Reduced Expression of miR-146a Potentiates Intestinal Inflammation following Alcohol and Burn Injury.

MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively regulate gene expression. Within the intestinal epithelium, miRNAs play a critical role in gut homeostasis, and aberrant miRNA expression has been implicated in various disorders associated with intestinal inflammation and barrier disruption. In this study, we sought to profile changes in intestinal epithelial cell miRNA expression after alcohol and burn injury and elucidate their impact on inflammation and barrier integrity. Using a mouse model of acute ethanol intoxication and burn injury, we found that small intestinal epithelial cell expression of miR-146a is significantly decreased 1 d following injury. Using in vitro studies, we show that reduced miR-146a promotes intestinal epithelial cell inflammation by promoting p38 MAPK signaling via increased levels of its target TRAF6 (TNFR-associated factor 6). Furthermore, we demonstrate that in vivo miR-146a overexpression significantly inhibits intestinal inflammation 1 d following combined injury and potentially supports intestinal barrier homeostasis. Overall, this study highlights the important impact that miRNA expression can have on intestinal homeostasis and the valuable potential of harnessing aberrant miRNA expression as a therapeutic target to control intestinal inflammation.

DSS-induced inflammation in the colon drives a proinflammatory signature in the brain that is ameliorated by prophylactic treatment with the S100A9 inhibitor paquinimod.

BACKGROUND: Inflammatory bowel disease (IBD) is established to drive pathological sequelae in organ systems outside the intestine, including the central nervous system (CNS). Many patients exhibit cognitive deficits, particularly during disease flare. The connection between colonic inflammation and neuroinflammation remains unclear and characterization of the neuroinflammatory phenotype in the brain during colitis is ill-defined. METHODS: Transgenic mice expressing a bioluminescent reporter of active caspase-1 were treated with 2% dextran sodium sulfate (DSS) for 7 days to induce acute colitis, and colonic, systemic and neuroinflammation were assessed. In some experiments, mice were prophylactically treated with paquinimod (ABR-215757) to inhibit S100A9 inflammatory signaling. As a positive control for peripheral-induced neuroinflammation, mice were injected with lipopolysaccharide (LPS). Colonic, systemic and brain inflammatory cytokines and chemokines were measured by cytokine bead array (CBA) and Proteome profiler mouse cytokine array. Bioluminescence was quantified in the brain and caspase activation was confirmed by immunoblot. Immune cell infiltration into the CNS was measured by flow cytometry, while light sheet microscopy was used to monitor changes in resident microglia localization in intact brains during DSS or LPS-induced neuroinflammation. RNA sequencing was performed to identify transcriptomic changes occurring in the CNS of DSS-treated mice. Expression of inflammatory biomarkers were quantified in the brain and serum by qRT-PCR, ELISA and WB. RESULTS: DSS-treated mice exhibited clinical hallmarks of colitis, including weight loss, colonic shortening and inflammation in the colon. We also detected a significant increase in inflammatory cytokines in the serum and brain, as well as caspase and microglia activation in the brain of mice with ongoing colitis. RNA sequencing of brains isolated from DSS-treated mice revealed differential expression of genes involved in the regulation of inflammatory responses. This inflammatory phenotype was similar to the signature detected in LPS-treated mice, albeit less robust and transient, as inflammatory gene expression returned to baseline following cessation of DSS. Pharmacological inhibition of S100A9, one of the transcripts identified by RNA sequencing, attenuated colitis severity and systemic and neuroinflammation. CONCLUSIONS: Our findings suggest that local inflammation in the colon drives systemic inflammation and neuroinflammation, and this can be ameliorated by inhibition of the S100 alarmin, S100A9.

A Caspase-1 Biosensor to Monitor the Progression of Inflammation In Vivo.

Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro-IL-1ß and pro-IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow-derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.

The associations between alcohol intake and cardiometabolic risk in African-origin adults spanning the epidemiologic transition.

