Adam M. Hammer

Burn Injury Alters the Intestinal Microbiome and Increases Gut Permeability and Bacterial Translocation

Sepsis remains one of the leading causes of death in burn patients who survive the initial insult of injury. Disruption of the intestinal epithelial barrier has been shown after burn injury; this can lead to the translocation of bacteria or their products (e.g., endotoxin) from the intestinal lumen to the circulation, thereby increasing the risk for sepsis in immunocompromised individuals. Since the maintenance of the epithelial barrier is largely dependent on the intestinal microbiota, we examined the diversity of the intestinal microbiome of severely burned patients and a controlled mouse model of burn injury. We show that burn injury induces a dramatic dysbiosis of the intestinal microbiome of both humans and mice and allows for similar overgrowths of Gram-negative aerobic bacteria. Furthermore, we show that the bacteria increasing in abundance have the potential to translocate to extra-intestinal sites. This study provides an insight into how the diversity of the intestinal microbiome changes after burn injury and some of the consequences these gut bacteria can have in the host.

Interleukin-22 Prevents Microbial Dysbiosis and Promotes Intestinal Barrier Regeneration Following Acute Injury

ABSTRACT Intestine barrier disruption and bacterial translocation can contribute to sepsis and multiple organ failure, leading causes of mortality in burn-injured patients. In addition, findings suggest that ethanol (alcohol) intoxication at the time of injury worsens symptoms associated with burn injury. We have previously shown that interleukin-22 (IL-22) protects from intestinal leakiness and prevents overgrowth of gram-negative bacteria following ethanol and burn injury, but how IL-22 mediates these effects has not been established. Here, utilizing a mouse model of ethanol and burn injury, we show that the combined insult results in a significant loss of proliferating cells within small intestine crypts and increases Enterobacteriaceae copies, despite elevated levels of the antimicrobial peptide lipocalin-2. IL-22 administration restored numbers of proliferating cells within crypts, significantly increased Reg3&bgr;, Reg3&ggr;, lipocalin-2 AMP transcript levels in intestine epithelial cells, and resulted in complete reduction of Enterobacteriaceae in the small intestine. Knockout of signal transducer and activator of transcription factor-3 (STAT3) in intestine epithelial cells resulted in complete loss of IL-22 protection, demonstrating that STAT3 is required for intestine barrier protection following ethanol combined with injury. Together, these findings suggest that IL-22/STAT3 signaling is critical to gut barrier integrity and targeting this pathway may be of beneficial clinical relevance following burn injury.

The Effects of Alcohol Intoxication and Burn Injury on the Expression of Claudins and Mucins in the Small and Large Intestines.

ABSTRACT Alcohol intoxication at the time of burn injury exacerbates postburn pathogenesis. Recent findings suggest gut barrier integrity is compromised after combined alcohol and burn insult, which could contribute to these complications. Tight junction proteins and mucins play critical roles in keeping the gut barrier intact. Therefore, the goal of this study was to examine the effects of alcohol and burn injury on claudin and mucin expression in the intestines. We also evaluated if the combined insult differentially influences their expression in the small and large intestines. Male C57BL/6 mice were given a single dose of 2.9 g/kg ethanol before an approximately 12.5% body area burn. One and three days after injury, we profiled expression of several tight junction proteins, mucin, and bacterial 16S rRNA genes in the small and large intestines, using qPCR. We observed >50% decrease in claudin-4 and claudin-8 genes in both ileal and colonic epithelial cells 1 day after injury. Claudin-2 was significantly upregulated, and occludin was downregulated in the small intestine 1 day after injury. Mucin-3 expression was substantially elevated (>50%) in the small intestine, whereas mucin-2 and mucin-4 were considerably diminished in the colon (>50%) 1 day after injury. Most of the parameters were normalized to sham levels on day 3, except for mucin-3 and claudin-8, which remained decreased in the large intestine. Neither alcohol nor burn alone resulted in changes in junction or mucin gene expression compared to shams. This was accompanied with increases in the family of Gram-negative bacteria, Enterobacteriaceae, in both the small and the large intestines 1 day after injury. These findings suggest that alcohol and burn injury disrupts the normal gut microbiota and alters tight junction and mucin expression in the small and large intestines.

