Some clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.In an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent DK values of 5.6–21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.Anti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease , National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891)  COVID-19 is caused by the novel coronavirus SARS-CoV-2 . Two yeaThere is significant evidence for preexisting IgG autoantibodies against type I IFNs playing a key role in mediating disease severity in COVID-19 , likely P = 0.0084 by Fisher’s exact test; https://doi.org/10.1172/jci.insight.158362DS1). In order to increase sample size and define the stability and kinetics of these antibodies, we assembled additional patients in whom serum was available from multiple laboratory blood draws taken across their hospitalization. This added 52 patients with COVID-19 for serum analysis: 38 in WHO ordinal groups 6 to 8 (31 ventilated and 7 dead) and 14 patients in ordinal group 4 , the host receptor for SARS-CoV-2 entry , could bP = 0.44 by Fisher’s exact test). Only 4/18 (22%) severe patients with anti-ACE2 IgM antibodies were also IgG positive  patients with severe COVID-19 (WHO 6–8) and 6/52 (11.5%) patients with moderate (WHO 3–5) disease    . While population average neutrophil counts (3) than in the anti-ACE2–negative group (4.95 K/mm3), these results did not reach statistical significance (P = 0.10). The elevated temperature observed in anti-ACE2 IgM–positive patients early after admission  In 3 patients , sampling spanned the development of anti-ACE2 IgM . In thesIn T cell–dependent immune responses, IgM levels generally decrease at the time of class-switching to IgG. We therefore examined whether decreasing IgM levels over time were associated with increasing anti-ACE2 IgG levels at later points. In 1 patient (CV-1), both IgG and IgM were present at the earliest point and remained constant over time . In 8 anP < 0.0001 by unpaired t test; P = 0.028; Mann-Whitney test; P = 0.017 by Fisher’s exact test; Multiple groups have noted that high levels of anti–SARS-CoV-2 S protein IgG occurring at the time of hospital admission are associated with more severe disease in COVID-19  to 21.7 nM (CV-134). In contrast, IgM from controls did not approach saturation binding to ACE2 even at the highest tested concentrations (representing the undiluted sample), and the DK values could thus not be determined , they benefit from avidity effects because of their pentameric construction . Patient IgM binding to immobilized ACE2 was saturable , with aptermined . It is itruction .Since hypomorphic ACE2 function has been associated with severity in ARDS, we investigated whether purified IgM from patients with COVID-19 affected the catalytic function of ACE2 against a fluorogenic substrate. Two purified IgMs used above in binding assays had no effect on ACE2 activity ..IgM antibodies are mainly found in the circulation, where they are the most effective antibody isotype in activating the classical complement cascade at surfaces expressing their cognate antigens . We founAutopsy studies have demonstrated prominent findings in the lungs of COVID-19 patients , 23, incP < 0.001)  for CV-1 IgM is about 80 minutes  . A decre minutes , we expeP = 0.0117). A similar increase in barrier function was observed after perfusion with 100 ng/mL of affinity-purified anti-ACE2 IgM (P = 0.008), providing further evidence that the effect on barrier function is mediated by ACE2 binding. It is noteworthy that some long-lived PMEC stimuli  cause an initial tightening of the endothelial barrier, followed by later barrier leakiness mediated through receptor downregulation , multiple signaling pathways can be initiated . These iThe ability of anti-ACE2 IgMs from patients with COVID-19 to bind C1q and induce C3 deposition on PMECs is consistent with their playing a role in inducing the complement pathway activation that is prominent in patients with severe COVID-19 , 21. DirThese IgM-activated pathways are potentially amenable to several readily available treatments applied in the acute inflammatory phase of severe illness, e.g., steroids and IVIG therapy –39, as wThe subset of patients (27%) with severe COVID-19 who developed IgM antibodies against ACE2  are almost certainly not overlapping with the 10% of patients with severe COVID-19 who had preexisting IgG antibodies against type I IFNs  a confirmed diagnosis of COVID-19; (b) survival to death or discharge; and (c) remnant specimens in the Johns Hopkins COVID-19 Remnant Specimen Biorepository, an opportunity sample that includes 59% of Johns Hopkins Hospital patients with COVID-19 and 66% of patients with length of stay ≥ 3 days between April 20, 2020, and May 14, 2020. Diagnosis of COVID-19 was defined as detection of SARS-CoV-2 using any PCR test with an Emergency Use Authorization from the US Food and Drug Administration. Selection and frequency of other laboratory testing were determined by treating physicians. The primary clinical data source was JH-CROWN, a Johns Hopkins Medicine COVID-19 registry that integrates all clinical data for COVID-19 patients, including demographics, medical history, comorbid conditions, symptoms, medications, laboratory results, medical images, and comprehensive bedside flow sheet data, including vital signs, respiratory events, and intravenous medication titration , to hospitalized with mild-moderate disease , hospitalized with severe disease , and 8 = death. For this study we combined adjacent WHO classes, dividing the inpatient population into 2 groups according to maximum WHO severity: patients who did not require mechanical ventilation (WHO classes 3–5) and those who required mechanical ventilation with or without additional support, such as intravenous pressors, continuous renal replacement therapy, and/or ECMO who survived (WHO classes 6–7) or died (WHO class 8). Serum samples were selected for timing within 24 hours of onset of the maximum WHO class; when multiple samples were available, the specimen closest to the WHO class onset was used. The initial analysis used a random sample of 12 to 20 unique patient specimens from each of the 4 classes meeting the criteria above . To determine biomarker trajectory, we analyzed an expanded cohort of patients who had 3 to 4 consecutive sera per patient across the course of their hospitalization. Patient selection was determined solely by specimen availability. Where available, additional serum for anti-ACE2 IgM–positive individuals was requested from the remnant biorepository.n = 25 patients with SLE from the Hopkins Lupus Cohort, (b) sera from n = 13 patients diagnosed with systemic sclerosis after evaluation at the Johns Hopkins Scleroderma Center, and (c) n = 15 patients with necrotizing myopathy defined by a positive anti-HMGCR antibody status evaluated at the Johns Hopkins Myositis Center. Serum from n = 30 patients with influenza diagnosed using the Cepheid Xpert Xpress Flu/RSV assay in the Johns Hopkins emergency departments or inpatient units was studied. Eleven were evaluated in the emergency department and discharged to outpatient care; an additional 19 patients were hospitalized and required oxygen therapy or assisted ventilation  sera from At the initiation of this study, we were struck by the similar clinical presentation of severe COVID-19 to a dermatopulmonary syndrome, characterized by skin rash, rapidly progressive interstitial lung process with frequent progression to a need for ventilatory support, and a unique vasculopathic phenotype including cutaneous ulcers and digital ischemia , 30, 42.ELISA plate wells were coated overnight with 50 ng of purified protein  diluted in PBS. For each serum assayed, duplicate wells were coated with protein, and an adjacent well was incubated overnight with PBS (to determine background specific to each sample). For anti-ACE2 IgM ELISA, wells were washed with PBS plus 0.1% Tween (PBST), then blocked with 3% milk/PBST. Primary antibody incubations were performed by diluting sera 1:200 in 1% milk/PBST . For area under the curve plots , serial Assays were performed as described for anti-ACE2 IgG ELISAs, using sera diluted 1:1200 .The CoronaChek serologic lateral flow assay (Hangzhou Biotest Biotech) detects IgM and IgG antibodies against the S protein and receptor binding domain of SARS-CoV-2. Studies on positive and negative control specimens from Maryland demonstrated sensitivity of 95%  in convalescent plasma donors an average of 50 days after symptom onset; sensitivity of 100%  in PCR-confirmed hospitalized individuals 15 days after symptom onset; specificity of 100%  in pre-pandemic patients infected with rhinoviruses and other coronaviruses.oC) with a mouse monoclonal anti-FLAG antibody  to detect CD9, a rabbit monoclonal anti-TMPRSS2 antibody , or a mouse monoclonal anti-CD81 antibody . Blotted proteins were detected using HRP-labeled secondary antibodies  and chemiluminescence (Pierce). Images were acquired using a Protein Simple Fluorochem-M digital imager. To screen for antibodies against CD9, CD81, and TMPRSS2 in patient sera, immunoblots were performed using lysates made from untransfected cells and from validated transiently transfected cell lysates . The sera were used at 1:1000 dilution , followed by a mixture of HRP-labeled anti-human IgG and anti-human IgM , with detection as described above.Complementary DNAs encoding full-length FLAG-tagged CD9 and TMPRSS2  and CD81 (GenScript OHu19408) were sequence verified. These DNAs were transiently transfected into HEK293T cells (purchased from ATCC) with ExpiFectamin_293 reagent (Thermo Fisher Scientific A14524). After 48 hours, the cells were lysed in buffer A  and a protease inhibitor cocktail. Robust expression of transiently expressed proteins was confirmed by immunoblotting equal protein amounts of lysates electrophoresed on 11% SDS-polyacrylamide gels. After blocking the membranes, primary antibody incubations were performed , and loaded onto the column. The column was washed twice with 5 CV of PBS. Bound IgM was eluted with 5 CV of 0.1 M glycine pH 3, neutralized with 0.5 CV of 1 M Tris-HCl (pH 8), exchanged into PBS, and concentrated to match the original serum volume. The 280 nm absorbance of the purified IgM was measured to calculate the IgM concentration, using the extinction coefficient for pentameric human IgM.We equilibrated 0.5 mL of POROS CaptureSelect IgM Affinity Matrix (Thermo Fisher Scientific) with 10 column volumes (CV) of PBS pH 7.2 in a Poly-Prep chromatography column (Bio-Rad). Patient sera (400 μL) were diluted 1:10 in PBS, filtered via centrifugation at 12,000Affinity purification of anti-ACE2 IgM from COVID-19 patient CV-1 was accomplished as follows. We incubated 3.5 μg biotinylated ACE2  