BACKGROUND: Cardiometabolic (CM) risk affects approximately 25% of adults globally, and is diagnosed by meeting 3 out of 5 of the following CM risk factors: elevated blood pressure, high triglycerides, elevated blood sugar, low high-density lipoprotein (HDL) level, and abdominal obesity. Adults with CM risk are approximately 22% more likely to have higher mortality rates, and alcohol consumption may be associated with higher CM risk. While previous studies have investigated this potential connection, the majority of them did not include African-origin adults. Therefore, the study aimed to explore the association between alcohol intake and CM risk in 5 African-origin cohorts, spanning the epidemiologic transition in Ghana, South Africa, Jamaica, Seychelles and the United States of America. METHODS: Measurements included clinical measures for CM risk and self-reported alcohol consumption. Each participant was categorized into one of three drinking categories: non-drinker, light drinker (1-3 drinks daily for men and 1-2 drinks daily for women) and heavy drinker (4 or more drinks every day for men and 3 or more drinks per day for women). Using non-drinker status as the reference, the association between alcohol consumption status and prevalence of each of the five CM risk factors and overall elevated CM risk (having 3 out of 5 risk factors) was explored, adjusting for site, age and sex. Associations were explored using logistic regression and significance was determined using odds ratios (OR) and 95% confidence intervals. RESULTS: Neither light nor heavy drinking was associated with increased odds for having higher CM risk compared to nondrinkers (OR = 1.05, p = 0.792 and OR = 1.11, p = 0.489, respectively). However, light drinking was associated with lower odds for having low high density lipoproteins (HDL) cholesterol (OR = 0.69, p = 0.002) and increased risk for high triglycerides (OR = 1.48, p = 0.030). Heavy drinking was associated with elevated blood pressure (OR = 1.59, p = 0.002), high triglycerides (OR = 1.73, p = 0.006) and decreased risk of low HDL-cholesterol (OR = 0.621, p < 0.0005). Finally, country-specific analyses indicated that the relationship between heavy drinking and elevated CM risk varied widely across sites. CONCLUSION: While several CM risk factors were associated with alcohol consumption, the associations were inconsistent and varied widely across five international cohorts of African-origin. Future studies should focus on understanding the individual site-related effects.

Dysregulation of microRNA biogenesis in the small intestine after ethanol and burn injury.

Ethanol exposure at the time of burn injury is a major contributor to post-burn pathogenesis. Many of the adverse effects associated with ethanol and burn injury are linked to an impaired intestinal barrier. The combined insult causes intestinal inflammation, resulting in tissue damage, altered tight junction expression, and increased intestinal permeability. MicroRNAs play a critical role in maintaining intestinal homeostasis including intestinal inflammation and barrier function. Specifically, miR-150 regulates inflammatory mediators which can contribute to gut barrier disruption. The present study examined whether ethanol and burn injury alter expression of microRNA processing enzymes (Drosha, Dicer, and Argonaute-2) and miR-150 in the small intestine. Male mice were gavaged with ethanol (~2.9g/kg) 4h prior to receiving a ~12.5% total body surface area full thickness burn. One or three days after injury, mice were euthanized and small intestinal epithelial cells (IECs) were isolated and analyzed for expression of microRNA biogenesis components and miR-150. Dicer mRNA and protein levels were not changed following the combined insult. Drosha and Argonaute-2 mRNA and protein levels were significantly reduced in IECs one day after injury; which accompanied reduced miR-150 expression. To further determine the role of miR-150 in intestinal inflammation, young adult mouse colonocytes were transfected with a miR-150 plasmid and stimulated with LPS (100ng/ml). miR-150 overexpression significantly reduced IL-6 and KC protein levels compared to vector control cells challenged with LPS. These results suggest that altered microRNA biogenesis and associated decrease in miR-150 likely contribute to increased intestinal inflammation following ethanol and burn injury.

Reduced neutrophil chemotaxis and infiltration contributes to delayed resolution of cutaneous wound infection with advanced age.

