked to participate in the study on the day of surgery, while biopsies were collected from patients visiting the Endoscopy Unit, both at the Royal London Hosptial, Whitechapel. All patients were told of the types of studies that were being carried out and how their contribution would benefit understanding of essential gut processes. Patients were not involved in the study design but were informed that results would be available by contacting reserachers directly  as well as in open access peer review publications.2 were invited to participate. Potential subjects were excluded if they had previous gastrointestinal surgery, major health problems and/or were taking medication for type 2 diabetes.We hypothesised that colonic delivery of endogenous GPR184 and GPR124 agonists would reduce food intake and increase circulating levels of PYY and GLP-1. The predefined primary outcome was change in calorific intake, and secondary outcomes included changes in levels of circulating hormones, PYY, GLP-1 and ghrelin. Men and women aged 18–60 years with a body mass index (BMI) of 30–40 kg/mThe study used a double-blind, randomised, placebo-controlled, cross-over design. Twenty subjects were randomised to receive either active treatment or placebo with a 4-week washout period to account for menstrual fluctuation that can influence appetite with sample size calculated based on previous studies examining similar outcomes to colonic nutrient treatment.et al.Active capsules contained a total of 500 mg 3′3 diindolylmethane (DIM) , 2100 mg alpha linolenic acid (ALA) contained within perilla oil  and 2400 mg lauric acid (LA) (Sigma Aldrich). Note: DIM (10 mM) and ALA (100 mM) both stimulated human ex vivo colonic tissue in Ussing chambers leading to activation of pERK and pCaMKII pathways, respectively (data not shown). Visually identical placebo capsules were filled with methylcellulose (Sigma). All capsules were coated with Phloral  to ensure capsule delivery and disintegration in the colon. Colonic arrival and release of contents coated with Phloral has been demonstrated in volunteers as described by Varum The first set of capsules was given 60 min prior to breakfast (time-point 0 hours) and the second 60 min before lunch (time-point 5 hours). The maximum calorific intake for breakfast was 903 kcal and lunch was 1340 kcal, and subjects were asked to eat as much as they desired up to this maximum. Following completion of the meal, the amount of food consumed was quantified and calorific intake calculated. All study visits commenced between 08:00 and 09:00 and were conducted at the Wingate Institute of Neurogastroenterology. Throughout the visit, the nurse/investigator maintained regular communication with the subject to encourage good compliance.Prior to blood collection, EDTA Vacutainer tubes were cooled on ice. Immediately after blood collection, 10 µL of DPPIV inhibitor (50 Nm), fluoxetine (100 Mm) and moclobemide (100 Mm) were added to prevent hormone/5-HT degradation. Plasma was collected by centrifugation (10 min at 1000×g), and 1 mL aliquots were stored at −20°C. Total GLP-1, total PYY, active ghrelin and gastric inhibitory polypeptide (GIP) were quantified using a human multiplex kit according to manufacturer’s instructions . 5-HT was measured using an ELISA  as previously described.Full thickness samples of human ascending colon were obtained from 4 to 6 patients  undergoing surgery for colon cancer at The Royal London Hospital . All specimens were obtained from patients with non-obstructive tumours that were not occuring in the context of inflammatory bowel disease. Specimens were taken with the permission of the histopathologist following macroscopic examination and were a minimum of 10 cm away from tumour, resection margins or lymphatic drainage field.Following surgical resection, tissues were fixed in 4% paraformaldehyde (4°C overnight). Tissues were cryoprotected in 30% sucrose/PBS then mounted in optimum cutting temperature medium. Tissue sections of 10 µm were cut on a Cryostat (Leica CM1860), incubated with blocking buffer  for 1 hour, before primary antibodies were applied overnight (4°C). Antibody details are provided in 10.1136/gutjnl-2020-323219.supp1Supplementary dataHuman colonic mucosal sheets (from cancer resections) were divided into 1×1 cm segments, and each segment was mounted in an Ussing flux chamber. We used a method developed by our group for optimised nutrient stimulation of human colonic mucosal sheets.As previously described,Nerve bundles running alongside mesenteric blood vessels that innervate the proximal region of the mouse colon were identified under a dissecting microscope and drawn into a borosilicate glass suction electrode  prefilled with Krebs buffer. These comprise both vagal and splanchnic afferent nerves, which were both therefore recorded. However, activation by nutrients and gut hormones is lacking in most studies of spinal afferents,For cell counting following immunohistochemistry experiments, five fields of view at 40× objective, covering the entire section so as to remove image acquisition bias, were analysed from 4 to 6 individual tissue samples. Cells clearly immunoreactive positive, found within colonic crypts and captured completely in field of view, were counted as previously described.In electrophysiology experiments, analysis was performed offline using the spike sorting function of Spike2 to discriminate afferent nerve activity of individual units.A sample size of 20 subjects was estimated based on calorific intake data from a previous study.2 and mean age: 48.9±2.0 years). To assess the effect of combined GPR84 and FFAR4 agonist stimulation, we used 3′3 diindolylmethane and LA as natural