with 50 μL of streptavidin dynabeads (Invitrogen 65305) in 1 mL of buffer A for 25 minutes. After removing unbound ACE2 and washing, the streptavidin dynabead/biotinylated ACE2 mix was added to 7.5 μg purified whole CV-1 IgM (prepared as described above) in 0.4 mL of buffer A. The mixture was rotated for 95 minutes, then washed, and the bound anti-ACE2 IgM was eluted with glycine and neutralized as described above. The eluted anti-ACE2 IgM was buffer exchanged into Endothelial Cell Growth Medium-2  and concentrated. Anti-ACE2 IgM binding activity of the affinity-purified preparation was confirmed by ELISA .BLI was performed using an Octet RED96 instrument (Molecular Devices) to measure the interaction of purified IgM to ACE2. Wells of a black, flat-bottom, polypropylene plate (Corning) were loaded with the following samples: 50 nM biotinylated human ACE2 ; 2-fold dilutions of purified patient IgM; PBSA (PBS pH 7.2 containing 0.1% BSA); and regeneration buffer . All samples and buffers were filtered before use. ACE2 and the IgM samples were diluted in PBSA. ACE2 was loaded onto hydrated streptavidin biosensor tips (Molecular Devices), and baseline measurements were collected in PBSA. Binding kinetics were then measured by submerging the ACE2-coated biosensors in wells containing 2-fold serial dilutions of each patient IgM sample for 300 seconds (association) followed by submerging the biosensor in wells containing only PBSA for 450 seconds (dissociation). Analysis and kinetic curve fitting (assuming a 1:1 binding model) were conducted using Octet Data Analysis HT software version 7.1 (Molecular Devices). Normalized equilibrium binding curves were obtained by plotting the response value after the 300 second association phase for each sample dilution and normalizing to the maximum value. Equilibrium binding curves were fitted to a 4-parameter logistic regression model in GraphPad Prism (v9.0) software.ACE2 activity was measured using a kit from BioVision (K897). Purified IgM (5 μg) or ACE2 inhibitor was preincubated with ACE2 in white 96-well Costar plates , followed by addition of fluorogenic ACE2 substrate per the manufacturer’s protocol. A PBS control was included for each assay. The positive control contained only ACE2 and substrate, and the negative control was ACE2 plus ACE inhibitor and substrate. Fluorescence was measured every 5 minutes after substrate addition in a BMG Labtech FLUOstar Omega plate reader . Fluorescence values for wells containing no ACE2 were subtracted from the readings.Polystyrene plates  were coated overnight with 50 ng/well of ACE2 protein (Abcam ab151852) in PBS or with PBS alone. After washing with PBST, the plates were blocked with 1% gelatin/PBST. Purified IgM (0.5 μg/well) diluted in 1% gelatin/PBST was then added to the wells . After washing with PBST, plates with attached IgM were used for 2 different assays. For C1q binding, human serum  was added as the complement source. After 1 hour at 37°C, wells were washed with PBST, and goat anti-human C1q (Comptech A200) diluted 1:20,000 in PBST was added . HRP-labeled anti-goat IgG (Thermo Fisher Scientific A16005) diluted 1:5000 in PBST was used as secondary antibody. To measure anti-ACE2 IgM, HRP-labeled anti-human IgM  diluted 1:5000 in PBST  was used. Following addition of SureBlue peroxidase reagent (KPL), reactions were stopped with HCl and absorbances (450 nM) were read.6 cells/mL in EC medium and seeded into microchannels under static conditions for 30 minutes to promote cell adhesion. Microchannels were then perfused at 3 dyne/cm2 for 48 hours in EC medium to form confluent pulmonary endothelium. To replicate the inflammatory state, microvessels were perfused with EC medium supplemented with 1000 U/mL recombinant human IFN-α2 (BioLegend) and 50 ng/mL recombinant human IFN-γ (R&D Systems) for 24 hours.PMECs (Lonza) were used to generate 3D pulmonary microvessels. PMECs were cultured in EC medium from passages 6 to 8 using routine procedures. Three-dimensional, 150 μm diameter microvessels were fabricated based on published protocols . Microch2). Microvessels were then washed out in the above medium minus IgM . After washout, microvessels were perfused with 200 μM Lucifer yellow (Thermo Fisher Scientific) or 2 μM fluorescently labeled 10 kDa dextran (Thermo Fisher Scientific) in EC medium supplemented with 10% normal human complement to assess paracellular permeability. Additional experiments were performed with CV-1 IgM at 3.33 μg/mL. Experiments were also performed using 100 ng/mL affinity-purified anti-ACE2 IgM; this concentration matches the low experimental dose  based on a predicted 3% prevalence of anti-ACE2 IgM in total IgM. Epifluorescence and phase contrast images at original magnification 10× were collected on an inverted microscope (Nikon Eclipse Ti-E) maintained at 37°C for 10 minutes before and 30 minutes after dye perfusion, with images collected every 2 minutes. Fluorescence intensity profiles of a region consisting of the microvessel lumen and surrounding ECM were obtained using ImageJ (NIH). Permeability was calculated as P = (d/4)(1/ΔI)(dI/dt), where d is the vessel diameter, ΔI is the initial increase in fluorescence intensity upon luminal filling, and (dI/dt)0 is the rate of increase in fluorescence intensity as solute exits into the gel , microvessels were treated with 100 μg/mL patient IgM in EC medium supplemented with 10% normal human complement (Quidel) for 30 minutes under low-flow conditions  with illumination provided by an MLC 400 monolithic laser combiner (Keysight Technologies). For ACE2 images, background subtractions were performed in ImageJ with the background subtract plugin and rolling ball radius set at 500 pixels.Microvessels were washed with PBS for 5 minutes, fixed with 3.7% paraformaldehyde (MilliporeSigma) for 15 minutes, blocked with 10% goat serum , then incubated with primary antibody . Primary antibodies used were rabbit anti-CD31 , goat anti-ACE2 , and FITC-conjugated anti-C3c complement rabbit polyclonal antibody . After washing with 10% goat serum for 2 hours, the microvessels were incubated  with secondary antibodies . Nuclei were visualized with DAPI (Thermo Fisher Scientific). Microvessel cross sections were reconstructed from confocal Autopsies of 23 patients infected with SARS-CoV-2, documented by PCR on a pre- or postmortem nasopharyngeal swab, were examined. Autopsies were consented for and performed on the clinical service with complete examination of chest organs and in situ sampling of remaining organs and tissues, with histology performed on all sites. Lung paraffin sections from COVID-19 autopsy patients were either stained with hematoxylin and eosin or processed as follows. After deparaffinization and rehydration, the sections were immersed in antigen retrieval solution (DAKO) . For IgM staining, the sections were blocked with goat serum , followed by incubation with HRP-labeled goat anti-human IgM (Jackson ImmunoResearch 109-035-043) diluted 1:500. Visualization was performed with a liquid DAB substrate-chromagen system (DAKO), and the sections were counterstained with hematoxylin.Patient data and autoantibody data will be stored in the JH-CROWN registry, which is housed on the Johns Hopkins Precision Medicine Analytics Platform. Requests for data or materials derived from human samples may be made available, subject to any underlying restrictions on such data and samples, and will require material transfer and data use agreements through Johns Hopkins University.The clinical measures used in this analysis are from the COVID-19 JH-CROWN Registry that is actively curated by a team of clinicians, informaticists, and statisticians to ensure data quality. For repeated measures outcomes , data were checked by making spaghetti plots  and visut test of the null hypothesis that the 2 population means are equal. When we detected a large deviation from Gaussian distribution (for S protein IgG), a nonparametric test (Mann-Whitney U) was used instead.To compare the rates of IgM antibody positivity between 2 subgroups, we estimated the ratio of the odds of positivity for one subgroup versus the other (OR) and 95% CI. Given the small numbers of patients in some comparator groups, we used a Fisher’s exact test of the null hypothesis that the odds were equal (OR = 1). To compare means of continuous variables with roughly Gaussian distributions (determined using a quantile-quantile plot), we estimated the mean difference and its standard error and used an unpaired 2-tailed 2 distribution with 4 degrees of freedom. The analysis was repeated using natural splines with 2 to 4 degrees of freedom to ensure that the findings were not sensitive to these assumptions.To compare the trajectory of clinical outcomes over time between IgM-positive and IgM-negative groups, we used a linear mixed effects model . Variablt test. These results were statistically significant . In this case, the mixed model reduced to ordinary linear regression, and the group effect was highly statistically significant   with a random intercept to account for a possible correlation among repeated observations for each person. The variance of the random intercepts was taken as 0, indicating no correlation among repeated observations. This assumption was confirmed by comparing the means of the permeability values for the 2 COVID-19 patients and the 3 healthy controls using a 2-tailed  < 0.01) .These studies on patients with COVID-19 were approved by the Johns Hopkins University Institutional Review Board (JHU IRB) , with a waiver of consent because all specimens and clinical data were deidentified by the Core for Clinical Research Data Acquisition of the Johns Hopkins Institute for Clinical and Translational Research; the study team had no access to identifiable patient data. Patient numbers per analysis are denoted in the figure legends. Informed consent for studies on various control populations was obtained following protocols approved by the JHU IRB .AR, LCR, SLZ, DRT, FA, JBS, PCS, MD, PMH, ALC, SCR, RER, AP, BTG, CEM, and OL designed research studies. LCR, FA, MITZ, JBS, EKL, NES, JB, SY, RW, AMV, AG, RML, ZG, PCS, JEH, CEM, CAM, OL, LG, LGA, QY, and DH conducted experiments. AR, LCR, DRT, FA, MITZ, JBS, EKL, NES, JB, SY, PCS, SD, LG, LGA, MD, PMH, ALC, SCR, RER, LMS, KZJF, JEH, BTG, CEM, LCS, CAM, and OL acquired data. AR, SLZ, NES, JB, SY, RW, AMV, AG, RML, ZG, PCS, SD, LG, LGA, MD, PMH, ALC, SCR, BTG, and ZW analyzed data. DRT, WAC, ALC, RER, AP, LMS, KZJF, BTG, LCS, and CAM provided data/reagents. AR, LCR, SLZ, DRT, FA, MITZ, JBS, EKL, NES, JB, SY, RW, AMV, AG, RML, ZG, PCS, SD, LG, MD, PMH, WAC, ALC, SCR, RER, AP, LMS, KZJF, JEH, BTG, CEM, LCS, CAM, OL, LGA, QY, and DH wrote/edited the manuscript. SLZ, DRT, and ZW curated data.