Advanced age is associated with alterations in innate and adaptive immune responses, which contribute to an increased risk of infection in elderly patients. Coupled with this immune dysfunction, elderly patients demonstrate impaired wound healing with elevated rates of wound dehiscence and chronic wounds. To evaluate how advanced age alters the host immune response to cutaneous wound infection, we developed a murine model of cutaneous Staphylococcus aureus wound infection in young (3-4 mo) and aged (18-20 mo) BALB/c mice. Aged mice exhibit increased bacterial colonization and delayed wound closure over time compared with young mice. These differences were not attributed to alterations in wound neutrophil or macrophage TLR2 or FcγRIII expression, or age-related changes in phagocytic potential and bactericidal activity. To evaluate the role of chemotaxis in our model, we first examined in vivo chemotaxis in the absence of wound injury to KC, a neutrophil chemokine. In response to a s.c. injection of KC, aged mice recruited fewer neutrophils at increasing doses of KC compared with young mice. This paralleled our model of wound infection, where diminished neutrophil and macrophage recruitment was observed in aged mice relative to young mice despite equivalent levels of KC, MIP-2, and MCP-1 chemokine levels at the wound site. This reduced leukocyte accumulation was also associated with lower levels of ICAM-1 in wounds from aged mice at early time points. These age-mediated defects in early neutrophil recruitment may alter the dynamics of the inflammatory phase of wound healing, impacting macrophage recruitment, bacterial clearance, and wound closure.

An alteration of the gut-liver axis drives pulmonary inflammation after intoxication and burn injury in mice.

Approximately half of all adult burn patients are intoxicated at the time of their injury and have worse clinical outcomes than those without prior alcohol exposure. This study tested the hypothesis that intoxication alters the gut-liver axis, leading to increased pulmonary inflammation mediated by burn-induced IL-6 in the liver. C57BL/6 mice were given 1.2 g/kg ethanol 30 min prior to a 15% total body surface area burn. To restore gut barrier function, the specific myosin light chain kinase inhibitor membrane-permeant inhibitor of kinase (PIK), which we have demonstrated to reduce bacterial translocation from the gut, was administered 30 min after injury. Limiting bacterial translocation with PIK attenuated hepatic damage as measured by a 47% reduction in serum alanine aminotransferase (P < 0.05), as well as a 33% reduction in hepatic IL-6 mRNA expression (P < 0.05), compared with intoxicated and burn-injured mice without PIK. This mitigation of hepatic damage was associated with a 49% decline in pulmonary neutrophil infiltration (P < 0.05) and decreased alveolar wall thickening compared with matched controls. These results were reproduced by prophylactic reduction of the bacterial load in the intestines with oral antibiotics before intoxication and burn injury. Overall, these data suggest that the gut-liver axis is deranged when intoxication precedes burn injury and that limiting bacterial translocation in this setting attenuates hepatic damage and pulmonary inflammation.

The role of aryl hydrocarbon receptor in interleukin-23-dependent restoration of interleukin-22 following ethanol exposure and burn injury.

OBJECTIVE: T-helper (Th)-17 lymphocytes play a crucial role in maintenance and regulation of gut immunity. Our laboratory has demonstrated that acute ethanol (EtOH) exposure before burn injury results in intestinal T cell suppression and enhanced bacterial translocation. BACKGROUND: To extend these studies, we examined the effects of EtOH exposure and burn injury on Th17 responses within intestinal lymphoid Peyer's patches (PP). We further investigated whether restitution of interleukin (IL)-23 enhances PP cell IL-17 and IL-22 after EtOH and burn injury. METHODS: Male mice, approximately 25 g, were gavaged with EtOH (2.9 mg/kg) before receiving an approximately 12.5% total body surface area full thickness burn. One day postinjury, PP mixed cells were cultured in the presence of plate-bound anti-CD3/soluble anti-CD28 in the presence or absence of IL-23 for 48 hours. Supernatants were harvested for IL-17 and IL-22 levels. RESULTS: When combined with EtOH intoxication, burn injury significantly decreased IL-17 and IL-22, as compared with sham injury. IL-23 treatment successfully increased levels of IL-22 but not IL-17. This restoration was prevented when PP cells were treated with CH-223191, an aryl hydrocarbon receptor inhibitor. To further delineate the mechanism of differential IL-17 and IL-22 suppression, PP cells were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, which signal via protein kinase C (PKC) and calcium flux. Treatment with PMA and ionomycin significantly prevented the decrease in IL-17 but not IL-22 after EtOH exposure and burn injury. CONCLUSIONS: These findings suggest that IL-23-mediated restoration of IL-22 is aryl hydrocarbon receptor dependent, whereas IL-17 requires activation of protein kinase C and intracellular calcium signaling.

