The role of mast cells (MCs) in fungal infection is largely unknown. This study was to explore a protective role and mechanism of MCs in fungal keratitis. Experimental fungal keratitis (FK) mouse model was developed. Mice untreated (UT) or receiving corneal wound without fungal infection (Mock) were used as controls. Large number of connective tissue MCs was found in normal mice. MC activation with degranulation was largely observed, and the percentage of degranulated/total cells was high in FK. Dilated limbal vasculature with increased permeability, as well as largely infiltrated neutrophils with stimulated ICAM-1 protein levels were observed in corneas of FK mice, when compared with Mock and UT mice. Interestingly, pretreatment with cromolyn sodium (Block) significantly blocked MC degranulation, dramatically suppressed vascular dilation and permeability, and markedly reduced neutrophil infiltration with lower ICAM-1 levels in FK mice at 6–24 hours. Furthermore, the Block mice manifested prolonged disease course, increased pathological damage, and vigorous fungus growth, with much higher corneal perforation rate than FK mice at 72 h. These findings reveal a novel phenomenon that MCs play a vital role in protecting cornea against fungal infection through degranulation that promotes neutrophil infiltration via stimulating ICAM-1 production and limbal vascular dilation and permeability. In the large sample of clinical epidemiology studies, Fusarium8, Aspergillus9, and Candida albicans were the main pathogens of FK, and Fusarium manifests with more serious symptoms and poor efficacy of treatment10.Fungal keratitis (FK), also known as keratomycosis or mycotic keratitis, is an infection caused by opportunistic pathogenic fungi. In the developing countries, it occurs mainly among male populations at low levels of education, who engage in agricultural activities, and is frequently accompanied by a history of plant trauma. Most patients eventually require surgery (59.45%), at a low level of pharmaceutical cure rate (40.55%)11. Neutrophils are attracted by chemokines in the affected area via neutrophil surface integrins. Then these immune cells subsequently transmigrate from blood vessels of limbus to the site of infection. Neutrophils can distinguish the site of the micro-organisms to initiate endocytosis and release extracellular traps, and final kill micro-organisms12.In fungal diseases, different types of immune cells are involved in the antifungal process with distinct mechanisms. Infiltrated inflammatory cells were mainly neutrophils followed by macrophages in corneas of patients and mice15, fibroblast proliferation, and scarring formation. MCs also induce the expression of ICAM-1 in endothelial cells through the release of cytokines to promote neutrophil chemotaxis16 and perform antimicrobial actions by secreting antimicrobial peptides defensins17. Studies on corneas infected by virus in MC-knockout mice manifest increased virus load and leukocyte infiltrations, but decreased corneal transparency18. When MCs encountered Candida albicans, they can digest spores by endocytosis19. After toe pads were inoculated with the fungus, MC activation-associated proteins can be detected in the serum of guinea pigs20. However, no direct data demonstrated the role of MCs or their anti-fungal mechanism in fungal keratitis.Mast cells (MCs) are bone marrow progenitor–derived immune cells that mature in tissues affected by the local microenvironment. MCs are mainly distributed in the transitional areas between the body and outside. MCs can participate in numerous biological processes, including infection control, wound healing, inflammation and immune tolerance. In infectious diseases, MCs play a critical role by releasing previously stored particulate materials as well as new synthetic substances in vascular permeability and angiogenesisFusarium Solani to explore possible functions and mechanisms of MCs during fungal infection in the cornea. We found that MCs activation initiated by fungi infection contributed to the neutrophil infiltration by influencing the expression of ICAM-1 to the infected cornea and result in protection in FK.To investigate how MCs influence corneal fungal infection, we established the mouse model of fungal keratitis with 23. To reveal the characteristics of these cells, we used classic staining methods—toluidine blue staining combining the whole-mount technique to verify the distribution of MCs in the cornea and conjunctiva. Consistent with previous studies, we found the presence of a large number of MCs in the conjunctiva  and mucosal mast cells (MMCs) based on to their locations and enzymes. Some researchers show both types of cells may co-exist in some areas. Studies show that mouse MC protease (mMCPT)-6 was specifically expressed in CTMC while mMCPT-1 existed only in MMCs. To determine the MC subtypes in the limbus, the corneal whole mounting and immunofluorescence staining technique was applied. Our results found that the MCs in limbus were mMCPT-6 positive but mMCPT-1 negative cells Fig. , indicat24. In order to observe whether the fungal infection trigger MC degranulation in the cornea, we established FK models to observe the presentation of activated (degranulated) MC in corneal limbus. We found that a large number of particles were around the MCs after 6 hours of corneal irritation by fungal infection or trauma  to detect changes in the vessel diameter and vessel permeability in the process of FK.P = 0.830), 12 h (P = 1.000) and 24 h (P = 0.250), respectively, in the Mock group, which was not statistically different. But it considerably widened to 29.53 ± 5.23, 30.14 ± 4.36, 34.09 ± 6.29 μm at 6 h (P < 0.01), 12 h (P < 0.01) and 24 h (P < 0.001) respectively, in FK mice. When MC activation was blocked, the vessel diameter remained at low levels with 23.96 ± 5.34 μm at 6 h, 23.02 ± 5.67 μm at 12 h, which was as same as the Mock group at 6 h (P = 0.706), 12 h (P = 0.108), but lower than that of the FK group . Although the vessel diameter of the Block group rose to 31.06 ± 5.97 μm at 24 h, which higher than that of the Mock group (P < 0.001), it was still lower than FK group (P = 0.028)  Figs  and 4A.FP < 0.001; 12 h, P = 0.001; 24 h, P < 0.001). The EB concentration of the FK group sustainably increased to 0.95 ± 0.26, 1.45 ± 0.39, 3.54 ± 0.17 μg/L at 6 h, 12 h and 24 h respectively in FK group, and higher than that the Mock group at 12 h and 24 h . When MC activation were blocked, the EB concentration were decreased to 0.95 ± 0.20, 2.47 ± 0.35 μg/L at 12 h and 24 h, lower than the FK group .The EB concentration used to analyze vascular permeability Fig.  was at a26. An enzyme-linked immune-sorbent assay was used to investigate whether ICAM-1 protein expression changed by MC activation.In addition to vascular changes, ICAM-1 is crucial for neutrophil migration into the sites of infection through the vascular wall−3 ± 9.24 × 10−3 ng/μg) in normal corneas, MC activation irritated by fungi triggered the expression of ICAM-1 to 61.15 × 10−3 ± 9.61 × 10−3, 63.53 × 10−3 ± 11.18 × 10−3, 69.28 × 10−3 ± 9.61 × 10−3 ng/μg at 6 h, 12 h and 24 h, respectively  and significantly lower than the FK group .Intercellular adhesion molecule varied following the vascular dilation and increased permeability during inflammation in FK model, Mock group, and Block group Fig. . We confely Fig. . After M16. They play a crucial role in killing fungi during infection27. To determine the role of MCs in the chemotaxis of neutrophils, we used cromolyn sodium, the mouse model of FK, and immunofluorescence staining to observe chemotaxis of neutrophil to the central cornea.Neutrophils are the earliest and the most innate immune cells infiltrating to the infected area during earlier stage of fungal keratitisP = 0.023) at 6 h; it was increased to 1847 ± 258 cells at 12 h (P = 0.015) and 5046 ± 258 cells at 24 h (P < 0.001), significantly higher than that of Mock group. When MC activation was blocked, the number of neutrophils was decreased to 511 ± 92 cells at 6 h, lower than the Mock group (P < 0.001) and FK group (P < 0.001), but similar to normal corneas  and FK group   Figs  and 4D.To investigate the effects of MC activation on the clinical process and pathological change of FK, we treated animals with cromolyn sodium and observed the corneal changes with a slit lamp microscope and pathological staining. Before (0 h) and after  establishing the fungal keratitis model, digital photos of the mouse corneas were captured under a slit-lamp microscopy Fig. .Figure 5In the Mock group, there was just slight edema in 6 h and 12 h, and corneal grey-white infiltration occurred after 24 h. In the FK group, the clinical manifestations were similar to the Mock group with scratching and coenobium only in the central cornea at 6 h and 12 h. The central and paracentral cornea had