GPR84 agonists and perilla oil  as a natural FFAR4 agonist. No adverse effects, including nausea or vomiting, were reported by the 20 volunteers when given the active or placebo capsules. Targeted colonic delivery of these compounds significantly reduced calorific intake of the lunch meal  and overall calorific intake , a 13.3% and 12.4% mean reduction (compared with placebo), respectively  were recruited to the study  30 but no10.1136/gutjnl-2020-323219.supp3Supplementary dataThis raised the question of whether different intracellular pathways may contribute synergistically to boost EEC responses to nutrient receptor activation. Incubation with different nutrient stimuli led to different patterns of EE mediator release from colonic biopsies. LA activated release of total PYY, active GLP-1 and 5-HT  but not 3, GLP-1 and Y2 receptors are expressed on vagal afferent fibres and mediate nutrient-related signals from the gut to the central nervous system  responded to both. The observation that individual single units are able to respond to each of these mediators indicated that receptors for each coexist on the same afferent endings . FurtherUsing a translational approach, we demonstrate that human colonic EECs are a key molecular target for maximising peripherally mediated appetite regulation and offer a novel therapeutic avenue for limiting food intake. We found EECs express multiple nutrient sensing GPCRs and that selective stimulation of GPR84 and FFAR4 in vitro results in activation of separate intracellular pathways leading to synergistic release of anorectic hormones PYY and GLP-1. These hormones may then activate peripheral nerve endings to increase neuronal activity. Importantly, in a proof-of-concept clinical trial in obese volunteers, we demonstrated that co-administration of GPR84 and FFAR4 agonists led to reduced food intake and increased levels of circulating PYY.1,3 receptors expressed on colonocytes.The EEC is becoming a key target in metabolic disease because of its role in orchestrating downstream responses in a range of neural, epithelial, endocrine and immune cells.2 receptors, respectively. Several studies show afferent endings innervate the mucosal epithelium and terminate in close apposition to EECs.3, Y2 and GLP-1 receptors on afferent endings and is supported by studies that have shown the receptors exist on vagal afferents.Our data show that nutrient mediated processes continue downstream of the EEC, with afferent nerves from the colon also showing convergent responses to EEC mediators GLP-1 and PYY acting on GLP-1 and YWe have shown two clear levels of convergence in this system: the first within the EEC itself, and the second in action of EEC mediators on afferent endings that subsequently signal to the central nervous system. Our proof-of-concept trial, mirroring findings from earlier ex vivo tissue experiments, demonstrated both reduced food intake and increased circulating levels of PYY. Other approaches to nutrient-induced changes to appetite include colonic delivery of short chain fatty acid propionate, which also reduces food intakeIn our obese volunteer study, there was no effect of MCFA capsules on subjective hunger ratings but modest yet significant effects on energy intake. This reduction in calorific intake of approximately 12% could result in weight loss of 12 kg over 24 weeks There are a number of limitations within the current study. For example, the absence of potent synthetic agonists for GPR84. We have used LA as it binds to the GPR84 to activate the receptor; however, this agonist may bind to other long-chain receptors. In addition, electrophysiology experiments in human colonic tissue would add a further translational advantage, while similar experiments in animal models of obesity and altered diet would provide insight to neuronal signalling pathways—these will be investigated in future studies. A clear limitation of the clinical trial using an acute administration paradigm is the lack of longer time-points for measurement of hormone release and effect on appetite perception. The capsule coating ensures colonic delivery, but this requires between 3 and 4 hours from ingestion to disintegration in the colon resulting from normal gastrointestinal motility time (as described in the Methods). As the active and placebo capsules are given over two points in a single day, it was not feasible to assess volunteers for more than 8 hours. Finally, although only CCK and ghrelin have been shown to be altered by oestradiolIn conclusion, we demonstrate that specific targeting of multiple nutrient-sensing receptors stimulates convergent activation of EEC and colonic afferent endings. Importantly, we show that targeting colonic L cells by costimulating specific receptors in obese volunteers has the capacity to reduce food intake and boost PYY levels, thereby supporting our preclinical findings and offering the potential development of a novel and effective obesity treatment without the drawbacks of surgery. With these principles in mind, we can devise more appropriate strategies for targeting nutrient sensing in the colon for the benefit of treatment of obesity and type 2 diabetes and move on to larger scale studies including body weight regulation.