Alteration in intestine tight junction protein phosphorylation and apoptosis is associated with increase in IL-18 levels following alcohol intoxication and burn injury.

Intestinal mucosal barrier is the first line of defense against bacteria and their products originating from the intestinal lumen. We have shown a role for IL-18 in impaired gut barrier function following acute alcohol (EtOH) intoxication combined with burn injury. To further delineate the mechanism, this study examined whether IL-18 alters intestine tight junction proteins or induces mucosal apoptosis under these conditions. To accomplish this, rats were gavaged with EtOH (3.2g/kg) prior to ~12.5% total body surface area burn or sham injury. One day after injury, EtOH combined with burn injury resulted in a significant decrease in total occludin protein and its phosphorylation in small intestine compared to either EtOH or burn injury alone. There was no change in claudin-1 protein content but its phosphorylation on tyrosine was decreased following EtOH and burn injury. This was accompanied with an increase in mucosal apoptosis (p<0.05). The treatment of rats with anti-IL-18 antibody at the time of burn injury prevented intestine apoptosis and normalized tight junction proteins following EtOH and burn injury. Altogether, these findings suggest that IL-18 modulates tight junction proteins and cause apoptosis leading to impaired intestinal mucosal integrity following EtOH intoxication combined with burn injury.

Intoxication by intraperitoneal injection or oral gavage equally potentiates postburn organ damage and inflammation.

The increasing prevalence of binge drinking and its association with trauma necessitate accurate animal models to examine the impact of intoxication on the response and outcome to injuries such as burn. While much research has focused on the effect of alcohol dose and duration on the subsequent inflammatory parameters following burn, little evidence exists on the effect of the route of alcohol administration. We examined the degree to which intoxication before burn injury causes systemic inflammation when ethanol is given by intraperitoneal (i.p.) injection or oral gavage. We found that intoxication potentiates postburn damage in the ileum, liver, and lungs of mice to an equivalent extent when either ethanol administration route is used. We also found a similar hematologic response and levels of circulating interleukin-6 (IL-6) when either ethanol paradigm achieved intoxication before burn. Furthermore, both i.p. and gavage resulted in similar blood alcohol concentrations at all time points tested. Overall, our data show an equal inflammatory response to burn injury when intoxication is achieved by either i.p. injection or oral gavage, suggesting that findings from studies using either ethanol paradigm are directly comparable.

TRAUMATIC BRAIN INJURY-INDUCED INFLAMMATION AND GASTROINTESTINAL MOTILITY DYSFUNCTION.

ABSTRACT: Background: Traumatic brain injury (TBI) is a significant cause of morbidity and mortality in the United States, with an annual cost of 60 billion dollars. There is evidence suggesting that in the post-TBI period, the gastrointestinal tract plays a central role in driving organ and immune dysfunction and may be the source of increased circulating proinflammatory mediators. In this study, we examined systemic inflammation and bacterial dysbiosis in patients who sustained a TBI with or without polytrauma. Using a mouse model of TBI, we further show how neuroinflammation after TBI is potentially linked to disruptions in gut homeostasis such as intestinal transit and inflammation. Methods: During a study of trauma patients performed from September 1, 2018, to September 1, 2019, at a single, level 1 trauma center, TBI patients aged 21 to 95 years were enrolled. Patients were categorized as TBI based on evidence of acute abnormal findings on head computed tomographic scan, which was a combination of isolated TBI and TBI with polytrauma. Blood and stool samples were collected between 24 h and 3 days after admission. Twelve plasma samples and 10 fecal samples were used for this study. Healthy control samples were obtained from a healthy control biobank. We examined systemic inflammation and bacterial changes in patients who sustained a TBI. In addition, TBI was induced in 9- to 10-week-old male mice; we assessed neuroinflammation, and intestine transit (motility) and bacterial changes 24 h after TBI. Results: When compared with healthy controls, TBI patients had increased systemic inflammation as evidenced by increased levels of IFN-γ and MCP-1 and a trend toward an increase of IL-6 and IL-8 ( P = 0.0551 and P = 0.0549), respectively. The anti-inflammatory cytokine, IL-4, was also decreased in TBI patients. Although there was a trend of an increase in copy number of Enterobacteriaceae and a decrease in copy number of Lactobacillus in both patients and mice after TBI, these trends were not found to be significantly different. However, TBI significantly increased the copy number of another potential pathogenic bacteria Bilophila wadsworthia in TBI patients compared with healthy controls. After a moderate TBI, mice had increased expression of TNF-α, IL-6 and IL-1ß, CXCL1, s100a9, and Ly6G and decreased IL-10 in the brain lesion after TBI. This accompanied decreased transit and increased TNF-α in the small intestine of mice after TBI. Conclusions: Our findings suggest that TBI increases systemic inflammation, intestinal dysfunction, and neuroinflammation. More studies are needed to confirm whether changes in intestinal motility play a role in post-TBI neuroinflammation and cognitive deficit.