a large area of gray-white infiltration with the iris visible, trauma in central cornea, and hypopyon occurred in the anterior chamber at 24 h. The clinical manifestations continued to worsen at 36 h, with corneal gray-white infiltrated lesions, iris not visible and increasing opacity. While corneal edema aggravated and hypopyon existed at 48 h, the central corneal lesions were reduced. And it showed only central corneal lesions at 72 h, with peripheral corneal transparent and no edema.P = 1, n = 6 for each time point of the cornea) between the FK group and Block group   Fig. .P < 0.001)  Fig. . After 7P = 1.000, P = 1.000), increased significantly at 24 h (P < 0.001), reached the peak at 36 h (P = 0.019), then significantly decreased to low level at 48 h, and very few at 72 h in the FK group. However, the strong growth of fungi continued and lasted from12 to 72 h in the Block group , which suggested that block of blocking MC activation decreased neutrophil suppression of fungal growth in keratitis.HRT3 confocal microscopy, GMS (Gomori’s methenamine silver) and immunofluorescence staining were performed to examine the growth of fungi. HRT3 images Fig.  showed toup Fig. , much hiThese findings suggest that MC activation by corneal central fungi may cause early neutrophil infiltration from the limbus site and thus lead to severe clinical manifestations in the early stage of fungal keratitis, indirect anti-fungal activity, and finally preventing the late tissue damage caused by fungi growth, decreasing the perforation rate in the late stage.29, however their role remains poorly understood in the process of corneal fungal infection. This research found that MCs, which are mainly distributed in the corneal limbus, play a dominant role in controlling the fungus infection of the cornea by several mechanisms. This conclusion is based on following evidence: (1) Fungi infection can induce activation and degranulation of MCs in the limbus; (2) cromolyn, the MC stabilizer for degranulation, can significantly prolong the course of disease, increase pathological damage, and increase the perforation rate of fungal keratitis; (3) cromolyn reduces neutrophil infiltration to the site of the fungal infection in the central cornea; (4) MC activation can increase ICAM-1 expression.MCs play a key role in the fight against bacterial infection and viral infection30. So we conjecture that fungi irritated corneal central nerve fibers to secrete nerve factors and inflammatory factors, then induction of MC degranulation via receptors on MC surface. However, detailed studies are required to establish the definitive mechanism.Bacteria, parasites, fungi and other physical stimuli can promote MCs activation through the recognition receptors on the cell surface. In the vitro studies, these microbes can directly stimulate the MC degranulation. In this study, the central cornea was irritated by fungi causing MC activation in limbus signaling there are some mechanisms during cell activation. Neurons can secrete nerve growth factors and inflammatory factors, which can bind to the receptors on MC surface following different stimuli26, and also kill bacteria indirectly by chemotaxis of neutrophils33. MCs can also make direct contact with the bacteria to produce a variety of antibacterial substances, and they can phagocytize and subsequently display the bacterial antigens on their cell surfaces34. Recent findings have revealed that MCs form the extra-cellular traps which can trap the bacteria to some location to facilitate the anti-bacterial reaction36. We demonstrated that MCs have an indirect effect on killing fungi in the pathogenesis of FK, via reducing fungal growth by promoting neutrophil infiltration. MC activation is needed for controlling the invasion of fungi was also similar to the study on rat model of acute invasive fungal rhino sinusitis37. In a HSV-1 keratitis animal model, research shows that MCs knock-out can reduce the infiltration of neutrophils in the cornea, so as to promote the elimination of virus tilter18. In our study, effects of MC activation blocked by cromolyn on neutrophil infiltration is also similar, but the effect on the elimination of fungi is opposite to that of HSV-1, and further studies are needed.Many immune cells participate in fungal infection. There are few evidences on MC in FK. Studies show that MCs can directly kill microorganisms38. Eric Pearlman research team confirmed that in the infectious keratitis, corneal haze formation was consistent with neutrophils infiltration39. The fungal load in neutrophil-knockout mice was higher than the controls40, showing that neutrophils are crucial to produce a fungicidal effect. Neutrophils engulf the spores and then kill the fungi or spore through oxygen-dependent or oxygen-independent mechanisms; it can also be formed by extracellular traps to limit fungal hyphae and spore wavering, and then ultimately digest the hyphae or spore through enzymes. In the present study, MCs were located only in the limbus, while the fungal hyphae in the cornea were mainly located in the central cornea. Neutrophils were attracted to the corneal center, where MCs were activated by the fungi. Therefore, it is clear that MCs play an important role in the chemotaxis of neutrophils in fungal keratitis, which have antifungal effects indirectly.MCs are necessary for corneal wound healing and coordinating neutrophil and platelet recruitment41. Changes of blood vessels were different when MCs activated by different stimuli42: After intraperitoneal injection of saline, there were no change in the diameter of the peritoneal blood vessels; after 48/80 stimulation, the vascular diameter decreased at the first 9 days, and then gradually return to normal. The different effect of the two stimuli on vessel diameter may be due to the degree of MC stimulation. Our results suggest that MC activation is less likely to be caused by trauma than by fungal stimulation. This may explain why trauma stimulus does not cause a change in the vessel diameter, whereas a persistent stimulation of the fungus causes increases. Experimental studies showed that 48/80 stimulation of MCs in the early tissue culture (6–8 days) did not increase the vascular permeability of macromolecules, while promotion of vascular permeability of macromolecules occurred in the late stage (9–10 days)43. Strbian et al. found that cromolyn could restrict hemorrhage formation induced by plasminogen activator44. Evans blue can bind with albumin to form Evans blue- albumin complex. In this study, vascular permeability detected by Evans blue concentration was increased by fungal irritation and was higher than trauma irritation but can be suppressed by cromolyn sodium in Block group.MCs which locate around the blood vessels can promote changes in vascular diameter46. The infiltration of neutrophils in the blood vessel is divided into three steps: rolling, adhesion, and penetrating. ICAM-1 is crucial for neutrophil amoeboid movement through the vascular wall to the damaged site. We confirmed that the increased expression of ICAM-1 in the cornea initially then decreases in the late stage in the FK group were coherent with the changes in the number of infiltrated neutrophils. The neutrophil infiltration and ICAM-1 expression were blocked by MC inhibitors during the early stage of fungal keratitis.MC activation promotes neutrophil infiltration, blood vessel diameter increases, and ICAM-1 changes. In fungal keratitis, corneal infiltration of inflammatory cells is mainly due to neutrophils. ICAM-1 plays an important role in the chemotaxis of neutrophils during inflammation47. Neutrophil chemotaxis was inhibited in the MC non-activation group, which made it unable to kill fungi. Consequently, the fungal virulence effect is far greater than the destructive effects of neutrophils on the tissue organization, which may be the reason that the perforation rate of the block group was higher than that of the FK group. This demonstrated that MC activation was required for controlling fungal keratitis.There are two factors inducing perforation in FK: one is the virulence or mechanical penetration of the fungus itself, and the other is the destruction of the inflammatory cells to the cornea. When neutrophils kill fungi, the intracellular enzyme is released, causing the destruction of corneal tissuein vivo effect48. Cromolyn may have an effect on the degranulation of other granule cells, such as neutrophils, basophils. To investigate this role, our next step will be to use MC knockout mice to verify this effect. In addition, the role of MCs in mice may be different with that in the human body, and the specific role in the human body still needs further experimental study.In this experiment, the choice of cromolyn sodium as a MC stabilizer, has certain limitations. Cromolyn sodium can affect macrophages and