Background: The lack of effective biomarkers makes it difficult to achieve early diagnosis and intervention for osteonecrosis of the femoral head (ONFH). Hence, we aimed to identify novel long noncoding RNA (lncRNA) biomarkers for ONFH.Methods: High-throughput RNA sequencing was performed to detect lncRNA and mRNA expression levels in subchondral bone samples from three patients with ONFH and three patients with femoral neck fractures. Integrated bioinformatics analyses were conducted to identify lncRNAs associated with ONFH development and their potential functions and signaling pathways. A co-expression network was constructed based on the gene time-series expression data in GSE113253. After selecting lncRNA GAS5 as a novel biomarker for ONFH, bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation assays were performed to verify the association between lncRNA GAS5 and osteogenic differentiation. Alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to measure the osteogenic phenotype and lncRNA GAS5 expression. Finally, for further validation, ONFH rat models were established, and lncRNA GAS5 expression in subchondral bone was detected by RT-qPCR.Results: We identified 126 and 959 differentially expressed lncRNAs and genes, respectively. lncRNA GAS5 expression level was significantly downregulated in patients with ONFH compared to the control group patients. The BMSC osteogenic differentiation assays showed that ALP activity increased gradually from days 3 to 7, while the lncRNA GAS5 expression level was significantly upregulated in the osteogenic differentiation induction groups. Furthermore, in vivo experiments suggested that the bone volume/tissue volume value and trabecular thickness significantly decreased in the ONFH rat model group compared to the control group, whereas the trabecular space significantly increased in the ONFH group compared to the control group. In addition, the lncRNA GAS5 expression level significantly decreased in the ONFH rat model group.Conclusion: The lncRNA GAS5 expression level was highly associated with BMSC osteogenic differentiation and was significantly downregulated in both the subchondral trabecular bone tissue of ONFH patients and ONFH rat models. Therefore, lncRNA GAS5 can serve as an ONFH osteogenic biomarker to provide an effective target for early diagnosis and molecular therapy of ONFH. Osteonecrosis of the femoral head (ONFH) is a severe and disabling orthopedic disease characterized by aseptic and avascular necrosis of the bone tissue, microfractures of the subchondral bone, and femoral head collapse  Mont et. It is min vitro and in vivo are necessary to achieve early diagnosis and investigate their molecular mechanisms.Long noncoding RNAs (lncRNAs) are noncoding RNAs (ncRNAs) greater than 200 nucleotides in length that have been suggested to play important roles in various biological processes and diseases  Yang et. Some prIn the present study, we annotated and classified lncRNAs and mRNAs based on the high-throughput RNA sequencing raw data included in our previous studies . Their eThe present study was approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University . All tissue donors provided written informed consent for this study. Finally, subchondral bone samples from six patients who underwent primary total hip arthroplasty  were obtained from the Second Affiliated Hospital of Xi’an Jiaotong University. All the samples were snap-frozen and stored in liquid nitrogen until further analysis.Frozen subchondral bone samples were rapidly ground in liquid nitrogen. Total RNA was extracted by TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), RiboZero Magnetic Gold Kit (Illumina), and KAPA Stranded RNA-Seq Library Prep Kit (Illumina) were used to achieve RNA enrichment and sequencing library generation. An Agilent Bioanalyzer 2100 system was used to conduct the quality control analysis. Finally, high-throughput RNA sequencing was performed based on the Illumina HiSeq 6000 sequencing platform (Illumina) using the TruSeq SR Cluster Kit (Illumina).p value < 0.05 .p value < 0.05.Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis, and gene set enrichment analysis were conducted by the “clusterProfiler” package with the threshold set at 2. When the BMSCs reached 70% confluence in six-well plates, osteogenic induction was performed using osteogenic medium .Human BMSCs were purchased from Procell  and cultured in minimum essential medium-α  supplemented with 10% fetal bovine serum  at 37°C in a humidified environment containing 5% CO2O, ALP stained with BCIP/NBT working solution at room temperature, and washed again with ddH2O. Finally, ALP staining image analysis was conducted using ImageJ software.We performed alkaline phosphatase (ALP) staining  on days In this study, we performed reverse transcription using StarScript II First-strand cDNA Synthesis Mix . Subsequently, 2× Universal SYBR Green Fast qPCR mix  was used to conduct the quantitative real-time PCR (RT-qPCR) according to the manufacturer’s instructions. The results were analyzed by using the 2−ΔΔCt method, and GAPDH was used as an endogenous reference gene to normalize the gene expression data. The sequences of primers are as follows:Human GAPDH , human GAS5 , Human RUNX2 , rat GAS5 , rat GAPDH primer .All animal experiments included in this study were approved by the Laboratory Animal Care Committee of Xi’an Jiaotong University. A total of 24 adult SPF Sprague–Dawley rats weighing 350–400 g were purchased from the Medical School of Xi’an Jiaotong University Animal Center. The rats were divided into two groups: the ONFH group and the control group. The rats were anesthetized with sodium pentobarbital , the fur was removed, and the skin was sterilized around the surgical area. An incision was made in the hip joint (right side) and then separated by layer to expose the femoral head. The hip joint was dislocated, the round ligament was removed, blood supply to the femoral head was stopped, the wound was disinfected, and then the incision layer was replaced. All the rats were killed after 9 weeks. Six femoral heads on the right side were collected for