Background: The endoplasmic reticulum (ER) is a multi-functional organelle responsible for cellular homeostasis, protein synthesis, folding and secretion. It has been increasingly recognized that the loss of ER homeostasis plays a central role in the development of autoimmune inflammatory disorders, such as rheumatic diseases. Purpose/Main contents: Here, we review current knowledge of the contribution of ER stress to the pathogenesis of rheumatic diseases, with a focus on rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We also review the interplay between protein folding and formation of reactive oxygen species (ROS), where ER stress induces oxidative stress (OS), which further aggravates the accumulation of misfolded proteins and oxidation, in a vicious cycle. Intervention studies targeting ER stress and oxidative stress in the context of rheumatic diseases are also reviewed. Conclusions: Loss of ER homeostasis is a significant factor in the pathogeneses of RA and SLE. Targeting ER stress, unfolded protein response (UPR) pathways and oxidative stress in these diseases both in vitro and in animal models have shown promising results and deserve further investigation. Rheumatic and musculoskeletal diseases (RMDs) are a heterogenous group of inflammatory conditions that usually affect joints, muscles, bones, tendons and ligaments, but can also affect any other organ of the body . There aDisease development in SLE is a result of a culmination of genetic, immunoregulatory, epigenetic, hormonal, and environmental influences. The disease is characterized by abnormal T cell signaling, B cell hyperactivity, production of autoantibodies, such as antinuclear (ANA) and anti-double-stranded DNA (dsDNA) antibodies, and formation of immune complexes (ICs), causing multisystem inflammation, and tissue and organ damage  ,3. RA isThe endoplasmic reticulum (ER) is a multi-functional organelle best known for its role in protein folding . When un2+) storage, and autophagic vacuole formation [The ER is responsible for synthesis and folding of approximately one third of the entire proteome in eukaryotic cells , it is aormation ,16. Specormation ,17. Howeormation ,19. In cormation , the ER 2+ levels, activates peptidylarginine deiminase (PAD) enzyme, which in turn facilitates protein citrullination [DRB1 allele was found to lead to in vivo generation of ACPAs, increase serum levels of tumor necrosis factor alpha (TNF-α) and bone erosions [ER stress has been implicated in the pathogenesis of several human leukocyte antigen (HLA)-associated disorders, including RA and AS ,22,23. Hlination . In the Another case in point, certain HLA-B27 alleles are major genetic risk factors in AS . One of When ER stress occurs, UPR is induced to resolve accumulation of misfolded proteins and restore homeostasis ,32. The Targeting protein aggregation and ER stress with the use of chemical chaperone 4-phenylbutyric acid (4-PBA) , a UniteSeveral other studies have targeted ER stress and specific UPR pathways in RA and SLE with encouraging results, indicating a pathogenic role for loss of ER homeostasis in these diseases. However, it is important to caution that although there is currently a wide range of molecules capable of modulating ER function in vitro and in animal models, substantial challenges remain in elucidating their precise mechanisms, as well as their specificities and safety. The currently available drug-like molecules aimed at targeting the UPR have been reviewed elsewhere .XBP1) mRNA. Spliced XBP1 (XPB1s) translocates to the nucleus and upregulates genes involved in mitigating the protein burden, including those responsible for increasing protein folding capacity , protein degradation  and transport pathways [When BiP is released, IRE1α oligomerizes and auto phosphorylates in trans, activates endoribonuclease and the splicing of X-box-binding protein 1  were downregulated for RA patients [IRE1α-XBP1s axis activation has been implicated in the pathogenesis of RA, by the demonstration that XBP1 splicing induces toll-like receptor-dependent cytokine secretion, which in turn induces XBP1 splicing in synoviocytes, in a autocrine loop that sustains synovial fibroblasts activation . In a stpatients . An addipatients . RA synopatients ,51,52. Spatients . TogetheXbp1s, while no modification in Xbp1t was observed. In B cells, it was observed that XBP1s-positive and CD19-positive B cells were significantly reduced in the pristane+ STF-083010 group in comparison to the pristane group. STF-083010 treatment suppressed pristane-induced B cell activation, plasma cell generation, immunoglobulin secretion, generation of B cell activating factors, and levels of TNF-α. No significant changes were observed in serum IL-6 levels among the groups. When considering levels of autoantibodies, STF-083010 treatment suppressed pristane-induced anti-dsDNA and anti-Smith antibody generation, with no differences observed among the three groups in relation to ANA. STF-083010 treatment also attenuated immunoglobulin deposition in the kidney and renal damage. No differential XBP1s expression was found in the kidneys among the three groups, suggesting that it is XBP1s activation in B cell, rather than in kidneys, that is driving renal damage [IRE-1 is a central regulator of B cell differentiation ,55,56,57l damage .Scd1 and Scd2) gene transcription and lipid accumulation in B cells of these patients, as compared to healthy controls [In vitro, Ire1α deletion in B cells decreased cell survival and growth, and greatly impaired B cell differentiation into plasma cells [lpr mice against nephropathy, pulmonary and hepatic lymphocyte infiltration, autoreactive antibody formation, plasma cell differentiation and dramatically reduced B cell lipid volumes. Levels of autoimmune antibodies were restored to pre-treatment levels 4 weeks after interruption of treatment [Another recent study revealed that the IRE-1α/XBP-1 axis is upregulated in B cells of SLE patients, and correlates with stearoyl-coenzyme A desaturase , involved in the activation of genes related to adaptation and relief of ER stress and oxidative stress [Atf4−/− cells are sensitive to oxidative stress and have impaired expression of genes related to glutathione synthesis [BiP release is associated with PERK dimerization and autophosphorylation. Once activated, PERK phosphorylates the eukaryotic initiation factor 2α (eIF2α), which can also be phosphorylated by PERK-independent mechanisms  and drame stress . ATF4 anynthesis . When ERynthesis ,68. Anotynthesis ,70. Nrf2ynthesis . PERK siynthesis .A chemical compound named salubrinal is known to reduce ER stress by selective inhibiting of eIF2α dephosphorylation . Salubriα-ATF4 axis in SLE has been underexplored, although impaired signaling in the PERK axis has been demonstrated in PBMCs from SLE patients, where downregulation of PERK was found, particularly in patient with severe condition (SLEDAI ≥ 12) [The role of the PERK-eIF2AI ≥ 12) . Lower eAI ≥ 12) . On the AI ≥ 12) . AnotherAI ≥ 12) .After BiP dissociation, ATF6 travels to the Golgi via coat protein complex II (COPII) vesicular transport, where in a process known as regulated intramembrane proteolysis (Rip), Golgi-localized proteases site-1 protease (S1P) and site-2 protease (S2P) cleave the cytoplasmic domain of ATF6, which then translocates to the nucleus, where it activates cytoprotective genes including those encoding the UPR, ERAD components, XBP1, and ER chaperone proteins  ,80,81. UATF6 in RAFLS [in vivo, including attenuation of ATF6 expression [There is a limited number of studies considering ATF6 in the context of RA. Although higher expression levels of ATF6 have been reported in RA synovium, and proinflammatory cytokines such as IL-1β and TNF have been shown to induced expression of in RAFLS ,87. Tacrin RAFLS , has beepression . There hpression ,76,77, hDRB1*03:01, the most significant genetic risk factor for SLE [DRB1*03:01 allele presented activation of UPR and proteasomal degradation, reduction of intracellular ATP levels, as well as enhanced TNF-α and nitrite production, as compared to the Tg mice carrying the RA predisposing DRB1*04:01 allele and the RA-protective DRB1*04:02 alleles. Intraperitoneal injection of IFN-γ in the mice culminated in increased serum levels of anti-dsDNA, glomerular IC deposition and histopathological renal changes that resemble human LN [One of the hypotheses put forward to explain the link between HLA alleles and disease pathogenesis has been the major histocompatibility complex (MHC) cusp theory, which states that in addition to presenting antigens, “HLA molecules encode ligands in one of their hypervariable regions, designated a “cusp” based on its three-dimensional cusp-like conformation. Under certain environmental and background gene conditions, these cusp-ligands can interact with non- MHC receptors thereby activating aberrant cell signaling events that cause disease development” ,90. In a for SLE , activat for SLE . Furtherhuman LN . These ahuman LN ,31,91.A recent study in AS, demonstrated that ATF6 mediates fibroblast growth factor 2 transcription in chondrocytes, thereby aggravating angiogenesis and osteogenesis, processes which are central in the pathogenesis