IL-22 promotes mucin-type O-glycosylation and MATH1+ cell-mediated amelioration of intestinal inflammation.

The interleukin (IL)-22 cytokine can be protective or inflammatory in the intestine. It is unclear if IL-22 receptor (IL-22Ra1)-mediated protection involves a specific type of intestinal epithelial cell (IEC). By using a range of IEC type-specific Il22Ra1 conditional knockout mice and a dextran sulfate sodium (DSS) colitis model, we demonstrate that IL-22Ra1 signaling in MATH1+ cells (goblet and progenitor cells) is essential for maintaining the mucosal barrier and intestinal tissue regeneration. The IL-22Ra1 signaling in IECs promotes mucin core-2 O-glycan extension and induces beta-1,3-galactosyltransferase 5 (B3GALT5) expression in the colon. Adenovirus-mediated expression of B3galt5 is sufficient to rescue Il22Ra1IEC mice from DSS colitis. Additionally, we observe a reduction in the expression of B3GALT5 and the Tn antigen, which indicates defective mucin O-glycan, in the colon tissue of patients with ulcerative colitis. Lastly, IL-22Ra1 signaling in MATH1+ progenitor cells promotes organoid regeneration after DSS injury. Our findings suggest that IL-22-dependent protective responses involve O-glycan modification, proliferation, and differentiation in MATH1+ progenitor cells.

Obesity and the microbiome in atopic dermatitis: Therapeutic implications for PPAR-γ agonists.

Atopic dermatitis (AD) is an inflammatory skin disease characterized by epidermal barrier disruption, Th2 immune responses to skin allergens and microbial dysbiosis within affected lesions. Studies within the past decade have revealed genetic and environmental factors contributing to AD in children. Obesity is a metabolic disorder that often manifests early in life and is associated with reduced bacterial diversity, leading to skin colonization with lipophilic bacteria and intestinal colonization with pro-inflammatory species. These changes impair epithelial barriers and promote Th17 responses, which may worsen the severity of AD symptoms. While few studies have examined the contribution of microbiota in obesity-induced allergies, there is emerging evidence that PPAR-γ may be an effective therapeutic target. This review discusses the microbiome in pediatric AD, treatment with probiotics, how disease is altered by obesity and potential therapeutic effects of PPAR-γ agonists. While healthy skin contains diverse species adapted for specific niches, lesional skin is highly colonized with Staphylococcus aureus which perpetuates the inflammatory reaction. Treatments for AD should help to restore microbial diversity in the skin and intestine, as well as epithelial barrier function. Pre-clinical models have shown that PPAR-γ agonists can suppress Th17 responses, IgE production and mast cell function, while improving the epidermal barrier and microbial homeostasis. Overall, PPAR-γ agonists may be effective in a subset of patients with AD, and future studies should distinguish their metabolic and anti-inflammatory effects in order to inform the best therapies.

GPR68 limits the severity of chemical-induced oral epithelial dysplasia.

Head and neck cancer is the sixth most common malignancy, and there is an urgent need to identify physiological processes contributing to tumorigenesis. Extracellular acidification caused by aerobic glycolysis within tumor microenvironments can stimulate proton-sensing receptors. GPR68, or ovarian cancer G protein-coupled receptor 1, responds to extracellular acidity and is highly expressed in head and neck squamous cell carcinoma (HNSCC) as well as normal esophageal tissue. To study the role of GPR68 in oral dysplasia, wild-type and GPR68-/- mice were treated with 4-Nitroquinoline N-oxide (4NQO) in drinking water for 11-13 weeks, followed by normal water for 11-12 weeks. 4NQO treatment resulted in 45 percent of GPR68-/- mice developing severe dysplasia or squamous cell carcinoma compared to only 10.5 percent of GPR68+/+ mice. This correlated with increased frequencies of regulatory T cells in the spleens of male GPR68-/- mice. Dysplastic regions of the tongue had increased CD31 staining compared to normal regions in both GPR68-/- and GPR68+/+ mice, suggesting that angiogenesis was GPR68-independent. RNA knockdown studies using HNSCC cell lines demonstrated no direct effect of GPR68 on survival or growth. Overall, we demonstrate that GPR68-deficiency worsens the severity of chemical-induced oral dysplasia, suggesting a protective role for this gene in tumorigenesis.

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment.

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

Dietary suppression of MHC class II expression in intestinal epithelial cells enhances intestinal tumorigenesis.

Little is known about how interactions of diet, intestinal stem cells (ISCs), and immune cells affect early-stage intestinal tumorigenesis. We show that a high-fat diet (HFD) reduces the expression of the major histocompatibility complex class II (MHC class II) genes in intestinal epithelial cells, including ISCs. This decline in epithelial MHC class II expression in a HFD correlates with reduced intestinal microbiome diversity. Microbial community transfer experiments suggest that epithelial MHC class II expression is regulated by intestinal flora. Mechanistically, pattern recognition receptor (PRR) and interferon-gamma (IFNγ) signaling regulates epithelial MHC class II expression. MHC class II-negative (MHC-II-) ISCs exhibit greater tumor-initiating capacity than their MHC class II-positive (MHC-II+) counterparts upon loss of the tumor suppressor Apc coupled with a HFD, suggesting a role for epithelial MHC class II-mediated immune surveillance in suppressing tumorigenesis. ISC-specific genetic ablation of MHC class II increases tumor burden cell autonomously. Thus, HFD perturbs a microbiome-stem cell-immune cell interaction that contributes to tumor initiation in the intestine.