further micro-CT analysis, and 18 subchondral bone samples from the femoral heads were collected for RNA extraction and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR).The main bone tissue parameters of the femoral heads were evaluated using high-resolution micro-CT scanning and reconstruction. Finally, the values of bone volume/tissue volume  were calculated to reflect the bone mass condition in the femoral heads, and the trabecular thickness  and trabecular space  were calculated to reflect the trabecular structure.p < 0.05 was considered statistically significant.Independent sample t-tests and Mann–Whitney U tests were conducted for the difference analysis according to the distribution of independent variables. IBM SPSS Statistics 22 software was used to perform all the statistical analyses, and p value < 0.05, a total of 126 differentially expressed lncRNAs (DELs) and 959 differentially expressed mRNA (DEGs) were identified .We performed lncRNA and mRNA annotation based on RNA-seq data, and the expression levels of 2966 lncRNAs and 13925 mRNAs were detected in clinical subchondral bone samples in this study. One sample was recognized as an outlier sample and removed through principal component analysis  and the AnnoLnc2 database (http://annolnc.gao-lab.org/) to investigate the potential functional route of GAS5. In total, 1870 lncRNA GAS5–mRNA interaction pairs and 523 lncRNA GAS5–protein interaction pairs were obtained  in GSE113253 datasets were downloaded from the GEO database . We furtp < 0.05 . Then, ap < 0.05 . The resobtained .p = 0.002). Finally, lncRNA GAS5 expression levels in the osteogenic differentiation induction and normal control groups were tracked at these two separate osteogenic differentiation points. The results showed that the expression level of lncRNA GAS5 was significantly upregulated (p < 0.05) in the osteogenic differentiation induction groups compared to the normal proliferation medium groups . lncRNA GAS5 is associated with angiogenesis and osteogenesis. It has been found that lncRNA GAS5 can regulate angiogenesis and activate the HIF1A/VEGF signaling pathway by binding to TAF15  and Tb.Th (mm) significantly decreased in the ONFH group compared to the control group, whereas Tb.Sp (mm) significantly increased in the ONFH group. Additionally, the lncRNA GAS5 expression level was significantly lower in the ONFH group than in the control group. To the best of our knowledge, this study is the first to identify lncRNA GAS5 as a novel biomarker for ONFH using high-throughput RNA-Seq and integrated bioinformatics analyses while comprehensively validating its expression both in vitro and in vivo. These results indicate that lncRNA GAS5 can serve as an ONFH osteogenic biomarker, providing an effective target for early diagnosis and molecular therapy of ONFH.It is widely accepted that lncRNAs play important roles in various biological processes and diseases . Howeverto TAF15 . Moreoveto TAF15 . Wang etto TAF15 –(Tingartto TAF15 . HIF 1α to TAF15 . In addito TAF15 . Howeverin vitro and in vivo validation have been conducted. lncRNA GAS5 expression levels were highly associated with BMSC osteogenic differentiation and were significantly downregulated in ONFH subchondral trabecular bone tissue.In conclusion, we identified lncRNA GAS5 as a novel biomarker for ONFH which may serve as a potential treatment target using high-throughput RNA-seq and integrated bioinformatics analyses. Comprehensive


Snakebite is a neglected tropical disease that causes high global rates of mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapeutic for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions.in vitro neutralisation profiles against each of the venoms. Despite variability of the immunogen mixtures, both experimental antivenoms exhibited broadly comparable in vitro venom binding and neutralisation profiles against the individual venom immunogens in immunological and functional assays. However, in vivo assessments using a murine preclinical model of antivenom efficacy revealed substantial differences in venom neutralisation. The experimental antivenom generated from the seven venom immunogen mixture outperformed the comparator, by providing protective effects against venom lethality caused by seven of the eight geographically diverse venoms tested, including three distinct venoms that were not used as immunogens to generate this antivenom. These findings suggest that a core set of venom immunogens may be sufficient to stimulate antibodies capable of broadly neutralising a geographically diverse array of haemotoxic snake venoms, and that adding additional venom immunogens may impact negatively on the dose efficacy of the resulting antivenom.In this study we explored the feasibility of generating globally effective pathology-specific antivenoms to counteract the haemotoxic signs of snakebite envenoming. Two different immunogen mixtures, consisting of seven and twelve haemotoxic venoms sourced from geographically diverse and/or medically important snakes, were used to raise ovine polyclonal antibodies, prior to characterisation of their immunological binding characteristics and Although selection of appropriate immunogens that encapsulate venom toxin diversity without diluting antivenom potency remains challenging and further optimisation is required, the findings from this pilot study suggest that the generation of pathology-specific antivenoms with global utility is likely to feasible, thereby highlighting their promise as future modular treatments for the world’s tropical snakebite victims. in vitro immunological cross-reactivity and venom functional-neutralisation findings demonstrated broadly comparable