of the disease. In vivo, ATF6 inhibition with ceapin-A7 slowed the progression of osteogenesis by preventing angiogenesis-osteogenesis coupling .In summary, several studies have targeted ER stress and specific UPR pathways in RA and SLE, indicating a pathogenic role for loss of ER homeostasis in these diseases. Molecular chaperones act to prevent aberrant protein aggregation by binding to unfolded and misfolded proteins ,17. In aGRP78 is a molecular chaperone and a key regulator of ER homeostasis, implicated in humoral and cellular autoimmune responses in RA, and a putative autoantigen in the disease ,100,101.GRP78 gene by CRISPR-Cas9 technique prevented anti-arthritic activity by azithromycin [Recently, it was demonstrated that azithromycin targets the UPR by inhibiting GRP78 activity, and that this treatment improved the severity of lesions comparably to the TNFα inhibitor etanercept in CIA mice. Additionally, deletion of the hromycin . When evhromycin . The samhromycin ,107,108.HLA–DRB1*04:05-restricted epitopes of BiP (BiP 336–355 and BiP 456–475) and found they were differently recognized by effector and regulatory T cells [336–355 induced PBMC proliferation and correlated with clinical arthritis activity and with the levels of circulating anti- BiP/citrullinated BiP antibodies, continuous oral administration of BiP 456–475 to mice with CIA resulted in improvement of joint inflammation and histologic scores, reduction of CD4+ T cell proliferation, increased numbers of CD4+CD25+FoxP3+ regulatory T cells and CD4+FoxP3+ T cells, and increased secretion of IL-10 from T cells. These findings illustrate the importance of the balance between effector and regulatory T cells in disease pathogenesis, as well as the immunomodulatory potential of BiP [Based on the previous observation that oral and nasal administration of specific peptides could induce Treg in animal models of autoimmune and inflammatory diseases , Shoda e T cells . While ol of BiP , highligl of BiP ,107,110.A study evaluating T lymphocyte cell death in SLE patients revealed that upon treatment with thapsigargin, a known inducer of ER stress, T cells from SLE patients responded aberrantly, showing reductions in expression levels of GRP78, increased apoptosis, and reduced autophagic response in comparison to healthy controls . Like RA2O2) [ROS are reactive species generated endogenously or exogenously due to incomplete oxygen reduction, and include oxygen radicals  and non-radical derivatives  . The mai2O2) ,113. Exo2O2) ,116,117.2O2) . Nonethe2O2) ,120. Oxi2O2) ,122.2+ signaling) and regulation of apoptosis. However, when overproduced, and depending on the redox state of its cellular environment, NO may become harmful and react with ROS to form highly reactive molecules such as peroxynitrite, thereby generating new epitopes with the potential to break immune tolerance [Also relevant to autoimmunity and RMDs are reactive nitrogen species (RNS)  and peroxynitrite, produced mainly by mitochondrial nitrogen oxide synthetase (mtNOS) . NO is aolerance . NO-induolerance .Epidemiologic, biologic, environmental, and genetic human studies, as well as animal models, have implicated oxidative stress in the pathogenesis of RA . ConsistHLA-DRB1 gene copies. For example, a 21-fold higher RA risk was found among patients who have had a smoking history and carried two SE-coding genes, compared to SE-negative nonsmokers [Cigarette smoking, a major culprit for the development of RA, increases protein citrullination and oxidative stress ,135, andnsmokers .2+ levels, activate PAD, and increase protein citrullination [Based on recent studies, the SE has been proposed as a signal transduction ligand  that intlination ,146,147,lination ,29. AddiIn SLE, the main source of ROS is overproduction by T-cell mitochondria due to mitochondrial hyperpolarization . In this2+ transfer, cell death and inflammation [2+ uptake by the mitochondria and increase in production of ATP for the adaption to the condition of stress [The ER and mitochondria form structural and functional intraorganellar connections aimed at maintaining cellular homeostasis; the organelles cross-talk and coordinate processes such as redox signaling , Ca2+ trammation ,160,161.ammation ,163. Calammation ,165. It f stress ,166. Conf stress .2O2), dysregulated disulfide bond formation/breakage can favor ROS accumulation and contribute to oxidative stress [Since disulfide bond formation in the ER is a relevant source of ROS (He stress ,168,169.e stress ,44,50. Ge stress ,172,173.DRB1*03:01 [Examples of the role of ROS-ER stress interplay in RMDs include the reduction of mitochondrial hydrogen peroxide generation in neutrophils upon IRE1α inhibition with 4μ8C , 4-PBA-iB1*03:01 . In addiB1*03:01 . The stuB1*03:01 .Loss of ER homeostasis is a significant factor in the pathogeneses of RA and SLE, and while the elucidation of the mechanisms involved is still a work in progress, targeting ER stress, UPR pathways and oxidative stress in these diseases both in vitro and in animal models have shown promising results and deserve further investigation.


In cancer, antigen-presenting cells (APC), including dendritic cells (DCs), take up and process proteins to mount adaptive antitumor immune responses. This often happens in the context of inflamed cancer, where reactive oxygen species (ROS) are ubiquitous to modify proteins. However, the inflammatory consequences of oxidized protein uptake in DCs are understudied. To this end, we investigated human monocyte-derived cell surface marker expression and cytokine release profiles when exposed to oxidized and native proteins. Seventeen proteins were analyzed, including viral proteins , inflammation-related proteins , matrix proteins , and vastly in the laboratory used proteins . The multifaceted nature of inflammation-associated ROS was mimicked using gas plasma technology, generating reactive species cocktails for protein oxidation. Fourteen oxidized proteins led to elevated surface marker expression levels of CD25, CD40, CD80, CD86, and MHC-II as well as strongly modified release of IL6, IL8, IL10, IL12, IL23, MCP-1, and TNFα compared to their native counterparts. Especially IL8, heme oxygenase 2, and vimentin oxidation gave pronounced effects. Furthermore, protein kinase phospho-array studies in monocyte-derived cells pulsed with native vs. oxidized IL8 and insulin showed enhanced AKT and RSK2 phosphorylation. In summary, our data provide for the first time an overview of the functional consequences of oxidized protein uptake by human monocyte-derived cells and could therefore be a starting point for exploiting such principle in anticancer therapy in the future. Inflammation is a process of local defense against harm inflicted by tissue injury, infection, and irritants. Irrespective of acute or chronic inflammation, a central trait is the release of reactive oxygen species (ROS) into the tissue by professional phagocytes, such as neutrophils and macrophages . There, 2O2) [Major ROS in the inflamed microenvironment are, for instance, superoxide, hydrogen peroxide, nitric oxide, and hypochlorous acid ,6,7. For2O2) . Myelope2O2) , which f2O2) . It is k2O2) ,20. ROS 2O2) . HoweverGas plasma technology was employed to generate and test an array of oxidized proteins. Exposure to gas plasma, a partially ionized gas generated at body temperature, is known to transfer a multitude of ROS from the gas phase into a liquid. This concept can be exploited by treating protein suspensions with gas plasma, generating a variety of biomolecule modifications and oxPTMs ,23,24. BIn this work, more than a dozen proteins were exposed to gas plasma in vitro. Subsequently, each of the proteins was added to monocyte-derived cells. After prolonged culture, the surface marker and cytokine and chemokine secretion signatures were analyzed across a range of markers and molecules. For specific proteins, protein phosphorylation was assessed, and monocyte-derived cells were found to react to the oxidation of proteins in distinct ways in several instances, suggesting that such processes may play a part in physiology and pathology alike.+ monocytes were separated via positive magnetic bead separation (BioLegend). Cell purity was assessed using flow cytometry, and viable cell counts were determined. 