Positioning Ganglioside D3 as an Immunotherapeutic Target in Lymphangioleiomyomatosis

Tumors that develop in lymphangioleiomyomatosis (LAM) as a consequence of biallelic loss of TSC1 or TSC2 gene function express melanoma differentiation antigens. However, the percentage of LAM cells expressing these melanosomal antigens is limited. Here, we report the overexpression of ganglioside D3 (GD3) in LAM. GD3 is a tumor-associated antigen otherwise found in melanoma and neuroendocrine tumors; normal expression is largely restricted to neuronal cells in the brain. We also observed markedly reduced serum antibody titers to GD3, which may allow for a population of GD3-expressing LAM cells to expand within patients. This is supported by the demonstrated sensitivity of cultured LAM cells to complement mediated cytotoxicity via GD3 antibodies. GD3 can serve as a natural killer T (NKT) cell antigen when presented on CD1d molecules expressed on professional antigen-presenting cells. Although CD1d-expressing monocyte derivatives were present in situ, enhanced NKT-cell recruitment to LAM lung was not observed. Cultured LAM cells retained surface expression of GD3 over several passages and also expressed CD1d, implying that infiltrating NKT cells can be directly cytotoxic toward LAM lung lesions. Immunization with antibodies to GD3 may thus be therapeutic in LAM, and enhancement of existing NKT-cell infiltration may be effective to further improve antitumor responses. Overall, we hereby establish GD3 as a suitable target for immunotherapy of LAM.

Alcohol and inflammatory responses: Highlights of the 2015 Alcohol and Immunology Research Interest Group (AIRIG) meeting

On September 27, 2015 the 20th 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 Raleigh, NC. The 2015 meeting focused broadly on adverse effects of alcohol and alcohol-use disorders in multiple organ systems. Divided into two plenary sessions, AIRIG opened with the topic of pulmonary inflammation as a result of alcohol consumption, which was followed by alcohols effect on multiple organs, including the brain and liver. With presentations showing the diverse range of underlying pathology and mechanisms associated with multiple organs as a result of alcohol consumption, AIRIG emphasized the importance of continued alcohol research, as its detrimental consequences are not limited to one or even two organs, but rather extend to the entire host as a whole.

Intestine immune homeostasis after alcohol and burn injury.

ABSTRACT Traumatic injury remains one of the most prevalent reasons for patients to be hospitalized. Burn injury accounts for 40,000 hospitalizations in the United States annually, resulting in a large burden on both the health and economic system and costing millions of dollars every year. The complications associated with postburn care can quickly cause life-threatening conditions including sepsis and multiple organ dysfunction and failure. In addition, alcohol intoxication at the time of burn injury has been shown to exacerbate these problems. One of the biggest reasons for the onset of these complications is the global suppression of the host immune system and increased susceptibility to infection. It has been hypothesized that infections after burn and other traumatic injury may stem from pathogenic bacteria from within the host’s gastrointestinal tract. The intestine is the major reservoir of bacteria within the host, and many studies have demonstrated perturbations of the intestinal barrier after burn injury. This article reviews the findings of these studies as they pertain to changes in the intestinal immune system after alcohol and burn injury.

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.