venom recognition and inhibition against the various venom immunogens, irrespective of whether they were included in the immunising mixture. However, assessments of murine in vivo protection against venom-induced lethality revealed that the antivenom generated with fewer immunogens outperformed the comparator, and provided broader preclinical efficacy, even against some venoms not used to raise the antibodies. These findings suggest that careful selection of venoms used as immunogens is crucial for maintaining antivenom potency, and that a small subset of venoms may be capable of generating an antivenom with potential global efficacy against a specific pathology commonly observed after snakebite.Snakebite is a major public health concern that causes extensive death and disability in the tropical world. Variation in venom composition causes major challenges for snakebite therapy by typically restricting antivenom efficacy to only the few snake species that each product was designed against, meaning that many geographically restricted therapeutics are manufactured for different parts of the world. In this study, we explored the feasibility of generating a global pathology-specific antivenom by using two different sets of haemotoxic venoms as immunogens, one including seven venoms and the other twelve venoms, to generate ovine polyclonal antibodies. Our  Snakebite envenoming is classified as a neglected tropical disease (NTD), and as such is most prevalent in tropical and subtropical regions of the world, particularly rural regions ,2. Of thViperinae and pit vipers, Crotalinae) primarily result in haemotoxic pathologies, typically characterised by local or systemic haemorrhage, including overt and internal bleeding , 3.75 ml 40% bis-acrylamide, 100 μl 10% SDS, 60 μl 10% ammonium persulfate [APS] and 7 μl N,N,N′,N′-Tetramethyl ethylenediamine [TEMED]); stacking gel . Next, 10 μl of each venom (1 mg/ml) was mixed 1:1 volume/volume with reducing buffer  and subsequently heated for 10–15 minutes at 100°C. Thereafter, 10 μl of each venom/buffer sample was added to the gel, alongside 5 μl of a molecular weight protein marker , and the samples run at 200 V for 55 minutes using a Mini-PROTEAN Electrophoresis System (Bio-Rad). Resulting gels were then stained at a final concentration of 0.1% (w/v) Coomassie blue R350  glacial acetic acid and 20% (v/v) methanol) overnight at RT. Gels were destained  for at least 2 hours at RT. For visualisation, gels were subsequently imaged using a Gel Doc EZ Gel Documentation System (Bio Rad).For gel electrophoresis, 15 well 15% SDS-PAGE gels were prepared using the following approach: resolving gel . Following confirmation of protein transfer by reversible Ponceau S staining, the membranes were blocked for non-specific binding using 5% non-fat dried milk in TBST , and left overnight at 4°C rocking at slow speed. Subsequently, blots were washed three times over 15 minutes with TBST, before the addition of primary antibodies , with all primary antibodies standardised to an initial concentration of 50 mg/ml, and then diluted to 1:5,000 in 5% non-fat dried milk in TBST, and incubated for 2 hours at RT. The immunoblots were then washed in triplicate with TBST as described above and incubated for two hours at RT with 50 ml of appropriate secondary antibodies diluted 1:2,000 in PBS: (i) horseradish peroxidase-conjugated donkey anti-sheep IgG  for the two EAVs and the ovine IgG control and (ii) horseradish peroxidase-conjugated Rabbit anti-horse IgG  for the SAIMR Polyvalent positive control antivenom and the equine IgG control. The immunoblots were then washed again with TBST and developed by the addition of DAB substrate  by placing the membrane into the substrate for 30 seconds, before washing with deionised water.Microtiter 96 well ELISA plates (Thermo Fisher Scientific) were coated with coating buffer  containing 100 ng of each venom or each venom immunogen mixture and incubated overnight at 4°C. The plates were then washed three times with TBST before the addition of 5% non-fat milk in TBST and incubated at RT for two hours, followed by washing another three times with TBST. Next, 120 μl of primary antibodies (as described for immunoblotting experiments) were added to the plate in duplicate at an initial dilution of 1:100 in 5% non-fat milk in TBST, followed by five-fold serial dilutions across the plate and incubation at 4°C overnight. The plates were then washed again with TBST and incubated for two hours at RT with appropriate secondary antibodies (as per immunoblotting experiments) diluted at 1:1,000 in PBS. The plates were then washed again with TBST before the addition of substrate  in citrate buffer  containing 0.015% hydrogen peroxide . Plates were gently mixed and incubated at RT for 15 mins, before the signal was read spectrophotometrically at 405 nm on a FLUOstar Omega microplate reader (BMG LabTech).4SCN), at a range of concentrations (0–8 M) in duplicate for 15 minutes at RT  readings). The antivenoms used consisted of the two EAVs (EAV 1 and EAV 2) and the SAIMR polyvalent antivenom as an antivenom comparator.