2 × 105 cells were seeded in 24-well plates in 500 µL Roswell Park Memorial Institute medium  containing 10% fetal bovine serum , 1% L-glutamine (Corning), 1% penicillin and streptomycin (Corning), 800 international units (IU) human granulocyte-macrophage stimulating factor  and 500 IU human interleukin (IL) 4  to generate immature human monocyte-derived cells. Cells were incubated at 37 °C with 95% humidity and 5% CO2.Peripheral blood mononuclear cells were isolated from buffy coats dedicated for research purposes and obtained from de-identified blood from the Institute of Transfusion Medicine  via Ficoll-Paque density gradient centrifugation as described before . Erythro2O. Cells and supernatants were collected 24 h later.Monocyte-derived cells were incubated with the following native and oxidized (ox) proteins: cytomegalovirus gB , human papillomavirus 16 , Epstein–Barr virus , heat shock protein 27 , interleukin-8 (IL8) (BioLegend), insulin , mumps virus nucleoprotein , calreticulin , hepatitis B virus e-antigen , high-mobility group box 1 , heme oxygenase 1 , heme oxygenase-2 , vimentin , collagen type II , ovalbumin , human albumin , and bovine serum albumin . Lyophilized proteins were reconstituted according to the manufacturer’s instructions and diluted in phosphate-buffered saline (PBS). For the screening, monocyte-derived cells were incubated with 10 ng/mL of the proteins, except for BSA, Alb, and Ova, which were added at a final concentration of 10 µg/mL. As a positive control, monocyte-derived cells were stimulated with 100 ng/mL lipopolysaccharide , while the addition of PBS alone served as vehicle control. For treatment, 100 µL of PBS-containing proteins were pipetted in a 96-well U-bottom plate . Oxidation was achieved using the atmospheric pressure plasma jet kINPen   operatedCytoFLEX LX device , and data were analyzed using Kaluza 2.1.3 software (Beckman-Coulter).Cells were collected in FACS tubes and washed three times with cold FACS washing buffer . Surface marker expression was investigated by incubating the cells with fluorochrome-conjugated antibodies  after inSupernatants of stimulated cells were collected after 24 h, and chemokines and cytokines were measured using bead-based cytokine detection technology according to the manufacturer’s instructions (BioLegend). The following targets were quantified against a 5-log standard curve: interleukin (IL) 1β, IL6, IL8, IL10, IL12p70, IL17A, IL18, IL23, IL33, interferon (IFN) α, IFNγ, monocyte-chemoattractant protein (MCP) 1, and tumor necrosis factor (TNF) α.7 monocyte-derived cells were cultured in the presence or absence of PBS, LPS, native or oxidized IL8, insulin, or Ova after 2 h starving. One hour later, cells were collected on ice and pelleted. Protein phosphorylation was analyzed using the Proteome Profiler Human Phospho-Kinase Array Kit  and Human/Mouse MAPK Phosphorylation Array  according to the manufacturer’s instructions. Signals were acquired after adding chemiluminescent reagent super signal WestPicoPlus  in a chemiluminescence detection system .At least 1 × 10prism 9.4.1 . Comparison of surface marker expression and cytokine secretion was made using Mann–Whitney Test. Principal component analysis (PCA) was performed with normalized surface marker expression and cytokine concentration data sets. Levels of significance were indicated as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).Graphing and statistical analysis were performed using + monocytes were isolated from blood donors and differentiated for three days using GM-CSF and IL4 before incubating them for 24 h with proteins , engulf proteins and differentiate after activation to promote T-cell response. In this study, moDC and moMac are collectively referred to as monocyte-derived cells. Three signals from monocyte-derived cells are required to activate T-cells: antigen presentation via MHC, high surface expression levels of co-stimulating ligands, and cytokine secretion. CD14proteins a. Our prproteins b. Despitproteins c. Unlikeproteins d–f. Howeproteins a–h. LPS proteins i. Furtheproteins a–d. InteInflammation elicits potent ROS formation affecting not only cells but also proteins. To this end, the next question was how monocyte-derived cells respond to the oxidized version of each protein individually compared to the native (non-oxidized) protein samples a. Gas plFinally, the goal was to infer signaling pathway alterations induced by oxidized proteins in monocyte-derived cells by performing protein phospho-kinase screening a. Given There is evidence that oxidized proteins correlate with immune cell activation and immune response, such as breaking immunological tolerance in autoimmune disorders ,34. Ther+ T-cell counts and paralleled oxidation [2H), and sulfonic acid (Cys-SO3H) were observed after gas plasma treatment of cysteine [Extracellular ROS can induce oxidative posttranslational modifications (oxPTMs) in proteins at the amino acid level, leading to structural or conformational changes. Several factors, such as the position of the amino acid side chains and the peptide backbone as well as the site selectivity of the radical attack reflect the complexity of the ROS-induced protein oxidation . The mosxidation . In plascysteine , and oxicysteine . In our Our study gives a rough overview of changes in the surface marker expression and cytokine secretion in monocyte-derived cells after recognizing oxidized proteins. Interestingly, activation of monocyte-derived cells was not necessarily associated with increased cytokine secretion. For instance, oxIL8 and oxIns did not show notable changes in CD25, CD40, and MHC II expression, except for a slight upregulation of CD80/86 , while bWhen interpreting the data, it must also be taken into account that experiments with human monocyte-derived cells from donors show a high variance due to, e.g., infection-related background activity, unique immune system, or previous illness of the individual donor. Such polymorphisms have previously been demonstrated in DC responses to TLR-mediated stimuli, which have also been suggested to be due to differential susceptibility to infectious pathogens or autoimmune diseases within the human population . However+ T-cells, thereby turning CD40-expressing DCs into more effective APCs. In combination with increased secretion of IL10 and downregulated IL12, oxidized vimentin seems to induce a tolerogenic immune response—which was unexpected, as modified vimentin is known as autoantigen and autoantibody production [Minor differences between oxidized vs. native proteins can still influence the immune response, e.g., interactions with other immune cells could lead to recruitment to differences in leukocyte recruitment. For instance, we saw only minor changes for CD40 after incubation with oxidized EBV and Vim. Inflammation also drives the upregulation of CD40L on CD4oduction ,46. Inteoduction ,47.Other investigated proteins are not associated with autoimmune disorders, such as calreticulin (CRT) and high-mobility group box 1 (HMGB1). However, they have been identified as DAMPs and secreted as danger signals from dead or stressed cells to elicit inflammation and subsequent immune responses. Therefore, it is not surprising that in our study DCs secrete pro-inflammatory cytokines when stimulated with oxHMGB1, as they can signal ROS-induced redox stress. Previously, HMGB1 was shown to promote DC maturation  when cells were activated with Toll-like receptors (TLR)-9 stimulating CpG . In lineStrikingly, a generalized activation could not be observed in our study, raising the question of identifying factors determining the immunogenicity of oxidatively modified proteins. However, delineating structural and biochemical requirements or treatment-related parameters is complex as production kinetics, and specificity of ROS are hard to control, while oxidative modifications are further challenging to track and require versatile, costly detection methods. This study’s increased activation of phagocytes points out an inherent damage-associated molecular patterns (DAMPs) activity of oxidized proteins. Similar observations have been reported for advanced glycosylation end-products (AGE) on naturally occurring carbohydrates activating APCs via scavengers, mannose receptors, RAGE, and galectin-3 . LikewisVarious receptors enable DCs to sense diverse extracellular stimuli, followed by activation, maturation, and cytokine secretion, driving differentiation and expansion of T helper cell subtypes . Once a 2O2 during an inflammatory response and is not nearly as reactive as H2O2. In contrast, short-lived species interact faster with a target structure, such as a protein. Physical gas plasma technology generates various short-lived and long-lived ROS, and its physical, medical, and biological applications are versatile [For instance, HOCl-modified Ova stimulates and activates APCs ,61. Otheersatile ,64,65. Tersatile , leadingersatile ,68,69,70ersatile ,72. HoweReactive oxygen and nitrogen species are important in pathophysiological conditions because they are generated by immune cells during an inflammatory response, thereby inducing oxidative modifications to proteins. Innate immune cells take up such extracellular oxidized proteins, become activated, and maturate to support an immune response. Using gas plasma technology as a tool to generate various ROS simultaneously, this study identified altered phenotypic alterations and cytokine secretion profiles in monocyte-derived cells after stimulation with 17 different oxidized proteins and LPS. IL8 and insulin were chosen to investigate signaling pathways in monocyte-derived cells. The observed changes in signaling pathways, activation/ maturational surface markers, and cytokine response help refine the complexity of oxidized proteins in an inflammatory microenvironment. Taken together, the results show different immunogenicities of exogenous ROS-induced oxidized proteins—whereby each protein, each species, each modification, but no differences in protein concentrations leads to an individual reaction—and functional consequences in activating myeloid cells can influence the course of the immune response.