other inflammatory cells, and it is unclear whether there is an immunosuppressive effect on other cells of the corneal tissue. In addition, studies have shown that in mice, effects of cromolyn sodium on MC membrane stability is lower than that of the other animal In the present study, we showed connective MCs are found in the corneal limbus and conjunctiva. Corneal fungal infection led to degranulation of these MCs. MC activation can promote vascular changes and increase ICAM-1 expression, thereby promoting neutrophil chemotaxis, as well as affect corneal transparency in the early stage and decrease the perforation rate. The specific reason for MC degranulation caused by fungi infection still needs further investigation. This study adds to evidence on the pathogenesis of fungal keratitis, and may provide information for the treatment of fungal keratitis in the future.All experimental protocols used in these studies were maintained and animals euthanized according to the protocols approved by the Ethical Committee of Experimental Animal Care of Henan Eye Institute. Eight-week-old C57BL/6 J male wild-type mice were purchased from the Model Animal Research Center of Nanjing University (MARC) . The mice were maintained on a 12:12-day cycle in a temperature-controlled room at 25 °C.49, and then additional injections were given every six hours for the duration of the experiment.Before injection, cromolyn sodium  was dissolved in phosphate-buffered saline  at a concentration of 4 mg/ml. The cromolyn sodium solution was protected from light during preparation. To stabilize the mast cells, cromolyn sodium  or vehicle (0.01 M PBS) was administered to the mice by a single intraperitoneal (i.p.) injection for 30–120 min before and after the modelFusarium solani (F. 3.1791) was built referring to previous methods50: using sterile knife to make crossing scratch on the corneal central with the depth of under Bowman’s membrane, then bamboo sticks with fungi hypha scratch 2 to 3 times following the crossing bottom to mimic the natural fungi infection in cornea. Corneas of the Mock group were processed the same procedure above, except fungi infection.The C57BL/6 J mice were randomly divided into three groups, which were the UT group, Mock group, FK group, and Block group, according to the littermate grouping method. The mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (80 mg/kg body weight), and l% (weight/volume) tetracaine hydrochloride eye drops were used for corneal surface anesthesia. Then, under a dissecting microscope, the FK model infected with 50. Parameters of lesion area, depth, corneal neovascularization were given a grade from 0  to 4 (very severe), and others that contained hypopyon, hyphema, descemetocele and perforation grade from 1 to 4. The fungi hypha and immune cells infiltration were observed by the HRT3 confocal microscope.Corneas of three groups were continuously observed and photographed at the corresponding time points under a slit-lamp microscope  and a HRT3 confocal microscope . Clinical scores were recorded under slit-lamp microscope according to lesion area, depth, corneal neovascularization and othersFor the histologic analysis, mice were euthanized by cervical dislocation at different time points, and the whole cornea and partial sclera were removed and then fixed in 10% neutral formalin (F) for 120 min. Two-micrometer sections were separately stained with Gomori methenamine silver (GMS), or hematoxylin-eosin (HE), and reviewed by light microscopy.The mice were euthanized by cervical dislocation at different time points. Whole eyes were removed and then fixed with an alcohol-formaldehyde solution (AF) for 120 min. The cornea was trimmed to retain the limbus, but the iris and ciliary body removed. After washing five times with 0.01 M PBS (10 min each), the corneas were dipped in the following solutions in sequence: a 0.2% Triton-BSA solution for 30 min, a 2% BSA solution for 30 min, and the primary antibodies  incubation for one night. Then, the secondary antibodies  were incubated for 6 hours, washed; incubated in CFW  for 30 min, washed; incubated with DAPI for 30 min, and washed. The corneas were then flat mounted with four or eight radial cuts . After multiple immunofluorescence and CFW staining procedures, the flat-mounted corneas of the model groups were photographed by confocal laser scanning microscopy . The image analysis was performed by visual inspection of the individual image sections. The different corneal layers were identified by their characteristic morphometric features. Representations of the morphological analyses of the neutrophils and fungal volumes were performed using the Imaris software, version 7.3.1 × 64 .Vessel diameter detection: The Vessel diameter detection was based on the corneal whole-mount technique. Vessel diameter was detected under fluorescent microscope. The limbal microartery in the four equal parts of the cornea was select and captured under 40× objective lens. Then the diameter of limbal microartery was detected on AR software. The limbal microartery is located between two capillary veins. There were four values per cornea, six corneas in every group.Limbal Vascular permeability: Evans Blue  in normal saline  was injected intravenously after operation. The mice were killed by cervical dislocation 1 h later and corneas were trimmed. After the tissue was then dried at 100 °C for 24 h, the samples were homogenized in methylformamide  with 200 μl per cornea, incubated for 24 h at 60 °C, and centrifuged for 5 min at 1000 g. The absorbance (A) of supernatants was analyzed at 632 nm using Envision Multi label plate reader 2104 . The amount of EB (μg/g) was calculated through standard curve established by known concentrations of EB.t) test or Kruskal-Wallis Rank Sum test using SPSS V.17.0. A P value of < 0.05 was considered significant. The data are expressed as the means ± STD.The statistical significance of the differences between groups at different times was determined by the one-way analysis of variance test, followed by the least significant difference t (LSD-


The aim of this study was to assess the prognostic value of chronic kidney disease (CKD) in relation to ischaemic stroke, intracranial haemorrhage, major bleeding, and all‐cause death in heart failure patients without atrial fibrillation.In this observational cohort study, heart failure patients without atrial fibrillation were identified using Danish nationwide registries. Risk of stroke, major haemorrhage, and death were calculated after 1 and 5 years to compare patients with and without CKD, ±dialysis . A total of 43 199 heart failure patients were included, among which 0.8% had CKD‐RRT and 5.9% had CKD‐no RRT. When compared with heart failure patients without CKD, both CKD‐RRT and CKD‐no RRT were associated with a higher 5 year rate of major bleeding (CKD‐RRT: adjusted hazard ratio (aHR): 2.91, 95% confidence interval (CI): 2.29 to 3.70; CKD‐no RRT: aHR: 1.28, 95% CI: 1.13 to 1.45) and all‐cause death . For the endpoints of ischaemic stroke and intracranial bleeding, only CKD‐no RRT was associated with significantly higher 5 year rates .Compared with patients without CKD, among incident heart failure patients without atrial fibrillation, CKD both with and without dialysis was associated with a higher rate of major bleeding and all‐cause death. Only CKD‐no RRT was associated with a higher rate of ischaemic stroke and intracranial bleeding. Heart failure (HF) and chronic kidney disease (CKD) often coexist.In the HF population, thromboembolic risk stratification is evolving to help identify ‘high‐risk’ subjects, who may be targeted for more regular review and follow‐up, as well as more intensified cardiovascular prevention strategies.The objective of the present observational cohort study was to assess the prognostic value of CKD in relation to the risk of ischaemic stroke, intracranial haemorrhage, major bleeding, and all‐cause death in HF patients without AF, using Danish nationwide administrative registries. We hypothesized that, compared with patients without CKD, in a population of incident HF patients without AF, CKD {both CKD with dialysis [CKD with renal replacement therapy (CKD‐RRT)] and without dialysis [CKD‐no RRT]} would be associated with a higher risk of ischaemic stroke, intracranial haemorrhage, major bleeding, and all‐cause death, also when controlling for concomitant cardiovascular risk factors.International Classification of Diseases (ICD‐10) since 1994; (ii) the National Prescription Registry,We used data from three nationwide registries: (i) the Danish National Patient Registry,ases ICD‐ since 19TableThe study population was identified as inpatient or outpatients aged >50 years, diagnosed with a primary discharge diagnosis of incident (first‐time diagnosis) HF in the period 1 January 2000 to 31 July 2014. Chronic