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Anas platyrhynchos) genome. Highly pathogenic VN1203 replicated in lungs and showed systemic dissemination, while BC500, like most low pathogenic strains, replicated in the intestines. VN1203 infection induced robust differential expression of genes all three days post infection, while BC500 induced the greatest number of differentially expressed genes on day 2 post infection. While there were many genes globally upregulated in response to either VN1203 or BC500, tissue specific gene expression differences were observed. Lungs of ducks infected with VN1203 and intestines of birds infected with BC500, tissues important in influenza replication, showed highest upregulation of pattern recognition receptors and interferon stimulated genes early in the response. These tissues also appear to have specific downregulation of inflammatory components, with downregulation of distinct sets of proinflammatory cytokines in lung, and downregulation of key components of leukocyte recruitment and complement pathways in intestine. Our results suggest that global and tissue specific regulation patterns help the duck control viral replication as well as limit some inflammatory responses in tissues involved in replication to avoid damage.Ducks are the natural host and reservoir of influenza A virus (IAV), and as such are permissive to viral replication while being unharmed by most strains. It is not known which mechanisms of viral control are globally regulated during infection, and which are specific to tissues during infection. Here we compare transcript expression from tissues from Pekin ducks infected with a recombinant H5N1 strain A/Vietnam 1203/04 (VN1203) or an H5N2 strain A/British Columbia 500/05 using RNA-sequencing analysis and aligning reads to the NCBI assembly ZJU1.0 of the domestic duck ( Anas playrhynchos) are the natural host and reservoir IAV, and as such are highly resistant to viral pathology or mortality while still being permissive to viral replication (Influenza A virus (IAV) causes disease in both humans and animals, resulting in periodic epidemics and potentially global pandemics. Mallard ducks  which detects IAV, and the mitochondrial antiviral-signaling protein (MAVS) signaling pathway. Notably, components of this pathway differ between ducks and chickens  and A/British Columbia 500/2005 (H5N2) upregulated key innate immune genes, and although we characterized only a limited number of genes using qPCR, the ducks rapidly cleared both viruses with robust early responses . Our aimHere we obtain transcriptome information from polyadenylated RNA of ducks infected with a reverse genetics version of the highly pathogenic H5N1 strain rgA/Vietnam/1203/04 (VN1203) and the low pathogenic H5N2 strain A/mallard/BC/500/05 (BC500). We look at the global differential expression (DE) in lung and spleen of Pekin ducks infected with VN1203; and lung, spleen and intestines of ducks infected with BC500. We have highlighted differences and similarities in differential expression of transcripts and identified sets of genes that have arisen by duplication and may contribute to host specific resistance. Our results suggest that tissue specific regulation mechanisms may play an integral role in both providing protection against IAV replication while limiting inflammatory responses.6 of 50% egg infectious doses (EID50) of VN1203 or BC500 using the natural route of infection by dripping virus in nares, eyes and trachea. Viral replication was tracked by taking tracheal and cloacal swabs from some ducks, and reported previously (SWIM6 gene (LOC101797738) in each RNA sample. SWIM6 is located on the W chromosome found in WZ females, but not ZZ males, and was established as a valid determinant of avian embryo sex by He and colleagues . Briefly, RNA-seq paired end libraries were created using Illumina’s TruSeq-stranded-mRNA sample preparation protocol . Integrity of RNA was checked using an Agilent Technologies 2100 Bioanalyzer. Two rounds of purification of poly(A) containing mRNAs were performed using oligo-dT magnetic beads. cDNA libraries were made and quality was assessed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing of the cDNA libraries was performed using llumina’s NovaSeq 6000 sequencing system. The sequencing resulted in paired 150 bp reads with approximately 6GB of data per run, resulting in a sequencing depth of approximately 40 million reads per sample.Library preparation and poly-adenylated RNA sequencing were performed by LC Sciences (Sequence data was submitted to NCBI sequence read archive (SRA) under the BioProject ID PRJNA767080.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used Trimmomatic  using HISAT2 version 2.20  analysis was performed in the R studio environment ng EdgeR . Libraryng EdgeR  or Vennyng EdgeR .The EdgeR function diffspliceDGE was used to assess alternative splicing events during infection. All alternatively spliced genes for both VN1203 and BC500 experiments can be found in Differentially expressed genes were combined for all three days of infection in each different tissue, infected with VN1203 or BC500. These genes were subjected to over-representation analysis (ORA) for gene ontology biological process (GO BP) terms, with the noRedundant filter added to reduce redundant enrichment terms on WebGestalt .Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) diagrams were made using Cytoscape version 3.8.0 . Enrichmhttps://www.extendoffice.com/documents/excel/1873-excel-find-and-replace-multiple-values-at-once.html). Any genes flagged in the datasets named with location numbers only were submitted to NCBI BLAST to search against both the bird and mammalian databases. For genes without significant BLAST orthologs, the location number identifier was not changed. Genes flagged as significant which had the same gene name as others in the list were investigated to determine if this was a genome mis-assembly, paralogous gene or misidentification through BLAST, using protein alignments, and chromosomal locations.Many of the annotations in the NCBI file had only numerical descriptions, and not annotated gene names. To assign names to those with significant hits from the DEG lists, we searched on NCBI and if available from the