The impact of prenatal alcohol exposure (PAE) varies considerably between individuals, leading to morphological and genetic changes. However, minor changes usually go undetected in PAE children. We investigated PAE’s effects on gene transcription of genes related to cardiac dysfunction signaling in mouse myocardium and morphological changes. C57Bl/6 mice were subjected to a 10% PAE protocol. In postnatal days 2 and 60 (PN2 and PN60), morphometric measurements in the offspring were performed. Ventricular samples of the heart were collected in PN60 from male offspring for quantification of mRNA expression of 47 genes of nine myocardial signal transduction pathways related to cardiovascular dysfunction. Animals from the PAE group presented low birth weight than the Control group, but the differences were abolished in adult mice. In contrast, the mice’s size was similar in PN2; however, PAE mice were oversized at PN60 compared with the Control group. Cardiac and ventricular indexes were increased in PAE mice. PAE modulated the mRNA expression of 43 genes, especially increasing the expressions of genes essential for maladaptive tissue remodeling. PAE animals presented increased antioxidant enzyme activities in the myocardium. In summary, PAE animals presented morphometric changes, transcription of cardiac dysfunction-related genes, and increased antioxidant protection in the myocardium. Ethanol/alcohol consumption can induce several cardiac dysfunctions, such as cardiomyopathy and arrhythmias in adults. Increased consumption among women of childbearing age is a worrying health problem . FurtherStructural changes such as congenital heart diseases are observed in 38% of newborns with Fetal Alcohol Spectrum Disorders (FASD), a group of conditions identified in a person exposed to alcohol during embryonic development, especially related to neurodevelopmental and neurobehavior disorders ,3. In adAntioxidant supplementation and early diagnosis are key to preventing congenital heart diseases induced by PAE . SeveralPAE can result in immediate and long-lasting toxic effects leading to impairments with lifetime repercussions. Adolescents with PAE are at risk for systemic inflammation . A growiAnimal models to study PAE repercussions are powerful tools to analyze alcohol toxicity to organs, including the heart . AlcoholTherefore, to investigate the effect of PAE on the transcription of several cellular pathways and possible repercussions on the adult myocardium, we analyzed changes in morphometric measurements, mRNA expression of 47 cardiac dysfunctions-related genes of nine signal transduction pathways , and activities of antioxidant enzymes in the myocardium of adult mice submitted to PAE.Twenty isogenic mice  of the C57Bl/ 6 strain, weighing 20–23 g, were used to generate the offspring. The animals were obtained at the Animal Facility of the Universidade Nove de Julho and kept confined in appropriate plastic boxes under a light cycle . The temperature 21 ± 2 °C) and humidity were controlled, and food and water were ad libitum  compared to the Control group [6.85 cm (6.71–6.95)]  PAE vs. 2.42 cm (2.34–2.49) in the Control group (1–6.95)] D.p < 0.0001) compared to the Control group. The heart weight mean was 139.5 mg (136–148) in the PAE and 134 mg (128–137) in the Control group  compared to the Control group [0.77 mg/mm (0.76–0.78); 18S rRNA gene. Genes that presented increased (upregulated) or decreased (downregulated) expression compared to the Control group are shown in The gene expression results in the experimental groups at PN60 were given by the relative transcript levels normalized to the Vascular endothelial growth factor A (VEGFA) and an increase of 2.1-fold in the expression of cell survival AKT serine/threonine kinase-1 genes were observed in PAE animals compared to the Control group. The cell apoptosis signal transduction pathway was evaluated by the quantification of mRNA expression of the genes BCL2-associated Protein X (Bax), death receptor cell surface Fas (Fas), mitogen-activated Protein Kinases 1 and 14 (Mapk1) and (Mapk14), and tumor -protein P53 (Tp53). The PAE group presented a significant upregulation in mRNA expression of all apoptosis-related genes, except for the Fas gene, which remained statistically unaltered. A decrease of 0.6-fold in the mRNA expression of mitogen-activated protein Kinase 14 showed an increase of 4.1-fold in mRNA expression compared to the Control group. The Tp53, Mapk1, and Bax genes also presented significant mRNA expression of 0.8-, 1.2-, and 1.7-fold, respectively, compared with the Control group.The gene coding for Calsequestrin 2 (Casq2) and Solute Exchanger Family (Na-Ca) 8, member A1 (Slc8a1) mRNAs was observed compared to the Control group. In turn, the PAE group showed considerable increases in mRNA expression of 2.6-, 2.4-, and 2.0-fold in ATPase, Ca2+ transport, cardiac muscle, slow contraction 2 (Atp2a2), Receptor 2 of ryanodine, (RyR 2) and phospholamban (Pln), respectively, compared to the Control group.Regarding calcium kinetics, in the PAE group, a significant reduction of 0.6-fold in both expressions of members 1A and 1B of the family of heat shock proteins—Hsp70 (Hspa1a/1b) and of antioxidants enzymes catalase (Cat), Superoxide Dismutase 1 (Sod1), and Glutathione Peroxidase 4 (Gpx4) were accessed. A significant increase was observed in all genes related to oxidative stress in the PAE group. The mRNA expression of the Hspa1a/1b gene showed an increase of 1.9- fold compared to the Control. The genes Gpx4, Sod1, and Cat present increased mRNA expression of 1.5-, 1.4-, and 1.1-fold, respectively, compared to the Control group.Alcohol, especially in the PAE, is an important event increasing oxidative stress in the body. The mRNA expression of Glyceraldehyde-3-Phosphate Dehydrogenase , Hexokinase 1 , and muscle Phosphofructokinase  mRNA expressions were also decreased when compared to the Control group. On the other hand, a significant increase in the mRNA expression of NAD+Ubiquinone Oxidoreductase Subunit A3 , Glucose transporter type 1 , Tafazzin , and Uncoupling Protein 2  genes were observed compared to the Control group.A significant reduction in the mRNA expression of glycolysis-related genes was observed. The IL-6 (2.0-fold), Tnfrsf1a (1.7-fold), and TNF (1.2-fold) were found compared to the Control group.The inflammation pathway was also assessed, and a significantly increased mRNA expression in all genes analyzed in the PAE group was observed. In addition, increased mRNA expressions of protein 2 similar to Calcineurin B (Chp2), Angiotensin II receptor, type 1a (Agtr1a), and α-myosin heavy chain (Myh6) genes by 0.8-, 0.8-, and 0.7-fold compared to the Control group. In contrast, the genes of the β-myosin heavy chain (Myh7) and protein 1 binding to Calcineurin (Cabin1) in the PAE group presented increments of 1.3-fold and 1.7-fold in mRNA expression, respectively, compared to the Control group. In addition, the mRNA expressions of activated T cell nuclear factor, cytoplasmic, dependent on Calcineurin 3 (Nfatc3), insulin-like growth factor 1 (Igf1), and protein Kinase, Kinase, Kinase, 2 activated by Mitogen (Map3k2) were increased by 1.6-, 1.2-, and 0.8-fold, respectively, compared to the Control group.Moreover, the PAE group presented downregulation of both mRNA expression of the natriuretic peptide B (Nppb) gene presented the second-highest increase in relative mRNA expression (3.2-fold) compared to the Control group. Furthermore, in comparison to the Control group, the genes of the natriuretic peptide A (Nppa), protein Kinase C beta (Prkcb), alpha (Prkca), and gamma (Prkcg) also presented elevated mRNA expression of 2.6-, 3.1-, 2.8- and 1.3-fold, respectively, in PAE group.The mRNA expressions of genes encoding natriuretic peptides and protein kinases C isoforms, important cardiac hypertrophy markers, were significantly increased in the PAE group compared to the Control group. Notably, the Col1a1, Col3a1, Tgfb1, and Mmp9 genes were evaluated to assess the PAE effect on cardiac extracellular matrix dynamics. In the PAE group, the type III collagen α 1 mRNA expression (Col3a1) was unaltered compared to the Control group. Oppositely, the PAE group presented an increment of 2.8-fold in the transforming growth factor β 1 (Tgfb1) mRNA expression. In addition, significant increases of 2.8-fold in the mRNA expression of the matrix metalloproteinase 9 (Mmp9) gene and 1.7-fold in the alpha 1 type I collagen (Col1a1) mRNA expression were observed in comparison to the Control group.Accordingly, like the genes related to cardiac hypertrophy, extracellular matrix genes showed significant changes in mRNA expression due to PAE. Therefore, the mRNA expressions of In agreement with the transcriptional changes of oxidative stress-related genes, augmented activities of antioxidant enzymes SOD, CAT, and GPx were found in PAE myocardium in PN60 .The toxic effects of alcohol on embryo development are widely reported. Alcohol readily crosses the placental and blood-brain barrier and impairs cell development in several organs . AlthougMoreover, the numerous molecular changes were similar to those observed in experimentally infarcted animals . In bothAlcohol consumption did not affect pre-pregnancy weight , as poinSeveral studies reported that PAE was sufficient at low to moderate doses to result in animal intrauterine growth restriction (IUGR) ,34. Inciv/v, [Cardiac hypertrophy after PAE has been reported, although controversial, in several studies in animals and humans. For example, using alcohol administration at dose of 6.36% v/v,  found thv/v,  found inv/v,  and in hv/v, . Notablyv/v, ,36,39. Cv/v, ,37,40.ANP (Nppa) and BNP (Nppb). These genes are mainly transcribed in the myocytes of the atria and ventricles, respectively [BNP, was observed in the PAE ventricular samples. To our best knowledge, modulation of natriuretic peptide expression has not been observed with PAE protocols. In addition to natriuresis, diuresis, and vasodilation, BNP acts directly on the heart [The natriuretic peptides, gold standards biomarkers in heart failure (HF) , are alsectively . Howeverectively ,45, whiche heart , providihe heart .v/v), even after 10 weeks. Furthermore, using lower alcohol concentration in our protocol, the Edn1 mRNA expression remained unchanged after PAEThe renin-angiotensin-aldosterone system (RAAS) contribution to cardiac hypertrophy is widely evidenced. A study by [angiotensin-converting enzyme (ACE) and Agtr1a (AT1R) genes may increase Ang II availability and effectivity, increasing heart stress.In our study, endothelin-1 (Edn1) did not present altered expression levels. Indeed,  did not study by  showed tAlpha-myosin heavy chain (Myh6) mRNA expression was negatively regulated in PAE mice. Hypertrophy is associated with an induction of β-MHC at the expense of α-MHC; therefore, this phenotype change is evidence of cardiac hypertrophy. Furthermore, a decreased Myh6 expression and the re-expression of beta-myosin heavy chain (Myh7) mRNAs directly impair myocardial contractile capacity after MI [after MI ,51.IGF-1 expression was related to a decrease in apoptotic cell death. A study by [IGF-1 mRNA upregulation caused accelerated maturation and consequent enlargement of fetal cardiomyocytes isolated from sheep exposed to alcohol during gestation and prevented apoptosis. Moreover, an increased mRNA expression of IGF-1 has already been described in other tissues undergoing apoptosis [IGF-1 mRNA expression augmentation observed in our study could be a compensatory response of the myocardium to alcohol exposure. Indeed, some studies have observed that increased IGF-1 expression after PAE is directly related to protein C kinase (PKC) expression [v/v significantly increase neuronal PKC-alpha and PKC-gamma gene expression [Prkca and Prkcb genes, together with augmented mitogen-activated protein kinases (MAPKs) mRNA expressions observed in our study, may play a critical role in transmitting and regulating intracellular signals. The MAPK signaling pathway includes the activation of kinases and their amplification actions [Studies on PAE attribute a cardioprotective role to IGF-1. For example, Chen and colleagues , using fstudy by  reportedpoptosis ,55. In fpression . Other apression . Moreovepression . These s actions , therefo actions ,61.matrix metalloproteinase-9 (Mmp-9) expression is associated with collagen I and III maturation in individuals with HF, indicating the importance of these enzymes in the activation and deposition of collagen in the fibrosis process [Mmp9 gene expression, along with an increased TGF-β1 mRNA expression—a persistent stimulus in the repair phase of hypertrophy and an important pro-fibrotic cytokine—reinforce the hypertrophic environment triggered by PAE. For instance, anti-TGF-β1 therapy attenuates cardiac remodeling [collagen I, TGF-β1, and matrix metalloproteinase 9 in PAE animals indicates a signal transduction mechanism that might suggest the cardiac repair process [The increase in cardiac  process . The incmodeling . The inc process . Indeed,HSP70 expression increased in PAE animals at PN21 and PN90. The authors observed, together with other data, that there was activation of a protective pathway in a tentative to reduce damage related to PAE. Accordingly, we might assume that increased gene expression and activities of antioxidant enzymes, along with increased HSP70 mRNA expression, may alleviate oxidative stress in the myocardium.Finally, the mRNA expression of antioxidant enzymes increased in PAE mice. A study performed by  found diAn interesting and comprehensive review  reportedThis study showed that PAE promoted morphometric changes, fetal gene reprogramming, altered mRNA expression of several genes related to cardiac dysfunction, and increased antioxidant enzyme activities in the myocardium from male mice in PN60. Two significant conclusions are possible to achieve based on our data. First, the evidence of a reprogrammed mRNA expression profile of genes encoding fetal contractile proteins and gold standards biomarkers for cardiac hypertrophy and increased cardiac indexes suggests a hypertrophic state of adult PAE animals. Then, the transcriptional activation of physiopathological biomarkers of different signal transduction pathways, together with the increased antioxidant activities, evidence the stressor role of alcohol when administered as PAE in the heart of adult mice. In addition, the dysfunctional profile of gene expression observed in the myocardium, combined with unhealthy habits commonly observed in adulthood, such as excessive alcohol consumption, smoking, abusive use of other drugs, and ingestion of ultra-processed foods, might facilitate the establishment and progression of cardiovascular diseases, worsening the prognosis.


The relationship between levels of AMR in livestock and its occurrence in humans is complex and is driven by many factors. We conducted whole genome sequencing of MCRPEC to analyze the molecular epidemiological characteristics, history, and relatedness of 50 isolates obtained in 2019 from different reservoirs in farms and markets in Ha Nam province, Vietnam. 34 sequence types (STs) with 3 new STs were identified in multilocus sequence typing analysis: ST12945 and ST12946 from chicken feces, and ST12947 from flies. The AMR phenotypes of 50 MCRPEC isolates were as follows: ampicillin , cefotaxime , gentamicin , amikacin , meropenem , ceftazidime , colistin  and ciprofloxacin . All 50 MCRPEC isolates were identified as MDR. 100% (50/50) isolates carried AMR genes, ranging from 5 to 22 genes. The most prevalent plasmid replicon types carrying mcr-1 were IncP-1 , IncX4 , and IncHI2/IncHI2A . These data suggest that the epidemiology of the mcr-1 gene is mostly determined by plasmid spreading instead of clonal dissemination of MCRPE strains. The co-occurrence of several STs such as ST10, ST48, ST155, ST206, ST2705 in various sample types, joined to the higher prevalence of a few types of Inc plasmids, confirms the dissemination of the mcr-1 carrying plasmids in E. coli clones established in livestock. 5 over 8 STs identified in flies  suggested the fly contribution in the transmission of AMR bacteria in environments. These popular STs also occur in human samples and 100% of the human samples were positive for the mcr-1 gene.Livestock has been implicated as a reservoir for antimicrobial resistance (AMR) genes that can spread to humans when antimicrobials are used in animals for food production to treat clinical diseases and prevent and control common disease events. In Vietnam,  Antimicrobial resistance (AMR) is a global public health and development threat. In 2019, WHO declared that AMR has been one of the top ten global health threats facing humanity that caused least 700,000 deaths globally per year, which could potentially increase to 10 million deaths globally per year by 2050 . LivestoEnterobacterales (CRE) have spread worldwide and present a serious public health threat with high mortality rates as high resistance also occurs to other clinically important antimicrobials, such as cephalosporins, fluoroquinolones, and aminoglycosides , especially the mcr-1 gene that spread rapidly in animals, travelers, foods, and environment around the world , flies, foods, and the environment  in a community and from clinical specimens in referral hospitals –16. HoweSamples were obtained in 2019 from various sources in Ha Nam, a province in Northern Vietnam including poultry and swine farms, farmers and markets. Samples including farmer feces (20), chicken feces (61), pigs feces (31), dogs feces (18), farm flies (36), wastewater (21), and market surface (15) were collected and kept at – 80°C in the Antimicrobial Resistance Laboratory of the National Institute of Hygiene and Epidemiology (NIHE) in Vietnam. All samples were pre-processed as previously described in 2022 .mcr-1 genes, the samples were kept overnight in tryptic soy broth at 37°C without shaking. We used 1.5 ml of enriched broth for total DNA extraction and detected mcr-1 by using PCR. Among mcr-1 positive samples, we randomly selected 50% of the samples for further analysis.To screen for samples containing E. coli-like colony represent for each sample for identification by MALDI-TOF MS . All confirmed E. coli isolates were selected for further screening for mcr-1. Next, DNA from E. coli isolates was subjected to confirm the presence of mcr-1 by PCR.The selected samples were cultured on MacConkey agar containing 0.5 mg/L colistin. We then picked up one Farm-scale is computed and classified by Decree 13/2020/ND-CP Detail Guideline of Livestock Law issued by the Vietnamese Government. Number of all types of food animals raised, including pigs, chickens, ducks, geese, quails, doves, ostriches, etc., is converted to “livestock unit” for each type of food animals, “total farm livestock unit” is sum of livestock units of all food animal types raised and is used to categorize farm-scale  .E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as controls.The minimum inhibitory concentrations (MIC) to antimicrobials commonly used in treatment were determined on 50 MCRPEC isolates to identify susceptible (S) or resistant (R) phenotypes. The MCRPECs were tested for resistance against 8 drugs of 6 class of antimicrobials. MICs were determined using the agar dilution method for ampicillin (AMP), cefotaxime (CTX), gentamicin (GEN), amikacin (AMK), meropenem (MER), ceftazidime (CAZ), ciprofloxacin (CIP) and the microdilution method for colistin (CST) (Sigma-Aldrich) according to CLSI 2018 and European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2018 , 20. E. mcr-1-positive E. coli isolates. Genomic DNA libraries of selected strains were prepared for WGS using the Nextera XT DNA Library Preparation Kit (Illumina), following the manufacturer's instructions. 