A critical review of recent knowledge of alcohol's effects on the immunological response in different tissues.

Alcohol misuse contributes to the dysregulation of immune responses and multiorgan dysfunction across various tissues, which are associated with higher risk of morbidity and mortality in people with alcohol use disorders. Organ-specific immune cells, including microglia in the brain, alveolar macrophages in the lungs, and Kupffer cells in the liver, play vital functions in host immune defense through tissue repair and maintenance of homeostasis. However, binge drinking and chronic alcohol misuse impair these immune cells' abilities to regulate inflammatory signaling and metabolism, thus contributing to multiorgan dysfunction. Further complicating these delicate systems, immune cell dysfunction associated with alcohol misuse is exacerbated by aging and gut barrier leakage. This critical review describes recent advances in elucidating the potential mechanisms by which alcohol misuse leads to derangements in host immunity and highlights current gaps in knowledge that may be the focus of future investigations.

Alcohol and Immunology: Mechanisms of multi-organ damage. Summary of the 2022 alcohol and Immunology research interest group (AIRIG) meeting.

On October 26th, 2022 the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite symposium at the annual meeting of the Society for Leukocyte Biology in Hawaii. The 2022 meeting focused broadly on the immunological consequences of acute, chronic, and prenatal alcohol exposure and how these contribute to damage in multiple organs and tissues. These included alcohol-induced neuroinflammation, impaired lung immunity, intestinal dysfunction, and decreased anti-microbial and anti-viral responses. In addition, research presented covered multiple pathways behind alcohol-induced cellular dysfunction, including mitochondrial metabolism, cellular bioenergetics, gene regulation, and epigenetics. Finally, the work presented highlighted potential biomarkers and novel avenues of treatment for alcohol-induced organ damage.

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury.

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1ß and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.

Activation of toll-like receptor 2 prevents suppression of T-cell interferon γ production by modulating p38/extracellular signal-regulated kinase pathways following alcohol and burn injury.

Recent studies indicate that toll-like receptors (TLRs) are expressed on T cells and that these receptors directly or indirectly activate the adaptive immune system. We have shown previously that acute alcohol/ethanol (EtOH) intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell interleukin-2 (IL-2) and interferon γ (IFN-γ) production. We examined whether direct stimulation of T cells with TLR2, 4, 5 and 7 agonists modulates CD3-mediated T-cell IL-2/IFN-γ release following EtOH and burn injury. Male mice were gavaged with EtOH (2.9 gm/kg) 4 h prior to receiving an ~12.5% total body surface area sham or full-thickness burn injury. Animals were killed on d 1 after injury and T cells were purified from MLN and spleens. T cells were cultured with plate-bound anti-CD3 in the presence or absence of various TLR ligands. Although TLR2, 4 and 5 agonists potentiate anti-CD3-dependent IFN-γ by T cells, the TLR2 agonist alone induced IFN-γ production independent of CD3 stimulation. Furthermore, T cells were treated with inhibitors of myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), p38 and/or extracellular signal-regulated kinase (ERK) to determine the mechanism by which TLR2 mediates IL-2/IFN-γ production. IL-2 was not influenced by TLR agonists. MyD88 and TIRAP inhibitory peptides dose-dependently diminished the ability of T cells to release IFN-γ. p38 and ERK inhibitors also abolished TLR2-mediated T-cell IFN-γ. Together, our findings suggest that TLR2 directly modulates T-cell IFN-γ production following EtOH and burn injury, independent of antigen-presenting cells. Furthermore, we demonstrated that MyD88/TIRAP-dependent p38/ERK activation is critical to TLR2-mediated T-cell IFN-γ release following EtOH and burn injury.