description, the gene name was manually assigned. Gene names were changed using a find and replace macro in excel .To differentiate genes in our results we applied certain rules to naming related genes. Genes with the “pseudogene” designation in NCBI were assigned a “-pseu” suffix. Likewise, genes classified as noncoding RNA (ncRNA) were given a “-ncRNA” suffix. Genes which were identified as “like” another gene on NCBI were given the “-L” suffix. In cases where multiple genes in one dataset were classified with the same name with the “-L” suffix, the genes were numbered as “-L#”, with the lowest number being closest in chromosomal location to the presumed ortholog or the annotated gene on NCBI. Genes which shared identical names, that could not be clarified by the above methods kept their gene names but were given a numerical suffix in the order of the genes on their respective chromosomes  clustered with similar tissue  DEGs on all three days of infection, while BC500 infected ducks had much less DE in lungs and spleen . In contTo determine how many genes were similarly upregulated or downregulated on all three days of infection, genes were filtered by FDR (>0.05) and log2(FC). Although each tissue had a number of unique altered genes there was considerable overlap of DE genes in lung and spleen on each day post infection  hits were reported in GO term enrichment resulting from downregulated DEGs found common terms in cell adhesion (GO:0031589) and cell growth (GO:0016049)  events in lung, spleen and intestine of infected ducks, we analyzed alternative splicing events by counting differences in reads mapped to individual exons between infected and control samples. As with total DE expression, VN1203 infected lungs and spleens had many more AS events than BC500 infected tissues , it is pTo determine the GO terminology associated with these AS events, all significant (FDR < 0.05) AS genes were submitted to the WebGestalt server. Only VN1203 infected ducks had enough genes to produce statistically significant (FDR < 0.05) GO results >1.5. Genes that reached these parameters on 1, 2 or 3 dpi were kept, combined into a list for each tissue, then compared. We removed the 65 genes found upregulated in all tissues during both VN1203 and BC500 infection to reduce redundancy in the datasets. Repeat non-coding RNA (ncRNA) and pseudogenes were also removed. A heatmap of most upregulated DEGs in VN1203 infection was plotted showing genes corresponding to different Reactome pathways placed together . Genes tDucks infected with both VN1203 and BC500 upregulate 65 genes in common, most of which produce proteins that respond to interferons or have immune function. VN1203 strongly upregulates a different subset of genes, including genes involved in the complement cascade, proinflammatory cytokines and interleukins and various other genes involved in immune responses. While VN1203 upregulates most immune genes on 1 dpi, BC500 upregulates these genes on 2 dpi, the delay likely due to the time needed for virus to reach the intestine for replication.Unfortunately, there were no available enrichment databases that accurately placed all genes in enrichment categories. Many genes that are involved in immunity, antiviral defense or are interferon inducible are not yet added to the Reactome databases or without enough additional terms to be considered enriched in this dataset. Genes such as IFIT5 –45, DDX6To determine which highly upregulated genes were uniquely expressed in lung or spleen of ducks infected with VN1203, we filtered significant DEGs by log2(FC)>1.5 for all three days post infection. Genes which were found in both tissues were removed from the dataset. We removed the 65 genes found upregulated in all tissues during both VN1203 and BC500 infection, as well as repeat ncRNA and pseudogenes, as described previously.IFNG. IFNG is primarily upregulated in lungs and has increased expression on both 1 and 3 dpi, but curiously not on 2 dpi. Lungs of ducks infected with VN1203 also see unique expression of many PPRs, including TLR1A, 1B, 2A, 2B and 4. All of these TLRs have the highest expression on 1 dpi. The other primary enrichment pathway in lungs is the “Reactome: Signal transduction” which includes “Reactome: Class A/1 (Rhodopsin-like receptors)”. The expression of these pathways is more varied, with some having highest expression at 1 dpi while others at 3 dpi.Many of the genes upregulated in lung have immune functions, however, only 20 out the total 54 “unique to lung” genes were found enriched in the “Reactome: Immune system” dataset >1.5 for all three days post infection  and log2(FC) (<-1). From this list, we manually compared genes of interest to infection in lung and spleen of VN1203 and BC500 infected ducks. Genes which were highly downregulated in intestines that may play a role in either immune responses or viral restriction were examined in each tissue and visualized using a heatmap  or interferon inducible genes . The top upregulated genes in BC500 however, are lacking high cytokine gene expression, but still have high expression of some interferon inducible genes (notably Mx).To identify the genes contributing to the peak immune response in the sites of replication, we examined gene expression patterns in infected ducks. Because many important immune genes were upregulated in the lungs of ducks infected with VN1203 on 1 dpi, and most gene regulation in intestines of ducks infected with BC500 was at 2 dpi, we filtered all statistically significant (FDR < 0.05) genes on these days by expression levels and compiled lists of the top 100 most up or downregulated genes. We removed genes designated on NCBI as ncRNA or pseudogenes to limit