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.

Heat shock factor 1 drives regulatory T-cell induction to limit murine intestinal inflammation.

The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of clusters of differentiation (CD)4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and transforming growth factor ß, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using chromatin immunoprecipitation (ChIP), we demonstrate the direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when wild-type Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T-cell component. SIGNIFICANCE STATEMENT: The heat shock response (HSR) is a canonical stress response triggered by a multitude of stressors, including inflammation. Evidence supports the role of the HSR in regulating inflammation, yet there is a paucity of data on its influence in T cells specifically. Gut homeostasis reflects a balance between regulatory clusters of differentiation (CD)4+ T cells and pro-inflammatory T-helper (Th)17 cells. We show that upon activation within T cells, heat shock factor 1 (HSF1) translocates to the nucleus, and stimulates Treg-specific gene expression. HSF1 deficiency hinders Treg development and function and conversely, HSF1 overexpression enhances Treg development and function. While this work, focuses on HSF1 as a novel therapeutic target for intestinal inflammation, the findings have significance for a broad range of inflammatory conditions.

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.

The Skin and Gut Microbiome in Hidradenitis Suppurativa: Current Understanding and Future Considerations for Research and Treatment.

Hidradenitis suppurativa (HS) is a chronic, inflammatory skin disease comprising painful abscesses, deep nodules, fistulas, and scarring predominantly in the axilla and groin. Bacterial colonization of HS lesions has been well characterized and may lead to chronic infection of lesions. While disease pathogenesis of HS is not fully understood, there is increasing evidence that microbial dysbiosis may be occurring in numerous locations, including the skin and gut. The skin-gut microbiome has been proposed as a mechanism by which inflammatory skin disorders, including HS, can be exacerbated. This is evidenced by HS patients being significantly more likely to develop inflammatory bowel disease as well as the well documented cutaneous manifestations in inflammatory bowel disease. In this review, we discuss the current literature regarding HS skin and gut microbiome research. Furthermore, we discuss further considerations for microbiome research in HS, including the potential role of bacterial metabolites in disease progression and future therapeutic avenues like probiotics.

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.

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.

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.

Gut Microbial Changes and their Contribution to Post-Burn Pathology.

ABSTRACT: Burn injuries are a common form of traumatic injury that leads to significant morbidity and mortality worldwide. Burn injuries are characterized by inflammatory processes and alterations in numerous organ systems and functions. Recently, it has become apparent that the gastrointestinal bacterial microbiome is a key component of regulating the immune response and recovery from burn and can also contribute to significant detrimental sequelae after injury, such as sepsis and multiple organ failure. Microbial dysbiosis has been linked to multiple disease states; however, its role in exacerbating acute traumatic injuries, such as burn, is poorly understood. In this article, we review studies that document changes in the intestinal microbiome after burn injury, assess the implications in post-burn pathogenesis, and the potential for further discovery and research.

6-Formylindolo (3, 2-b) Carbazole (FICZ)-mediated protection of gut barrier is dependent on T cells in a mouse model of alcohol combined with burn injury.

6-Formylindolo (3, 2-b) Carbazole (FICZ) is a ligand of aryl hydrocarbon receptor (AHR) which regulates Th17 release of IL-17 and IL-22 production. Earlier, we showed that ethanol combined with burn injury suppresses Th17 responses and disrupts intestinal barrier leading to increased gut bacterial growth and translocation. Since IL-22 is known for its role in intestinal barrier maintenance, we determined whether treatment of mice with FICZ restores T cell IL-22 release and protects intestine barrier following ethanol and burn injury. Wildtype and Rag1-/- mice were gavaged with ~2.9 g/kg ethanol or water, and given a ~12.5% total body surface area burn. Mice were given FICZ (5 µg) in resuscitation fluid. FICZ treatment of wildtype mice normalized IL-22 and IL-17 in lamina propria and spleen T cells, as well as increased CYP1A1 expression in spleen T cells. This was accompanied by improved gut motility, decreased copy number of small intestine total bacteria and Enterobacteriaceae, attenuation of intestinal tissue levels of IL-6, KC, IL-18, decreased apoptosis, and prevention of gut leakiness following ethanol and burn injury. However, FICZ treatment of Rag1-/- mice did not improve any of the parameters listed after ethanol and burn injury. Additional data generated using mice treated with recombinant IL-22 alone or in combination with anti-IL-18 antibody suggest that full protection of gut barrier integrity requires both IL-18 inhibition and IL-22 restoration following ethanol and burn injury. Together our findings suggest that AHR ligand FICZ may have better therapeutic potential for maintenance of gut barrier function after ethanol and burn injury.

miR-106a deficiency attenuates inflammation in murine IBD models.

Pro-inflammatory cytokine TNFα antagonizes regulatory T cell (Treg) suppressive function with a measurable reduction of IL-10 protein secretion. Tregs are critical to suppress excessive immune activation, particularly within the intestine where high antigenic loads elicit chronic subclinical immune activation. Employing a TNFα-driven murine inflammatory bowel disease (IBD) model (TNFΔARE/+), which mirrors the Treg expansion and transmural ileitis seen in Crohn's disease, we demonstrate that the TNFα-mediated loss of Treg suppressive function coincides with induction of a specific miRNA, miR-106a in both humans and mice, via NFκB promoter binding to suppress post-transcriptional regulation of IL-10 release. Elevation of miR-106a and impaired Treg function in this model recapitulate clinical data from IBD patients. MiR-106a deficiency promotes Treg induction, suppressive function and IL-10 production in vitro. MiR-106a knockout attenuated chronic murine ileitis, whereas T cell restricted deficiency of miR-106a attenuated adoptive transfer colitis. In both models, attenuated inflammation coincided with suppression of both Th1 and Th17 cell subset expansion within the intestinal lamina propria. Collectively, our data demonstrate impaired Treg suppressive function in a murine IBD model consistent with human disease and support the potential for inhibition of miR-106a as a future therapeutic approach to treat chronic inflammatory conditions including IBD.

Prospects for Therapeutic Targeting of MicroRNAs in Human Immunological Diseases.

MicroRNAs (miRNAs) are endogenous oligoribonucleotides with exciting therapeutic potential. Early studies established a clear role for miRNAs in leukocyte biology. The first miRNA-based therapy, miravirsen, is now in phase 2 clinical trials, making the reality of these therapies undeniable. The capacity for miRNAs to fine-tune inflammatory signaling make them attractive treatment targets for immunological diseases. Nonetheless, the degree of redundancy among miRNAs, coupled with the promiscuity of miRNA binding sites in the transcriptome, require consideration when designing miRNA-directed interventions. Altered miRNA expression occurs across a range of inflammatory conditions, including inflammatory bowel disease, arthritis, and diabetes. However, very few studies successfully treated murine models of immunological diseases with miRNA-based approaches. While discussing recent studies targeting miRNAs to treat immunological conditions, we also reflect on the risks of miRNA targeting and showcase some newer delivery systems that may improve the pharmacological profile of this class of therapeutics.