Ethanol Intoxication and Burn Injury Increases Intestinal Regulatory T Cell Population and Regulatory T Cell Suppressive Capability.

ABSTRACT: Traumatic injuries, such as burn, are often complicated by ethanol intoxication at the time of injury. This leads to a myriad of complications and post-burn pathologies exacerbated by aberrant immune responses. Recent findings suggest that immune cell dysfunction in the gastrointestinal system is particularly important in deleterious outcomes associated with burn injuries. In particular, intoxication at the time of burn injury leads to compromised intestinal T cell responses, which can diminish intestinal immunity and promote bacterial translocation, allowing for increased secondary infections in the injured host and associated sequelae, such as multiple organ failure and sepsis. Regulatory T cells (Treg) have been identified as important mediators of suppressing effector T cell function. Therefore, the goal of this study was to assess the effects of ethanol intoxication and burn injury on Treg populations in small intestinal immune organs. We also evaluated the suppressive capability of Tregs isolated from injured animals. Male C57BL/6 mice were gavaged with 2.9 g/kg ethanol before receiving a ∼12.5% total body surface area scald burn. One day after injury, we identified a significant increase in Tregs number in small intestine Peyer's patches (∼×1.5) and lamina propria (∼×2). Tregs-producing cytokine IL-10 were also increased in both tissues. Finally, Tregs isolated from ethanol and burn-injured mice were able to suppress proliferation of effector T cells to a greater degree than sham vehicle Tregs. This was accompanied by increased levels of IL-10 and decreased levels of pro-proliferative cytokine IL-2 in cultures containing ethanol + burn Tregs compared with sham Tregs. These findings suggest that Treg populations are increased in intestinal tissues 1 day following ethanol intoxication and burn injury. Tregs isolated from ethanol and burn-injured animals also exhibit a greater suppression of effector T cell proliferation, which may contribute to altered T cell responses following injury.

IL-23 Promotes Neutrophil Extracellular Trap Formation and Bacterial Clearance in a Mouse Model of Alcohol and Burn Injury.

Our previous studies have shown that ethanol intoxication combined with burn injury increases intestinal bacterial growth, disrupts the intestinal barrier, and enhances bacterial translocation. Additionally, studies show that Th17 effector cytokines IL-17 and IL-22, which are dependent on IL-23, play important roles in maintaining intestine mucosal barrier integrity. Recent findings suggest neutrophils are a significant source of IL-17 and IL-22. We determined the effect of ethanol and burn injury on neutrophil IL-17 and IL-22 production, as well as their ability to phagocytose and in bacterial clearance, and whether these effects are modulated by IL-23. Mice were given ethanol 4 h prior to receiving ∼12.5% total body surface area burn and were euthanized day 1 after injury. We observed that intoxication combined with burn injury significantly decreases blood neutrophil phagocytosis and bacteria killing, as well as their ability to produce IL-17 and IL-22, compared with sham vehicle mice. The treatment of neutrophils with rIL-23 significantly increases IL-22 and IL-17 release and promotes expression of IL-23R, retinoic acid-related orphan receptor γt, Lipocalin2, and Nod-like receptor 2 following ethanol and burn injury. Furthermore, IL-22- and IL-17-producing neutrophils have enhanced neutrophil extracellular trap formation and bacterial killing ability, which are dependent on IL-23. Finally, although we observed that peritoneal neutrophils harvested after casein treatment are functionally different from blood neutrophils, both blood and peritoneal neutrophils exhibited the same response to rIL-23 treatment. Together these findings suggest that IL-23 promotes neutrophil IL-22 and IL-17 production and their ability to kill bacteria following ethanol and burn injury.

IL-22 and Lactobacillus delbrueckii mitigate alcohol-induced exacerbation of DSS-induced colitis.