Effects of caspofungin, tolcapone and other FDA-approved medications on MRSA susceptibility to vancomycin.

OBJECTIVES: Vancomycin is a first-line antibiotic for the treatment of invasive infections in humans caused by methicillin-resistant Staphylococcus aureus (MRSA). Based on the premise that antibiotic combinations can exhibit synergistic and antagonistic interactions, medications used for the treatment of infection and other medical conditions were evaluated for their ability to alter MRSA susceptibility to vancomycin. METHODS: A chemical library comprised of 1237 pharmacological agents was evaluated in a 96-well plate format for its ability to inhibit MRSA growth in combination with half the minimum inhibitory concentration (MIC) of vancomycin. Caspofungin and tolcapone were further assessed for synergistic potential by isobologram (checkerboard) and flow cytometric analysis. In addition, the antibacterial activity spectrum and effects of growth conditions of the two drugs were delineated by MIC determination. RESULTS: The study identified 17 nonantibiotic library members with synergistic or additive potential, including caspofungin and tolcapone. Further analyses revealed that the respective medications for invasive candidiasis and Parkinson disease were bactericidal and bacteriostatic inhibitors of S. aureus growth. Flow cytometric analysis of viability further demonstrated that caspofungin in combination with vancomycin increased MRSA cell death in an additive manner, whereas tolcapone appeared to suppress the bactericidal action of vancomycin. CONCLUSION: Overall, this proof of concept study concluded that nonantibiotic drugs can alter the pharmacodynamic properties of vancomycin, with potential clinical implications in patients with a MRSA infection receiving medications for other medical conditions.

Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors.

Th17 cells contribute to host defense on mucosal surfaces but also provoke autoimmune diseases when directed against self-antigens. Identifying therapeutic targets that regulate Th17 cell differentiation and/or cytokine production has considerable value. Here, we study the aryl hydrocarbon receptor (AhR)-dependent transcriptome in human CD4 T cells treated with Th17-inducing cytokines. We show that the AhR reciprocally regulates IL-17 and IL-22 production in human CD4 T cells. Global gene expression analysis revealed that AhR ligation decreased IL21 expression, correlating with delayed upregulation of RORC during culture with Th17-inducing cytokines. Several of the AhR-dependent genes have known roles in cellular assembly, organization, development, growth and proliferation. We further show that expression of GPR15, GPR55 and GPR68 positively correlates with IL-22 production in the presence of the AhR agonist FICZ. Activation of GPR68 with the lorazepam derivative ogerin resulted in suppression of IL-22 and IL-10 secretion by T cells, with no effect on IL-17. Under neutral Th0 conditions, ogerin and the Gq/11 receptor inhibitor YM254890 blunted IL-22 induction by FICZ. These data reveal the AhR-dependent transcriptome in human CD4 T cells and suggest the mechanism through which the AhR regulates T cell function may be partially dependent on Gq-coupled receptors including GPR68.

Contributions of the intestinal microbiome in lung immunity.

The intestine is a critical site of immune cell development that not only controls intestinal immunity but extra-intestinal immunity as well. Recent findings have highlighted important roles for gut microbiota in shaping lung inflammation. Here, we discuss interactions between the microbiota and immune system including T cells, protective effects of microbiota on lung infections, the role of diet in shaping the composition of gut microbiota and susceptibility to asthma, epidemiologic evidence implicating antibiotic use and microbiota in asthma and clinical trials investigating probiotics as potential treatments for atopy and asthma. The systemic effects of gut microbiota are partially attributed to their generating metabolites including short chain fatty acids, which can suppress lung inflammation through the activation of G protein-coupled receptors. Thus, studying the interactions between microbiota and immune cells can lead to the identification of therapeutic targets for chronic lower respiratory diseases.

Aspergillus fumigatus Preexposure Worsens Pathology and Improves Control of Mycobacterium abscessus Pulmonary Infection in Mice.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Mutations in this chloride channel lead to mucus accumulation, subsequent recurrent pulmonary infections, and inflammation, which, in turn, cause chronic lung disease and respiratory failure. Recently, rates of nontuberculous mycobacterial (NTM) infections in CF patients have been increasing. Of particular relevance is infection with Mycobacterium abscessus, which causes a serious, life-threatening disease and constitutes one of the most antibiotic-resistant NTM species. Interestingly, an increased prevalence of NTM infections is associated with worsening lung function in CF patients who are also coinfected with Aspergillus fumigatus We established a new mouse model to investigate the relationship between A. fumigatus and M. abscessus pulmonary infections. In this model, animals exposed to A. fumigatus and coinfected with M. abscessus exhibited increased lung inflammation and decreased mycobacterial burden compared with those of mice infected with M. abscessus alone. This increased control of M. abscessus infection in coinfected mice was mucus independent but dependent on both transcription factors T-box 21 (Tbx21) and retinoic acid receptor (RAR)-related orphan receptor gamma t (RORγ-t), master regulators of type 1 and type 17 immune responses, respectively. These results implicate a role for both type 1 and type 17 responses in M. abscessus control in A. fumigatus-coinfected lungs. Our results demonstrate that A. fumigatus, an organism found commonly in CF patients with NTM infection, can worsen pulmonary inflammation and impact M. abscessus control in a mouse model.