kidney disease was defined as a diagnosis of CKD‐RRT  or CKD‐no RRT  between 1994 and time of HF diagnosis . All‐cause death was also included as an outcome, because of the high mortality among HF patients and the appertaining possibility of distortion from competing risks, and the fact that in administrative registries some deaths may be due to undiagnosed stroke or bleeding.Baseline characteristics were described separately for patients without CKD, CKD‐no RRT, and CKD‐RRT using means and standard deviation for continuous variables, and proportions for categorical variables. Time‐to‐event analysis was used to describe the association between CKD status (CKD‐no RRT or CKD‐RRT) and the risk of ischaemic stroke, intracranial haemorrhage, major bleeding, or all‐cause death. Time at risk was measured from baseline date (date of HF diagnosis) and until an event of ischaemic stroke, intracranial haemorrhage, major bleeding, all‐cause death, emigration, or end of study (31 December 2014), whichever came first. Additionally, patients were censored if they initiated anticoagulant therapy during the follow‐up period.et al.Crude cumulative incidence curves for all endpoints according to CKD status  were constructed based on the Aalen–Johansen estimator2DS2‐VASc  score; thus, we basically adjusted for components of this score. Additionally, as antiplatelet therapy may modify the association between CKD and risk of the endpoints, we also included use of antiplatelet therapy in the main adjusted Cox model.For the main analysis, Cox regression was used to calculate 1 and 5 year HRs of the endpoints according to the status of CKD (CKD‐no RRT or CKD‐RRT), with patients without CKD as reference group. Concomitant baseline co‐morbidities such as hypertension, diabetes, vascular disease, and prior ischaemic stroke are well‐known prognostic factors of stroke and bleeding in other heart diseases.Analyses were performed using Stata version 13 . Results are reported with 95% confidence intervals (CIs).As some patients might have a diagnosis of AF shortly after the HF diagnosis, a sensitivity analysis was performed by repeating the HR calculations after extending the definition of concomitant AF at baseline; AF patients were identified if they had a prior diagnosis code for AF at baseline (at time of HF diagnosis) or received a diagnosis code for AF within 30 days after the HF diagnosis. Furthermore, some patients are diagnosed with AF during follow‐up; thus, to investigate whether the inclusion of AF patients affects our main results, we performed another sensitivity analysis after censoring patients who are diagnosed with AF during follow‐up.In a supplementary analysis, to investigate whether the chosen well‐known risk factors are correctly included in the main adjusted model, additional models were constructed adjusting for age and sex only (model 1) and age, sex, and each individual risk factors (Models 2–6). The results of these additional models are presented in the No ethical approval is required for studies based on data from administrative Danish registries. The study was approved by the Danish Data Protection Agency (J. No. File No. 2015‐57‐0001). The study was conducted and reported in accordance with the STROBE  recommendations.FigureThe study population comprised 43 199 HF patients aged >50 years, among which 0.8% had CKD‐RRT and 5.9% had CKD‐no RRT at baseline Figure1. The TableBaseline patient characteristics are summarized in TableFigureThe number of events and the absolute risk of ischaemic stroke, intracranial haemorrhage, major bleeding, and all‐cause death in each patient group after 1 and 5 years of follow‐up are shown in FigureIn the Cox regression analysis adjusted for concomitant prognostic factors, when compared with HF patients without CKD, both CKD‐RRT and CKD‐no RRT were associated with a higher 5 year rate of major bleeding  and all‐cause death , which may reduce their thrombotic risk. Thus, in patients with CKD and HF, individualized risk assessment according to disease severity is necessary to optimize cardiovascular prevention strategies.We provided both risk/probability (risk ratio) and rate (HR) assessments of the associations.The large sample size uniquely possible with this type of cohort study minimizes the risk of random error. Selection into the study was not an issue, as we investigated a nationwide population cohort of incident HF patients without AF using administrative data, which also implies very limited loss to follow‐up.The diagnosis of HF has previously been validated with a sensitivity of 29%, a specificity of 99%, and a positive predictive value of 81–100%Patients with CKD were identified using ICD‐10 codes in the Danish registries, which have also been used in previous registry‐based studies of patients with CKD.2DS2‐VASc score.We investigated whether the presence of CKD was associated with ischaemic stroke, intracranial haemorrhage, major bleeding, and all‐cause death in patients with HF, and therefore, we adjusted for well‐known cardiovascular prognostic factors for stroke and bleeding. This was not an attempt to adjust for confounding and hereby explore the potential causal relationship between the exposure and outcomes, but to elucidate the potential added predictive ability of the exposure in relation to risk stratification of patients with HF after adjustment for other possible prognostic factors, essentially those included in the previously investigated CHAThe study population consisted only of patients aged >50 years, and HF in persons aged <50 years might represent a different group of patients, for example, patients with congenital heart disease. Accordingly, our findings may not apply to younger HF patients. Additionally, we were unable to distinguish between HF with preserved and reduced ejection fraction or to estimate the functional classification, as we did not have access to echocardiograms, so we cannot be sure that the results apply to patients with preserved and reduced ejection fraction. Lastly, the study was carried out as a nationwide study in the Danish population, which ethnically is fairly homogeneous; thus, future studies are needed to evaluate if our findings hold in more ethnically diverse populations.In conclusion, compared with patients without CKD, among incident HF patients without AF, CKD, despite severity, was associated with a higher rate of major bleeding and all‐cause death. Only CKD‐no RRT was associated with higher rates of ischaemic stroke and intracranial bleeding. Chronic kidney disease may have prognostic value when considering cardiovascular prevention strategies in the HF population without AF.http://www.icmje.org/coi_disclosure.pdf and declare the following.All authors have completed the ICMJE uniform disclosure form at G.Y.H.L. is consultant for Bayer/Janssen, BMS/Pfizer, Biotronik, Medtronic, Boehringer Ingelheim, Microlife, and Daiichi‐Sankyo; and speaker for Bayer, BMS/Pfizer, Medtronic, Boehringer Ingelheim, Microlife, Roche, and Daiichi‐Sankyo. No fees are received personally.T.B.L. is an investigator for Janssen Scientific Affairs, LLC, and Boehringer Ingelheim; and speaker for Bayer, BMS/Pfizer, Janssen Pharmaceuticals, Takeda, Roche Diagnostics, and Boehringer Ingelheim. No fees are received personally.All other authors declare no conflict of interest.Statistics Denmark provided the data for this study. No additional data are available beside the Table S1. ICD10‐codes and ATC‐codes used in the study.Table S2. Relative risks of all endpoints after 1 year of follow‐up, according to chronic kidney disease status (reference group: patients without chronic kidney disease).Table S3. Relative risks of all endpoints after 5 years of follow‐up, according to chronic kidney disease status (reference group: patients without chronic kidney disease).Table S4. Sensitivity analysis excluding patients with an atrial fibrillation diagnosis at baseline or within 30 days after the heart failure diagnosis: Hazard rate ratios of all endpoints after 5 years of follow‐up, according to chronic kidney disease status (reference group: patients without chronic kidney disease).Table S5. Sensitivity analysis censoring patients diagnosed with atrial fibrillation during follow‐up: Hazard rate ratios of all endpoints after 5 years of follow‐up, according to chronic kidney disease status (reference group: patients without chronic kidney disease).Table S6. Hazard rate ratios of all endpoints after 1 year of follow‐up (using different adjusted models), according to chronic kidney disease status (reference group: patients without chronic kidney disease).Table S7. Hazard rate ratios of all endpoints after 5 years of follow‐up (using different adjusted models), according to chronic kidney disease status (reference group: patients without chronic kidney disease).Click here for additional data file.