Summary of the 2014 Alcohol and Immunology Research Interest Group (AIRIG) meeting

On November 21, 2014 the 19th annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at Loyola University Chicago Health Sciences Campus in Maywood, Illinois. The meeting focused broadly on inflammatory cell signaling responses in the context of alcohol and alcohol-use disorders, and was divided into four plenary sessions focusing on the gut and liver, lung infections, general systemic effects of alcohol, and neuro-inflammation. One common theme among many talks was the differential roles of macrophages following both chronic and acute alcohol intoxication. Macrophages were shown to play significant roles in regulating inflammation, oxidative stress, and viral infection following alcohol exposure in the liver, lungs, adipose tissue, and brain. Other work examined the role of alcohol on disease progression in a variety of pathologies including psoriasis, advanced stage lung disease, and cancer.

IL‐23 restoration of Th17 effector function is independent of IL‐6 and TGF‐β in a mouse model of alcohol and burn injury

T cells play a critical role in host defense against intestinal bacteria. We have shown that ethanol combined with burn injury suppresses Peyers patch (PP) Th17 cytokines 1 d after injury. We assessed the mechanism of suppressed Th17 effector functions. Mice were gavaged with ethanol 4 h before burn injury and euthanized 1, 3, and 7 d after injury. Mesenteric lymph nodes (MLNs), PPs, and spleen Th1 and Th17 cytokines were assessed. A significant decrease in IL‐17, IL‐22, IL‐2, and IFN‐γ were observed in all 3 lymphoid organs 1 and 3 d after injury. We used splenic cells to study the role of IL‐6, IL‐23, TGF‐β, and aryl hydrocarbon receptor (AHR) in suppressing Th17 cytokines. We also assessed whether the AHR agonist 6‐formylindolo (3, 2‐b) carbazole (FICZ) modulates Th17 cytokines. We found a significant decrease in IL‐6 and TGF‐β after ethanol and burn; IL‐23 was undetectable. The reconstitution of IL‐23 in culture medium increased IL‐17 by 2‐fold and IL‐22 by 20‐fold in cells from burn ethanol mice. The restoration of IL‐6 and TGF‐β combined did not influence the release of Th17 cytokines. We observed that AHR was necessary for IL‐23 restoration of IL‐22 after ethanol and burn injury. The AHR agonist FICZ enhanced IL‐22, but not IL‐17. None of these treatments influenced the release of Th1 cytokines. Together, these results suggest that IL‐23 plays a critical role in regulation of Th17 cytokines. Furthermore, IL‐6 and TGF‐β do not appear to influence IL‐23‐mediated restoration of Th17 cytokines after ethanol and burn injury.

Effects of Mesalamine Treatment on Gut Barrier Integrity After Burn Injury.

Gut barrier disruption is often implicated in pathogenesis associated with burn and other traumatic injuries. In this study, the authors examined whether therapeutic intervention with mesalamine (5-aminosalicylic acid [5-ASA]), a common anti-inflammatory treatment for patients with inflammatory bowel disease, reduces intestinal inflammation and maintains normal barrier integrity after burn injury. Male C57BL/6 mice were administered an approximately 20% TBSA dorsal scald burn and resuscitated with either 1 ml normal saline or 100 mg/kg of 5-ASA dissolved in saline. The authors examined intestinal transit and permeability along with the levels of small intestine epithelial cell proinflammatory cytokines and tight junction protein expression 1 day after burn injury in the presence or absence of 5-ASA. A significant decrease in intestinal transit was observed 1 day after burn injury, which accompanied a significant increase in gut permeability. The authors found a substantial increase in the levels of interleukin (IL)-6 (by ~1.5-fold) and IL-18 (by ~2.5-fold) in the small intestine epithelial cells 1 day after injury. Furthermore, burn injury decreases the expression of the tight junction proteins claudin-4, claudin-8, and occludin. Treatment with 5-ASA after burn injury prevented the burn-induced increase in permeability, partially restored normal intestinal transit, normalized the levels of the proinflammatory cytokines IL-6 and IL-18, and restored tight junction protein expression of claudin-4 and occludin compared with that of sham levels. Together these findings suggest that 5-ASA can potentially be used as treatment to decrease intestinal inflammation and normalize intestinal function after burn injury.