the lists to genes that presumably code for protein. VN1203 induces a much more robust response than BC500  compared to lymphoid tissues (spleen). Ducks respond to VN1203 infection with a high interferon signature at 1 dpi in lung, yet soon down regulate key proinflammatory cytokines. BC500 infection stimulates the highest gene upregulation at 2 dpi in intestine together with downregulation of leukocyte recruitment cytokines. A global picture emerges of a robust and rapid interferon response to VN1203 in lung, and a significant response to BC500 in intestine, yet both responses were tempered to limit damage. As part of the sequencing effort for the  strains . They sa strains . The GraIFIH1/MDA5) and toll-like receptor 3 (TLR3). VN1203 infection induces a much more rapid and robust response of IFN inducible genes (such as RSAD2/Viperin and IFIT5) in the duck than BC500 infection does. The interferon response peaks at 1 dpi for VN1203, while the response to BC500 peaks at 2 dpi. The expression of genes in the RIG-I pathway from the RNAseq data mostly match their expression profiles we previously determined using qPCR  expression in VN1203 infected ducks. In our previous studies, the qPCR results suggested these two genes were robustly expressed on 1 dpi, and rapidly dropped to basal levels by 3 dpi, while our RNA-seq analysis suggests these genes exhibit sustained expression across all 3 dpi. Likely these discrepancies are due to the different techniques of normalization used between the two studies, with the qPCR experiments normalized to a single gene (GAPDH) and the RNA-seq data normalized using TMM and library size. Additionally, RNA-sequencing data will still count splice variants in its read counts, while qPCR may miss some of these variants due to primer design and placement. Some of the changes in gene expression seen in tissues are likely due to infiltrating immune cells responding to infection. We previously reported aggregates of leukocytes in lung tissues and leukocyte depletion of spleen tissues in VN1203 infected ducks  which can detect RNA viruses were highly upregulated in all tissues. This includes RIG-I and the related interferon induced with helicase C domain 1/melanoma differentiation-associated protein 5  and TRIM25  upregulated by both VN1203 and BC500 in all tissues sampled. In mammals, DDX60 binds RIG-I and promotes RNA binding and downstream type I IFN production during viral infection (RSAD2 or Viperin), 2′5′-oligoadenylate snythetase-like (OASL), interferon induced protein with tetratricopeptide repeats 5  and Mx. RSAD2, OASL and Mx are all highly upregulated by VN1203 and BC500 in all tissues sampled. Recent experiments establish the antiviral function of these duck homologues. Overexpression of duck IFIT5 reduces viral titre at early timepoints but appears to inhibit innate immunity later . Of interest, PARP12 can inhibit replication of RNA viruses . The shorter gene, PARP12-L is only significantly upregulated in lungs of VN1203 infected ducks, suggesting it may play a tissue specific role in viral inhibition. PARP12 was also upregulated in a previous study in both lungs and ileums of VN1203 and BC500 infected Gray mallard ducks (PARP12 or PARP12-L) has yet been characterized in birds, and it is unknown if either can restrict RNA viral replication. PARP14 appears to be duplicated in the duck, with two forms of PARP14 sharing equal percent identities to human PARP14 (~43%), but only 52% identity to each other (data not shown). PARP14 deletion reduces proinflammatory responses in murine macrophages  is upregulated by both BC500 and VN1203 infection in ducks, while PARP14.2 (LOC101798744) is only upregulated by VN1203 in lungs and spleens of infected ducks. Members of the PARP gene family appear to be expanded in the duck, making them interesting candidates for further study of proteins which may play lineage specific roles in immune responses to IAV in the duck.We found gene duplications unique to ducks particularly interesting, especially if the mammalian homologue has known antiviral activity. The poly-ADP-ribose polymerase (PARP) family of genes is largely understudied but is often associated with DNA repair and transcription. We found members of this family upregulated by both VN1203 and BC500 infection in ducks  and IL8-L (LOC101803817). Both genes show strong upregulation in the spleen, particularly on 1 dpi. However, in lungs there is significant downregulation on 2 and 3 dpi of both IL8 and IL8-L. In humans, IL8 is secreted by alveolar epithelial cells infected by IAV , a chemoattractant for neutrophils and macrophages  can be found below: The animal study was reviewed and approved by the Animal Care and Use Committee of St. Jude Children’s Research Hospital and performed in compliance with relevant institutional policies, National Institutes of Health regulations, and the Animal Welfare Act.LC performed the computational analysis and originally drafted and edited the manuscript. XF-C prepared RNA samples for sequencing. RW provided expertise and facilities for all infections and tissue collection. KM performed infections, collected tissues and RNA from infected ducks and edited the manuscript.Our research is funded by the Canadian Institutes of Health Research (PJT 159442) and the Natural Sciences and Engineering Research Council (to KM). All duck infections and isolation of infected tissues were supported by the National Institute of Allergy and Infectious Diseases, National Institute of Health under Contract No. HHSN27220140006C and by the American Lebanese-Syrian Associated Charities (ALSAC) to RW and conducted in Memphis Tennessee, USA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.