CN‐105, in a microglial cell line and murine model of ischemic stroke.At present, the absence of a pharmacological neuroprotectant represents an important unmet clinical need in the treatment of ischemic and traumatic brain injury. Recent evidence suggests that administration of apolipoprotein E mimetic therapies represent a viable therapeutic strategy in this setting. We investigate the neuroprotective and anti‐inflammatory properties of the apolipoprotein E mimetic pentapeptide, BL6 mice underwent transient middle cerebral artery occlusion and were randomly selected to receive CN‐105 (0.1 mg/kg) in 100 μL volume or vehicle via tail vein injection at various time points. Survival, motor‐sensory functional outcomes using rotarod test and 4‐limb wire hanging test, infarct volume assessment using 2,3,5‐Triphenyltetrazolium chloride staining method, and microglial activation in the contralateral hippocampus using F4/80 immunostaining were assessed at various time points. In vitro assessment of tumor necrosis factor‐alpha secretion in a microglial cell line was performed, and phosphoproteomic analysis conducted to explore early mechanistic pathways of CN‐105 in ischemic stroke.Ten to 13‐week‐old male C57/CN‐105 demonstrated improved survival, improved functional outcomes, reduced infarct volume, and reduced microglial activation. CN‐105 also suppressed inflammatory cytokines secretion in microglial cells in vitro. Phosphoproteomic signals suggest that CN‐105 reduces proinflammatory pathways and lower oxidative stress.Mice receiving CN‐105 improves functional and histological outcomes in a murine model of ischemic stroke via modulation of neuroinflammatory pathways. Recent clinical trial of this compound has demonstrated favorable pharmacokinetic and safety profile, suggesting that CN‐105 represents an attractive candidate for clinical translation. Ischemic stroke is a major cause of morbidity and mortality worldwide.The postischemic brain demonstrates a proinflammatory cascade, characterized by glial activation and release inflammatory mediators, neuronal excitotoxicity, and oxidative stress.Apolipoprotein E (apoE) is the primary apolipoprotein produced in the CNS, where its glial secretion is upregulated after injury.CN‐105 is an apoE‐mimetic 5 amino acid peptide  derived from the apoE receptor binding region by linearizing the polar face of the amphipathic helix involved in receptor interaction.This study evaluates the therapeutic efficacy of CN‐105 in a murine model of transient focal cerebral ischemia and reperfusion. We test the hypothesis that intravenous administration of CN‐105 reduces mortality, results in improvement of functional and histological outcomes after experimental ischemic stroke, and reduces microglial activation. We also explore the mechanisms by which CN‐105 exerts its immunomodulatory and neuroprotective effects by performing a phosphoproteomic analysis in animals exposed to cerebral ischemia.We performed a series of experiments to assess the safety and determine the physiological effects of CN‐105 using both males and females 8‐week‐old Crl:CD(SD) rats . These experiments were independently conducted  prior to the conduct of further experiments in this study. These animals received an implanted femoral vein catheter exteriorized between the scapulae for intravenous dosing purposes. 0.9% Sodium Chloride for injection was used as vehicle. CN‐105 was synthesized by Polypeptide Inc  to a purity of >99%. CN‐105 was dissolved in vehicle to the concentration of 1.25 mg/mL. Repeated intravenous administration of 5 mg CN‐105/kg/dose or vehicle, four times per day (20 mg/kg/day) for 14 days 6 h apart . Compared to the subsequent experiments, doses of CN‐105 used in these physiological studies were 50 times higher per dose and 200 times higher per day. Animals were randomized prior to any experiments and outcome assessments were performed in a blinded fashion.Multiple physiological parameters were measured in these experiments. These included body temperature, hematological (platelet count and coagulation profile), biochemistry , and respiratory (respiratory rate and minute ventilation) evaluations. Full experimental details are available in Data μL volume and administered via tail vein injection.CN‐105 used in subsequent animal experiments was similarly synthesized by Polypeptide Inc  to a purity of >99%. However, CN‐105 was dissolved in 1× phosphate‐buffered saline (PBS), reconstituted to 0.1 mg/kg concentration in 100 In all experiments, mice were randomized to treatment and vehicle group before injury and all animals were treated with allocation concealment. In each experiment, mice were randomized to treatment or vehicle groups prior to injury. Animals were treated with blinded concealment. All procedures and assessments were performed in blinded fashion. Animal procedures were approved by the Duke University Institutional Animal Care and Use Committee in keeping with established guidelines.Ten‐ to 13‐week‐old C57Bl/6J male mice  were used. Focal cerebral ischemia was induced by filamentous transient middle cerebral artery occlusion (tMCAO), as previously described.Vestibulomotor performance was assessed daily postinjury with the accelerating rotarod , as previously described.Motor coordination and limb muscle strength was assessed with the 4‐limb wire hanging test.Coronally sectioned (1‐mm thick) brain slices were stained in 2% 2,3,5‐triphenyletrazolium chloride  according to protocol.μm thick) were stained with anti‐rat F4/80 antibody  specific for activated microglia and cells of the monocyte lineage.Frozen coronal brain sections (40 α) produced by increasing concentrations of CN‐105  using fixed lipopolysaccharide (LPS)  stimulation (10 ng/mL), and by fixed concentration of 1 μM/mL of CN‐105 using increasing concentrations of LPS stimulation . Cell supernatant was analyzed using Mouse TNF‐α DuoSet ELISA kit  per manufacturer's protocol. All experiments were repeated six times and mean TNF‐α concentrations were recorded at 4 h post incubation.For all in vitro experiments, C8‐B4 mouse microglia cells  were used. We investigated the change in production of tumor necrosis factor‐alpha  analysis Data . Peptidet‐test was performed for infarct volume, microglial density analysis, and TNF‐α concentration of microglial cells. Repeated measures analysis of variance (ANOVA) was performed for rotarod and 4‐limb wire hanging latencies, and Dunnett's post hoc test for multiple comparisons was used. Log‐rank test was performed for survival evaluation. Principal components analysis was performed between sample groups to screen for outliers in the phosphoproteomic data. The independent two‐tailed t‐test was performed on logP < 0.05. All data were analyzed using SPSS version 20 .Independent two‐tailed Repeated intravenous administration of 5 mg CN‐105/kg/dose 4× per day (20 mg/kg/day) for 14 days over 5 min did not produce mortality and was without significant clinical effect on body temperature, hematology and coagulation, biochemistry, and respiratory parameters. Systemic exposure to CN‐105 was independent of sex and did not appear to change following repeated administration. Full results on physiological testing of CN‐105 are found in Supplemental Material 1.P = 0.013)  and hanging latency (P < 0.001). The improvement was observed as early as the first day post injury and the benefit was maintained up to 7 days. No significant functional improvement was observed when CN‐105 was administered beyond 30 min post reperfusion  and 15 min ischemic occlusion time  , supporting the mortality benefit of CN‐105.We observed that the survival in the vehicle arm in this experiment with 30‐min ischemic occlusion‐time (6 of 12 mice) was better than the preceding mortality experiment (3 of 12 mice). However, the survival of the CN‐105 arm was the same for both experiments (9 of 12 mice). Moreover, a post hoc analysis of survival at 72‐h, by combining the survival data of both experiments and using a two‐tailed chi‐squared test, still demonstrated significant survival benefit (15/24 (62.5%) vs. 9/24 (37.5%), 4/mm3 vs. 3.5 ± 0.7 × 104/mm3, P = 0.016). There was significant reduction in microglial cell density by CN‐105 compared to vehicle   using 10 ng/ml LPS stimulation measured at 4 h. Although suppression of TNF‐α production was not significant at lower concentrations of CN‐105, the percentage of suppression of TNF‐α by CN‐105 is concentration‐dependent with 20%, 34% and 53% suppression observed with 0.1, 0.3 and 1 μM/mL of CN‐105, respectively. CN‐105 was also able to suppress microglial TNF‐α secretion up to a concentration of 50 ng/mL of LPS stimulation , immunity (tyrosine kinase Lyn), and BBB integrity (cyclophilin A) Table . BiologiOur study has demonstrated that CN‐105, an apoE‐mimetic pentapeptide, reduces mortality, infarct volume, microgliosis, and improves long‐term functional outcome in a murine reperfusion model of ischemic stroke. We have previously demonstrated that larger peptides derived from the apoE receptor‐binding region (apoE133‐149) retain the neuroprotective and anti‐inflammatory propertiesα secretion from C8‐B4 microglial cells. These immune‐modulatory effects on microglial by CN‐105 are consistent with apoEWe demonstrated that CN‐105 downregulates microglial activation in ischemic stroke and suppress TNF‐Our study revealed a novel finding of increased microglial activation in the uninjured contralateral hippocampus after experimental ischemic stroke. Although the blood supply is not interrupted, it has been previously reported that the “healthy” contralateral hemisphere of an ischemic brain is associated with radiographic changesA possible mechanism by which CN‐105 exerts its anti‐inflammatory and neuroprotective effect may be through the preservation of the BBB. After ischemic stroke, BBB disruption occurs and persists for days. ApoE appears to protect cerebrovascular integrity in acute brain injury through maintenance of the BBB, as demonstrated in apoE‐deficient mice, which was associated with an increase in disruption of BBB after injury.Reduction in early mortality 24–72 h) after ischemic stroke was observed in our study with CN‐105. Early mortality in acute ischemic stroke is associated with initial clinical stroke severity, the size of the infarct and subsequent cerebral edema. h after Other differentially phosphorylated proteins may also provide clue toward CN‐105′s mechanism of action. For example, CN‐105 may suppress microglial activation through the deactivation of Lyn. Lyn belongs to the Src family of tyrosine kinases predominantly expressed in granulocytesOur study was limited by the inability to demonstrate improved functional outcome when CN‐105 was administered at 0.1 mg/kg beyond a latency of 30 min from ischemia. Although the protective effect of CN‐105 appears to be time‐dependent, our study was focused on delineating the early mechanistic effects of CN‐105 in ischemic stroke, and did not test the effects of higher drug dosage or multiple dosing, which have increased efficacy in prior studies.In summary, we demonstrate that intravenous administration of CN‐105 conferred durable sensorimotor functional improvements, reduced mortality, histological inflammation, and inflammatory phosphoproteomic signals following cerebral ischemia. In vitro application of CN‐105 also reduced microglial activation. With safety already demonstrated in a clinical trial, CN‐105 may be an attractive candidate for further clinical evaluation in ischemic stroke.T.M.T. designed the study, performed the in vivo experiments and functional testing, analyzed the data, and wrote the manuscript. B.J.K. designed the study and wrote the manuscript. E.J.S designed, collected and analyzed data, and wrote the manuscript. V.C. contributed to the in vivo experiments, immunohistochemistry assessments and wrote the manuscript. P.F. performed the in vitro experiments and wrote the manuscript. H.W. designed the study and contributed to the animal experiments. M.A.M. had scientific oversight of the proteomic component. H.N.D. assisted the study design, immunohistochemistry assessment, and edited the manuscript. D.T.L. designed the study, acquired grant, supervised the work, and wrote the manuscript. All authors reviewed the full manuscript.D.T.L, B.J.K, and H.N.D are co‐inventors of CN‐105 with US patent 9,303,063. D.T.L serves as an officer for Aegis‐CN, LLC. Aegis‐CN, LLC supplied the drug, but had no editorial control over study design, conduct, or writing of the manuscript.Data S1. Physiological studies of CN‐105.Click here for additional data file.Data S2. Supplemental methods.Click here for additional data file.Data S3. Supplemental results.Click here for additional data file.Table S1. Principal biological process of differentially significant phosphoproteins.Click here for additional data file.Table S2. Known cell signaling pathways of differentially expressed phosphoproteins.Click here for additional data file.Table S3. Biological functions of phosphoproteins that are significantly different between CN‐105 and vehicle in ischemic stroke. Click here for additional data file.