300 bp paired-end sequencing was performed on an Illumina MiSeq platform . Raw sequence reads were de novo assembled into contigs using Shovill v1.1.0 with SPAdes v3.14.1 as the assembler. NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to annotate genes. AMR genes from ResFinder databases of the Center for Genomic Epidemiology were identified using ABricate and Staramr  were used to identify replicon typess, and MLST following Achtman sequence definitions database (https://pubmlst.org/bigsdb?db=pubmlst_ecoli_achtman_seqdef) was used to identify sequence type for the isolates. A maximum-likelihood phylogenetic trees based on the core genome single nucleotide polymorphisms (SNPs) were constructed from WGS data of the 50 MCRPEC isolates utilizing Prokka and Roary and IQ-TREE v1.6.11.The procedure was performed as previously reported . Whole gAll isolates were assigned to phylogenetic groups  based on Clermont phylogenetic typing schemes .https://cge.cbs.dtu.dk/services/VirulenceFinder/).Virulence genes were identified by VirulenceFinder V2.0  using descriptive statistics as appropriate.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the National Institute of Hygiene and Epidemiology, Vietnam .Informed consent was obtained from all human subjects involved in the study.mcr-1 positive: 100% (20/20) of farmer feces samples, 62.3% (38/61) of chicken feces samples, 41.9% (13/31) of pig feces samples, 11.1% (2/18) of dog feces samples, 55.6% (20/36) of farm fly samples, 22.6% (7/31) of wastewater samples, and 26.7% (4/15) of market surface samples. We selected 50% (n = 52) from 104 mcr-1 positive samples for further analysis. Among 52 selected samples, 50 samples appeared E. coli like colonies and all of them were confirmed E. coli by MALDI-TOF-Biotyper. Next, DNA of E. coli isolates was subjected to confirm the presence of mcr-1 by PCR. Whole genome sequencing was performed on DNA from all mcr-1-positive E. coli isolates (n = 50) which included feces from farmer feces (n = 17) chicken feces (14), pig feces (4), dog feces (1), farm flies (11), wastewater (2), and market surface (1).We found that 51.5% (104/202) of samples were Examining antimicrobial resistance of MCRPEC isolates, per drug and sample type, we observed that the resistance proportions to tested antimicrobials in the isolates were as follows: 100% (50/50) resistance against ampicillin, 10% (5/50) against cefotaxime, 60% (30/50) against gentamicin, 8% (4/50) against amikacin, 18% (9/50) against ceftazidime, 24% (12/50) against colistin, and 80% (40/50) against ciprofloxacin . In partHouseholds which provided 78% (39/50) MCRPECs have resistance to more antimicrobials with 8 chosen antimicrobials than 18% (9/50) strains from medium-scale farms, 2% (1/50) strains from small-scale farms, and 2% (1/50) strains from market. 46% (23/50) and 44% (22/20) MCRPECs in this study belonged to poultry and mixed farms. Notably, there was one MCRPEC strain collected on the surface market but resistant to 8/8 antimicrobials tested in this study.Based on the PCR screening results, WGS was conducted to analyze AMR genes for further characterization of these species. The results of the MIC tests and WGS analysis are presented in mcr-1 but 76%  strains had MIC value at 2 mg/L. Among which 24% (12/50) of isolates were resistant to colistin, of which 33% (4/12) were resistant at an MIC value >16 mg/L. Especially, three isolates showed reduced susceptibility to meropenem (3/50) at 2 mg/L.In our study, all isolates bearing mcr-1), aminoglycosides (aac(3)-, aac(6')-, ant(3”)-IIa, aph(3”)-Ib, aph(3')-Ia, aph(6)-Id, aadA), fosfomycin (fosA), β-lactams , sulfonamides , fluoroquinolones , tetracyclins [tet(A)], trimethoprim (dfrA), phenicols , and macrolides  (Results from whole genome sequencing showed that 100% (50/50) of isolates harbored antimicrobial resistance genes (ARGs) with the range from 5 to 22 genes responsibility for resistance to colistin  .n = 4; 8%); ST206 , ST48 , ST2705 , ST10 , ST 4015 , ST101  and ST189 . The minimum spanning tree based on multi-locus sequence types (mcr-1 carrying plasmids in E. coli clones established in livestock.Thirty four sequence types (ST) of 50 selected MCRPECs were identified  and clasce types  showed tThe phylogenetic tree indicated two major clades: one clade  containeThe core gene phylogeny revealed the close relation among MCRPECs isolated from different sample types and sources: ST2705, ST155, ST683, and ST206 have the close relation; ST2705 isolated from human, pig feces and flies, ST155 and ST683 from human, chicken feces, flies, ST206 from human, dog feces, market, wastewater.All 50 MCRPECs harbored genes conferring resistance to various other antimicrobials: aminoglycosides, β-lactams, fosfomycin, macrolides, phenicols, fluoroquinolones, sulfonamide, tetracyclin, trimethoprim also presented on the csgG, entA, entE, entS, fepC, fepD, fes and ompA were detected in ≥ 98% the MCRPEC isolates. Other virulent genes that were commonly prevalent include; aslA, csgB, csgD, csgF, entB, entC, entD, entF, fdeC, fepA, fepB, fepG, fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH, fimI, gspC, gspD, gspE, gspF, gspG, gspH, gspI, gspJ, gspK, gspL, gspM, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, and ykgk/ecpR. The less prevalent (≤ 30%) were astA, chuS, chuT, chuU, chuV, chuW, chuY, fyuA, iroN, iutA, kpsD, kpsM, and PapC. The proportion of virulence genes from humans, animals, flies and environment was presented in A total of 59 different virulence genes were detected from the MCRPECs. The virulence genes; valent ≤ 0% were amcr-1 in the genomes, mcr-1-containing contigs were extracted from the complete genomes of MCRPEC isolates. We detected that mcr-1 is located on both the chromosome (n = 7) and on plasmids (n = 43) isolates  followed by IncX4 , and IncHI2/IncHI2A . Other replicons were IncHI2/IncHI2A/IncN/p0111 , IncFIA(HI1)/IncHI1A/IncHI1B(R27) , IncHI2/IncHI2A/IncN , IncFIB(AP001918)/IncHI2/IncHI2A , p0111 , and IncY . Plasmid replicons of six isolates were unable to identify.To classify the locations of isolates . Among pmcr-1 with the presence of insertion sequence ISApl1 on both chromosomes (n = 7) and plasmids (n = 8). Among 7 isolates carrying mcr-1 on their chromosome, 3 contained the full Tn6330 transposon sequence ISApl1-pap2-mcr-1-ISApl1. These strains were collected from human feces, pig feces and wastewater. Although, no full transposon was found on plasmids, 5 plasmids contained indication of the presence of ISApl1 with a full copy downstream and 2 plasmids with a partial downstream ISApl1 element. Among plasmids carrying the full copy of downstream ISApl1, 3 plasmids were IncP1 replicon, which has been known as an important plasmid type in antimicrobial transmission between gram negative bacteria.We observed 15 different genetic contexts of mcr-1 selected for sampling in this study had a high diversity of specimen types with MCRPECs; including fecal samples of humans and farm animals, such as chickens, dogs, fly samples, wastewater samples from farms and market surface samples. A study conducted in more than 25 nations on four continents also indicate the existence of mcr-1 gene in animals, food, human, and the environment  to important antimicrobials such as colistin  and genes conferring resistant to aminoglycosides, fosfomycin, β-lactams, sulfonamides, fluoroquinolones, tetracyclins, trimethoprim, phenicols and macrolides, suggesting that the impact of MCRPECs on the transmission of AMR genes should not be underestimated.The results of WGS confirmed this statement when they observed various AMR genes with a minimum of 5 resistance genes and a maximum of 22 AMR genes. transfer . 22 AMR E. coli strains as the study conducted in the Southern region of Vietnam, with colistin resistance rates in chickens and pigs of 22.2 and 24.4%, respectively  , 29. In colistin . Possibl EN.CITE , 32Althoommunity . This camcr-1 positive Enterobacteriaceae with MIC values of ≤ 2 mg/L  in this study had the MIC value at 2 mg/L coresponding with multiple reports of ≤ 2 mg/L . Recent ≤ 2 mg/L . The omnmcr-1 from animals and the environment to humans, but there was no specific genetic evidence to verify this hypothesis . Phylogenetic groups A (76%) and B1 (18%) were predominant among the tested isolates while group C and group F had low prevalence 2 and 4%, respectively. This result is in agreement with those reports that indicated c groups –37. Commc groups , 38–41. c groups . It is aorldwide . Three norldwide , 45. Thiorldwide . MCRPECsorldwide .E. coli strains can possess different virulence markers to invade and colonize inner layers of the intestinal lining causing infections with different clinical symptoms , the less prevalent virulence genes was reported the close related to ESBL genes such as iutA; fmH, papC encode the adhesion subunit of type 1 fimbriae and related to colonization. Overal, the presence of MCRPECs bearing different virulence genes which are reported in extraintestinal pathogenic E. coli (ExPEC)  . This camcr-1 gene showed that there were many types of plasmids that could carry mcr-1 belonging to incompatibility groups IncX4, IncI2, InHI2, IncF, IncFIB, IncY and IncP-1 , followed by IncX4 . IncP-1 plasmid group is widely distributed in feces, soil, wastewater treatment plants and in both pathogenic and opportunistic species of bacteria, playing an important role in encoding multidrug resistance and bacterial adaptation by horizontal gene transfer  flanked by two ISApl1 insertion sequences. In this study, we found full genetic context of the Tn6330 transposon, which was found on only chromosome of three E. coli isolates. In line with previous studies, these findings suggested that the insertion of mcr-1 into the chromosome of E. coli occurred recently. However, we also detected a loosening of ISApl1 insertion sequences on both chromosome and plasmid harboring mcr-1, indicating that the mcr-1 is being stabilized on the core genetic backbone in the ecological niches .The data of this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB55625  under grant number 108.02-2017.320, GCRF OneHealth Poultry Hub  under grant number BB/S001269/1, the Japan Agency for Medical Research and Development (AMED) under grants numbers JP22gm1610003, JP22fk0108133, JP22fk0108139, JP22fk0108642, JP22wm0225004, JP22wm0225008, JP22wm0225022, JP22wm0325022, JP22wm0325003, and JP22wm0325037, and from IRD and LMI DRISA.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. 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