Ulcerative colitis (UC) is characterized by cycles of active disease flare and inactive disease remission. During UC remission, IL-22 is up-regulated, acting as a hallmark of entrance into UC remission. Recently, we found that in our mouse model of binge alcohol and dextran sodium sulfate (DSS)-induced colitis, alcohol increases severity of UC pathology. In this study, we assessed not only whether alcohol influenced IL-22 expression and thereby perpetuates UC, but also whether recombinant IL-22 (rIL-22) or treatment with a probiotic could alleviate exacerbated symptoms of UC. Levels of large intestine IL-22 were significantly decreased ∼6.9-fold in DSS ethanol compared with DSS vehicle. Examination of lamina propria (LP) cells in the large intestine revealed IL-22+ γδ T cells in DSS vehicle-treated mice were significantly increased, while IL-22+ γδ T cells in DSS ethanol mice were unable to mount this IL-22 response. We administered rIL-22 and found it restored weight loss of DSS ethanol-treated mice. Colonic shortening and increased Enterobacteriaceae were also attenuated. Administration of Lactobacillus delbrueckii attenuated weight loss (p < 0.01), colon length (p < 0.001), mitigated increases in Enterobacteriaceae, increased levels of IL-22, and increased levels of p-STAT3 back to that of DSS vehicle group in DSS ethanol mice. In contrast, sole administration of L. delbrueckii supernatant was not sufficient to reduce UC exacerbation following alcohol. Our findings suggest L. delbrueckii contributes to repair mechanisms by increasing levels of IL-22, resulting in phosphorylation of STAT3, thus attenuating the alcohol-induced increases in intestinal damage after colitis.

IL-27 Promotes Intestinal Barrier Integrity following Ethanol Intoxication and Burn Injury.

Alcohol intoxication combined with burn injury can lead to life-threatening complications, including sepsis, multiple organ failure, and death. After an acute burn, the gastrointestinal system becomes hypoxic because of fluid loss and reduction of intestinal blood flow. This can cause perturbations in the intestinal epithelial barrier, immune function, and the composition of the gut microbiome. Increased gut permeability leads to proinflammatory signaling, contributing to further damage to the intestinal barrier. Recent studies have suggested that IL-27 plays an anti-inflammatory role, which may be beneficial in intestinal barrier repair. Therefore, in this study, we examined the effect of ethanol and burn injury on IL-27 in the small intestine, as well as the potential beneficial role of IL-27 in restoring the intestinal barrier after intoxication and burn. Male C57BL/6 mice were gavaged with 2.9 g/kg ethanol before receiving a ∼12.5% total body surface area scald burn with or without rIL-27 in resuscitation fluid. Our results demonstrate that IL-27-producing cells are reduced in the small intestine after injury. When IL-27 is supplemented in resuscitation fluid, we were able to restore intestinal barrier integrity and transit, mediated through increased intestinal epithelial cell proliferation, reduced inflammatory cytokines, and increased anti-inflammatory cytokine IL-10. We also observed increased gene expression of tight junction proteins. These findings suggest that IL-27 may be a contributor to maintaining proper intestinal barrier function after injury through multiple mechanisms, including preventing excess inflammation and promoting intestinal epithelial cell proliferation and tight junction integrity.

New insights into the mechanism of alcohol-mediated organ damage via its impact on immunity, metabolism, and repair pathways: A summary of the 2021 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On November 19th, 2021, the annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at Loyola University Chicago Health Sciences Campus in Maywood, Illinois. The 2021 meeting focused on how alcohol misuse is linked to immune system derangements, leading to tissue and organ damage, and how this research can be translated into improving treatment of alcohol-related disease. This meeting was divided into three plenary sessions: the first session focused on how alcohol misuse affects different parts of the immune system, the second session presented research on mechanisms of organ damage from alcohol misuse, and the final session highlighted research on potential therapeutic targets for treating alcohol-mediated tissue damage. Diverse areas of alcohol research were covered during the meeting, from alcohol's effect on pulmonary systems and neuroinflammation to epigenetic changes, senescence markers, and microvesicle particles. These presentations yielded a thoughtful discussion on how the findings can lead to therapeutic treatments for people suffering from alcohol-related diseases.