Abstinent AUD patients displayed a high prevalence of comorbid mental disorders (72%) and other substance use disorders (45%). Plasma concentrations of chemokines CXCL12/stromal cell-derived factor-1 (p < 0.001) and CX3CL1/fractalkine (p < 0.05) were lower in AUD patients compared to controls, whereas CCL11/eotaxin-1 concentrations were strongly decreased in female AUD patients (p < 0.001). In the alcohol group, CXCL8 concentrations were increased in patients with liver and pancreas diseases and there was a significant correlation to aspartate transaminase  and gamma-glutamyltransferase . Focusing on comorbid psychiatric disorders, we distinguish between patients with additional mental disorders (N = 61) and other substance use disorders (N = 38). Only CCL11 concentrations were found to be altered in AUD patients diagnosed with mental disorders (p < 0.01) with a strong main effect of sex. Thus, patients with mood disorders (N = 42) and/or anxiety (N = 16) had lower CCL11 concentrations than non-comorbid patients being more evident in women. The alcohol-induced alterations in circulating chemokines were also explored in preclinical models of alcohol use with male Wistar rats. Rats exposed to repeated ethanol  had lower CXCL12 (p < 0.01) concentrations and higher CCL11 concentrations (p < 0.001) relative to vehicle-treated rats. Additionally, the increased CCL11 concentrations in rats exposed to ethanol were enhanced by the prior exposure to restraint stress (p < 0.01). Concordantly, acute ethanol exposure induced changes in CXCL12, CX3CL1, and CCL11 in the same direction to repeated exposure. These results clearly indicate a contribution of specific chemokines to the phenotype of AUD and a strong effect of sex, revealing a link of CCL11 to alcohol and anxiety/stress.Recent studies have linked changes in peripheral chemokine concentrations to the presence of both addictive behaviors and psychiatric disorders. The present study further explore this link by analyzing the potential association of psychiatry comorbidity with alterations in the concentrations of circulating plasma chemokine in patients of both sexes diagnosed with alcohol use disorders (AUD). To this end, 85 abstinent subjects with AUD from an outpatient setting and 55 healthy subjects were evaluated for substance and mental disorders. Plasma samples were obtained to quantify chemokine concentrations [C–C motif (CC), C–X–C motif (CXC), and C–X Substantial evidence suggests that the immune system modulates behaviors through specific actions of inflammatory signals in the central nervous system (CNS) . Among tAdditionally, alterations in circulating chemokines {e.g., chemokine C–C motif ligand 2 , chemokine C–C motif ligand 11 , chemokine C–X–C motif ligand 8 , and chemokine C–X–C motif ligand 12 } have been recently associated with psychiatric disorders such as cocaine use disorders , mood diConcerning substance use disorders, a growing literature indicates that the pharmacodynamic actions of alcohol and other drugs of abuse involve the central activation of immune signaling . Thus, s3–C motif ligand 1  are correlated to cocaine symptom severity, which allows stratifying the cocaine addicts in patients with different incidence of comorbid psychiatric disorders  and neuroinflammation through the blood stream and the blood–brain barrier . In lineisorders , 21. Howisorders , 22. TakIn the present study, we tested the hypothesis that lifetime AUD are associated with altered circulating chemokine concentrations that could be observed during abstinence in male and female patients. Furthermore, variables associated with alcohol addiction  and psychiatric comorbidity  could be associated with alterations in the expression of certain chemokines in the plasma of patients with AUD in outpatient treatment. Additional studies in rats were performed to test the impact of alcohol administration on the chemokine expression, exploring also the combined association of stress and alcohol exposure.Hospital Universitario 12 de Octubre in Madrid (Spain) for a period of 18 months (November 2013–May 2015). Fifty-five healthy individuals with no history of substance use disorders or pathological use of substances were matched with the alcohol group for age, sex ratio, and body mass index (BMI). The average participant was a man (65%) of 46.6 years and a BMI of 25.5 kg/m2.The present cross-sectional study was performed in a total of 140 White Caucasian subjects who were divided into alcohol and control groups. Eighty-five abstinent subjects diagnosed with AUD  were recruited from outpatient programs for alcohol use disorder at inclusion criteria: age ≥18 years up to 60 years of age, lifetime AUD, and at least 4 weeks of abstinence before testing; (B) exclusion criteria: presence of infectious diseases, incapacitating cognitive alterations to complete psychiatric interviews, and pregnancy for female participants. A breathalyzer was used daily for estimating blood alcohol content from a breath sample. Because of high incidence of alcohol-induced diseases in accessory organs (liver and pancreas), we included these individuals in the alcohol group. A description of the sample is presented in Table The participants in the alcohol group had to meet eligibility criteria based on (A) N = 35), anxiolytics (N = 26), anticonvulsants (N = 29), and antipsychotics (N = 7).Alcohol use disorder patients were being treated with pharmacological approaches and psychosocial techniques. Sixty-one subjects were treated with disulfiram during the last year. Regarding psychiatric medication, 60 patients were treated with psychiatric medication during the last year: antidepressants , Recommendation No. R (97) 5 of the Committee of Ministers to Member States on the Protection of Medical Data (1997), and Spanish data protection act .Written informed consent was obtained from each participant after a complete description of the study and discussing any question or issue. The study and protocols for recruitment were approved by the Ethics Committee of the Patients from outpatient programs for alcohol use disorder were evaluated according to “Diagnostic and Statistical Manual of Mental Disorders-4th Edition-Text Revision” (DSM-IV-TR) criteria, using the Spanish version of the “Psychiatric Research Interview for Substance and Mental Diseases” (PRISM) . DiagnosControl subjects were initially evaluated by PRISM (for substance screening and abuse and dependence) and subsequently by the Spanish version of the “Composite International Diagnostic Interview” (CIDI) to detect psychiatric disorders . All the2-EDTA tubes  and was immediately processed to obtain plasma. Blood samples were centrifuged at 2,200 × g for 15 min (4°C) and individually assayed for detecting infectious diseases by three rapid tests for HIV (HIVTOP®), hepatitis B (HBVTOP®), and hepatitis C (HCVTOP®) purchased from ALL.DIAG . Samples displaying infection were discarded following safety protocols. Plasma samples were individually characterized, registered, and stored at −80°C until further analyses.Blood samples were obtained in the morning (9:00–11:00 a.m.) after fasting for 8–12 h (previous to the psychiatric interviews). Venous blood was extracted into 10 mL K3CL1 , CCL2 (MCP-1), CCL3 (MIP-1α), and CCL11 (eotaxin-1). The measurements of these analytes in plasma were performed following the manufacturer’s instructions . We examined the following parameters: aspartate transaminase (AST/GOT), alanine transaminase (ALT/GPT), gamma-glutamyltransferase (GGT), pancreatic α-amylase, and pancreatic lipase. The reference ranges from the laboratory were established as follows: 0–40, 0–40, 0–45, 5–100, and 0–67 IU/L, respectively.ad libitum throughout the course of these studies.Animal studies were conducted on 5- to 8-week-old male Wistar rats  weighing 200–250 g at the beginning of the experiments. Rats were kept in clear plastic cages under a 12-h light/dark cycle (lights off at 8:00 p.m.) in a room at ambient temperature (23°C) and humidity (55%) in the Animal Resource Center at the University of Málaga (Spain). Unless otherwise indicated, water and chow pellets were available Experiments and procedures were conducted under strict adherence to the European Directive 2010/63/EU on the protection of animals used for scientific purposes and with Spanish regulations (RD 53/2013 and 178/2004). All efforts were made to minimize unnecessary suffering. All protocols were approved by the Committee on the Ethics of Universidad de Málaga .For ethanol experiments, rats were accustomed to the experimental conditions including handling and intragastric gavage (i.g.) procedure for a period of 1 week using saline. The number of animals for each experimental group was ranged 7–8.Rats were gavaged daily with a solution of ethanol  or saline as vehicle during 4 weeks. An additional group of rats were individually restrained for a period of 90 min in a Plexiglas cylindrical restrainer (diameter × length: 6 cm × 20 cm) with breathing holes in a separate testing room 48 h before beginning the ethanol or saline i.g. treatment. All rats were sacrificed by decapitation 1 week after the last ethanol or saline i.g. session in a room provided with low-intensity white noise.t = 0 min) and at the end (t = 240 min) of the experiment with no ethanol exposure.Another set of rats were acutely treated with a solution of ethanol  and returned to the home cages immediately. Rats were sacrificed by decapitation at 30, 60, 120, and 240 min after the ethanol administration in a room provided with low-intensity white noise. To evaluate the effect of time on chemokine expression, two groups of naive rats were sacrificed at the beginning (g for 15 min) and plasmas were kept at −80°C for immunoassay analyses. CXCL12 (SDF-1), CX3CL1 , and CCL11 (eotaxin-1) were measured in rat plasma with commercial enzyme-linked immunosorbent assay (ELISA) kits in 96-well plate format following the manufacturer’s instructions: ELISA Kit for SDF-1 , Rat Fractalkine ELISA Kit (CX3CL1) , and Rat Eotaxin 1 (Eotaxin 1/CCL11/ECF) ELISA Kit . The plates were read at 450 nm within 30 min at a set wavelength of 540 nm or 570 nm. Data are expressed as nanograms of protein per milliliter of plasma.Blood samples from the ethanol experiments in rats were immediately collected and centrifuged ] or mean and SD of concentrations [mean (SD)]. The significance of differences in categorical and normal continuous variables was determined using Fisher’s exact test (chi-square test) and Student’s t-test, respectively.All clinical data in the tables are expressed as number and percentage of subjects [post hoc comparisons in ANCOVA were performed using Sidak’s correction test. Estimated marginal means [95% confidence intervals (95% CI)] of chemokine concentrations were expressed and represented in the figures after back-transformation if that was the case. In addition, correlation analyses were performed using the Pearson’s coefficient (r). Regarding data from rat studies, Student’s t-test and analysis of variance (ANOVA) with multiple post hoc tests (Dunnett and Tukey tests) were conducted for statistical analyses and chemokine concentrations in rat plasma are expressed as mean and SEM.Statistical analysis of chemokine concentrations was performed using multiple analysis of covariance (ANCOVA) to indicate the relative effect of explanatory variables and their interactions on the chemokine expression in the plasma, controlling for additional covariates. Log(10)-transformation was used to ensure statistical assumptions for positive skewed distributions. The p-value < 0.05 was considered statistically significant. Statistical analyses were performed using the GraphPad Prism version 5.04 software  and IBM SPSS Statistical version 22 software .A N = 92 men and N = 48 women) as factors, and “age” and “BMI” as covariates. Concentrations were examined to ensure that statistical assumptions were met and a base-10 logarithmic transformation of chemokine concentrations was conducted for CXCL8, CXCL12, CX3CL1, CCL2, and CCL3. Estimated marginal means for “history of AUD” were represented after back-transformation in Figure The impact of lifetime diagnosis of AUD on the plasma levels of chemokines in men and women was investigated using a two-way ANCOVA with “history of AUD” and “sex”  = 11.589, p < 0.001]  were differentially affected. Thus, although plasma CXCL8 concentrations were not affected by “history of AUD”  = 6.124, p = 0.015]  = 6.679, p = 0.011]  = 4.922, p = 0.028] . Furthermore, the “age” was significantly related to CCL11 concentrations  and correlation analysis indicated a positive association between “age” and CCL11 concentrations  in the total sample. Additional correlation analyses between “age” and CCL11 concentrations were conducted in different groups according to each factor (“history of AUD” or “sex”) and we observed an enhanced association in the control group  and in male participants .Regarding the CC chemokine expression , plasma concentrations were not affected by “history of AUD” as observed in the Figures N = 20 disease and N = 65 no disease), “sex” (N = 58 men and N = 27 women), “AUD symptom severity,” length of “abstinence,” and “age.” For all analyses, “psychiatric medication” was included as confounding variable. All variables were examined to ensure that statistical assumptions were met.We evaluated the relative effects of variables associated with AUD and their interactions on chemokine concentrations in the alcohol group using a two-way ANCOVA. The independent variables that we included were as follows: alcohol-induced “liver and pancreas diseases”  criteria for AUD, 9.2 (SD = 11.8) months of abstinence, and 47.2 (SD = 7.3) years. Plasma concentrations of CXCL12, CXF = 11.409, p < 0.001]  = 5.773, p = 0.019]  compared to female patients with no diseases (N = 22) (p < 0.05). In addition, “psychiatric medication” was significantly related to CXCL8 concentrations  and patients treated with medication  displayed higher CXCL8 concentrations relative to patients with no medication  during the last year.CXCL8 concentrations were significantly affected by “liver and pancreas diseases”  Figure A but the] Figure B. Thus, r = +0.456, p < 0.001) and GGT  after multiple testing correction with Bonferroni. Although the subclinical affectation of the liver and pancreas in AUD patients could induce changes in the expression of other chemokines, we observed no associations in the total sample  with the exception of CXCL8 with AST/GOT  and CXCL8 and GGT .Because alcoholic liver and pancreas damage are characterized by changes in certain circulating markers we examined AST/GOT, ALT/GPT, GGT, pancreatic α-amylase, and pancreatic lipase levels in patients and their degree of association with chemokine concentrations. We found an overall increase of plasma levels of these markers in AUD patients diagnosed with cirrhosis, hepatitis, and/or pancreatitis compared to AUD patients with no diseases, whereas control subjects had lower levels of these circulating markers than AUD patients Table . RegardiF = 8.298, p = 0.005]  = 4.239, p = 0.041] and we found a negative correlation between CCL11 concentrations and length of abstinence but this association failed to reach statistical significance. Similar to previous analysis of CCL11 concentrations in the total sample, the “age” was also significantly related to CCL11 concentrations .Regarding CCL11, despite there was no main effect of “liver and pancreas diseases” Figure C, concenSince chemokines have been linked to psychiatric disorders, we studied the high prevalence of psychiatric comorbidity (76%) diagnosed in abstinent AUD patients. Thus, the alcohol group was divided into comorbid and non-comorbid patients according to lifetime diagnosis of substance use disorders 45%) and mental disorders (72%) for characterization Table . Additio5% and meN = 31 men and N = 7 women) (Table As is shown in Table n) Table , we obseRegarding patients diagnosed with other comorbid mental disorders [mainly mood disorders (69%)], we found no differences in age and sex compared to patients with no disorders Table . HoweverFinally, a more precise diagnosis of common mental disorders according to sex is shown in Table The impact of psychiatric comorbidity on chemokine expression was analyzed using a two-way ANCOVA with the following factors: the diagnosis of “comorbid substance use disorders” or diagnosis of “comorbid mental disorders” and “sex.” These independent variables were controlled for “age.” For all analyses, “psychiatric medication” was also included as confounding variable. All variables were examined to ensure that statistical assumptions were met.F = 11.132, p < 0.001] with no interaction between both factors  = 5.572, p = 0.021].Plasma concentrations of chemokines were not found to be affected by any of the variables evaluated, with the exception of CCL11. We observed no main effect of “comorbid substance use disorders” on CCL11 concentrations Figure A but theF = 7.559, p = 0.007] on CCL11 concentrations as shown in Figure F = 6.524, p = 0.013] on CCL11 concentrations  = 6.752, p = 0.011].Regarding the diagnosis of comorbid mental disorders, we observed significant effects on CCL11 concentrations. Thus, statistical analysis revealed a significant main effect of “comorbid mental disorders”  and the estimated CCL11 mean was lower in patients diagnosed with mood disorders than in patients with no mood disorders .We analyzed the effects of these psychiatric disorders on CCL11 including “sex” and controlling for “age” and “psychiatric medication” Figure E. Thus, F = 4.941, p = 0.028] and the estimated marginal mean of CCL11 was lower in patients diagnosed with lifetime anxiety than in patients with no anxiety .Plasma CCL11 concentrations were also affected by “anxiety” [N = 5) and/or displayed the lowest concentrations of CCL11 [26.38 (95% CI = 16.47–36.29) pg/mL].Not surprisingly, the statistical analysis of the effects of these common mental disorders on CCL11 expression revealed a significant main effect of “sex” with no interaction. Therefore, female patients displayed lower concentrations than male patients . Therefore, female abstinent patients diagnosed with lifetime anxiety  = 2.99, p = 0.005]  = 27.02, p < 0.001] with higher CCL11 concentrations in alcohol-exposed rats compared to vehicle rats and a main effect of early “stress”  with higher CCL11 concentrations in rats exposed to stress compared to rats without restraint. There was no interaction between both factors.In humans, we found that the CCL11 expression was significantly affected by lifetime mental disorders, especially mood disorders and anxiety disorders. In order to investigate the potential link of this chemokine to emotional behaviors, we investigated whether these effects on CCL11 concentrations were also observed in male Wistar rats exposed to acute stress induced by immobilization and treated with ethanol. As shown in Figure 3CL1, and CCL11 concentrations were determined in the plasma during 4 h at different times . Data were analyzed using one-way ANOVA with post hoc comparisons.To study the impact of acute ethanol on these chemokines, another set of male Wistar rats were treated with ethanol (i.g.) and CXCL12, CXF = 3.871, p = 0.007] but the post hoc tests only found a significant decrease in CXCL12 at 240 min after receiving ethanol compared to the t = 0 min (p < 0.05). An additional group of rats with no ethanol exposure was examined at 240 min after beginning the experiment and this group displayed higher CXCL12 concentrations than the t = 0 min . Consequently, ethanol-treated rats showed lower CXCL12 concentrations relative to naive rats at 240 min .As shown in Figure 3CL1, one-way ANOVA revealed a significant main effect of “time” in the subgroups  . In this case, we found no differences in the CX3CL1 expression when naïve groups at 0 and 240 min were compared. Furthermore, ethanol-treated rats and naïve rats did not differ in CX3CL1 concentrations at 240 min.Regarding CXgroups  . Regarding the naïve groups, rats with no ethanol exposure showed no differences in CCL11 at t = 0 and 240 min. There were also no differences in CCL11 concentrations when ethanol-treated and naïve rats were compared at 240 min.Similar to previous analyses, there were significant differences in the CCL11 expression among groups according to “time” after ethanol exposure [F,65 = 2.93CL1 compared to control subjects whereas CCL11 concentrations were strongly decreased in female AUD patients; (ii) CXCL8 concentrations were elevated in patients with cirrhosis, steatosis, or pancreatitis, particularly in female AUD patients, and there was a positive correlation to AST/GOT and GGT; (iii) CCL11 concentrations were found to be decreased in abstinent AUD patients with comorbid mental disorders ; (iv) there was a strong and permanent effect of sex and age on plasma CCL11 concentrations in the alcohol group throughout the present study; (v) studies conducted in male rats exposed to repeated ethanol revealed similar changes in plasma CXCL12 concentrations than those observed in humans during abstinence during abstinence and acute ethanol exposure also induced a decrease in CXCL12; and (vi) rats exposed to repeated ethanol and early stress (restraint) displayed changes in CCL11 concentrations, although the changes were opposite to those observed in humans.While the association of immune signaling and psychiatric disorders is widely investigated, its association with addiction is still a field under exploration. We lack essential information concerning how immune signaling in the brain contributes to the neuroadaptations imposed by chronic substance use. This is especially relevant in the case of alcohol that can directly activate neuroimmune processes . In the 3CL1 relative to control subjects with no pathological use of substances or substance use disorders. Various studies have reported an association between both chemokines and abused drugs, mainly with cocaine. Recently, our group has reported a decrease in the plasma CXCL12 concentrations and a positive correlation between CX3CL1 concentrations and the cocaine symptom severity in abstinent cocaine-addicted subjects from outpatient setting  with ethanol  and the AUD severity in the clinical studies. Our study has been performed in patients diagnosed with AUD in a prolonged abstinence (about 9 months) and a history of heavy alcohol consumption, whereas the above cited studies in humans were performed in high cardiovascular risk patients reporting moderate alcohol consumption. This discrepancy might indicate that both chemokines fluctuate regarding the duration of abstinence and intensity of alcohol use.Therefore, although increased circulating levels of CXCL12 and CX3CL1, differences in CCL11 concentrations were found between men and women but only in the alcohol group, which indicated the presence of a sexual dimorphism in the circulating CCL11 concentrations in response to a history of pathological alcohol exposure and/or AUD. By contrast, we observed differences in the expression of this chemokine in male rats after ethanol exposure. Thus, we found increased levels of CCL11 in rats treated with acute and repeated ethanol compared to control rats.In addition to CXCL12 and CXAge and sex are critical variables in the expression of inflammatory signals –36 and f3CL1 and cocaine symptom severity in abstinent cocaine addicts  and alcohol had no effects on any of the chemokines evaluated, the diagnosis of mental disorders had a primary effect on the plasma expression of CCL11. Several studies have reported the association between blood levels of CCL11 and psychiatric disorders, for example in schizophrenia , bipolarInterestingly, a clinical study in young adults from general population by Magalhaes and colleagues reported no effects of the diagnosis of substance use disorders as predictor of CCL11 concentrations although both bipolar and depression disorders and sex influenced significantly the expression of this chemokine , which iThe present findings support the importance of monitoring chemokines in the context of complex disorders such as substance use disorders including AUD. We are aware of the limitations of this exploratory study, which need to be borne in mind in interpreting the data. First, the validity of the results presented here needs to be assessed in a larger sample of patients and include psychiatric patients with no history of substance use disorders or pathological use of substances. Second, additional studies to determine plasma chemokines in active alcohol users are necessary to test the effect of alcohol on these inflammatory signals considering the differences and effects observed in preclinical data with rats. Third, the impact of sex differences in both the prevalence of psychiatric disorders and plasma concentrations of chemokines  requires a higher effort on recruiting female participants and a better clinical characterization  because the average age of the women seeking treatment for alcohol problems usually belongs to the menopause transition years. In fact, the lack of data in female rats exposed to ethanol represents a limitation that will need to be addressed in future investigations.Although CCL11 could be proposed as a potential indicator of psychiatric comorbidity in abstinent subjects with AUD, there are no data about other drugs of abuse. Further clinical and preclinical research is necessary to elucidate the role of chemokines and sex in the etiology of alcoholism and psychiatric disorders.FF, FP, and AS were responsible for the study concept and design. NG-M, GP, PA, MP, and GR coordinated recruitment of participants. NG-M, PA, and MP contributed to the acquisition of psychiatric data by means of interviews and generated the database. AS, EC-O, and JD processed, registered, and stored human blood samples. VB, JC, and JA supervised and performed the quantification of chemokines in human plasma. LS-M, AG, JD, DS, and AS performed rat studies and ELISAs. FP, AS, and PR-S assisted with data analysis and interpretation of findings. FF and FP drafted the manuscript. MT, JA, and GR provided critical revision of the manuscript for important intellectual content. All the authors critically reviewed content and approved the version for publication.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.