Allicin-inspired pyridyl disulfides as antimicrobial agents for multidrug-resistant Staphylococcus aureus.

A chemical library comprised of nineteen synthesized pyridyl disulfides that emulate the chemical reactivity of allicin (garlic) was evaluated for antimicrobial activity against a panel of pathogenic bacteria. Gram-positive species including vancomycin-intermediate and vancomycin-resistant Staphylococcus aureus (VISA, VRSA) demonstrated the highest level of susceptibility toward analogs with S-alkyl chains of 7-9 carbons in length. Further biological studies revealed that the disulfides display synergy with vancomycin against VRSA, cause dispersal of S. aureus biofilms, exhibit low cytotoxicity, and decelerate S. aureus metabolism. In final analysis, pyridyl disulfides represent a novel class of mechanism-based antibacterial agents that have a potential application as antibiotic adjuvants in combination therapy of S. aureus infections with reduced vancomycin susceptibility.

Antiinflammatory effects of bromodomain and extraterminal domain inhibition in cystic fibrosis lung inflammation.

Significant morbidity in cystic fibrosis (CF) results from chronic lung inflammation, most commonly due to Pseudomonas aeruginosa infection. Recent data suggest that IL-17 contributes to pathological inflammation in the setting of abnormal mucosal immunity, and type 17 immunity-driven inflammatory responses may represent a target to block aberrant inflammation in CF. Indeed, transcriptomic analysis of the airway epithelium from CF patients undergoing clinical bronchoscopy revealed upregulation of IL-17 downstream signature genes, implicating a substantial contribution of IL-17-mediated immunity in CF lungs. Bromodomain and extraterminal domain (BET) chromatin modulators can regulate T cell responses, specifically Th17-mediated inflammation, by mechanisms that include bromodomain-dependent inhibition of acetylated histones at the IL17 locus. Here, we show that, in vitro, BET inhibition potently suppressed Th17 cell responses in explanted CF tissue and inhibited IL-17-driven chemokine production in human bronchial epithelial cells. In an acute P. aeruginosa lung infection murine model, BET inhibition decreased inflammation, without exacerbating infection, suggesting that BET inhibition may be a potential therapeutic target in patients with CF.

Pulmonary Th17 Antifungal Immunity Is Regulated by the Gut Microbiome.

Commensal microbiota are critical for the development of local immune responses. In this article, we show that gut microbiota can regulate CD4 T cell polarization during pulmonary fungal infections. Vancomycin drinking water significantly decreased lung Th17 cell numbers during acute infection, demonstrating that Gram-positive commensals contribute to systemic inflammation. We next tested a role for RegIIIγ, an IL-22-inducible antimicrobial protein with specificity for Gram-positive bacteria. Following infection, increased accumulation of Th17 cells in the lungs of RegIIIγ(-/-) and Il22(-/-) mice was associated with intestinal segmented filamentous bacteria (SFB) colonization. Although gastrointestinal delivery of rRegIIIγ decreased lung inflammatory gene expression and protected Il22(-/-) mice from weight loss during infection, it had no direct effect on SFB colonization, fungal clearance, or lung Th17 immunity. We further show that vancomycin only decreased lung IL-17 production in mice colonized with SFB. To determine the link between gut microbiota and lung immunity, serum-transfer experiments revealed that IL-1R ligands increase the accumulation of lung Th17 cells. These data suggest that intestinal microbiota, including SFB, can regulate pulmonary adaptive immune responses.

Therapeutic Role of Interleukin 22 in Experimental Intra-abdominal Klebsiella pneumoniae Infection in Mice.

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout (Il22Ra1(Hep-/-)) and Stat3 knockout (Stat3(Hep-/-)) mice were generated and subjected to intra-abdominal infection with Klebsiella pneumoniae, which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3. Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae, which was dependent on lipocalin 2 (LCN2). However, in vivo, rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti-K. pneumoniae bactericidal activity in vitro. These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections.

The role of PGE2 in intestinal inflammation and tumorigenesis.

Release of the free fatty acid arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2) and its subsequent metabolism by the cyclooxygenase and lipoxygenase enzymes produces a broad panel of eicosanoids including prostaglandins (PGs). This study sought to investigate the roles of these mediators in experimental models of inflammation and inflammation-associated intestinal tumorigenesis. Using the dextran sodium sulfate (DSS) model of experimental colitis, we first investigated how a global reduction in eicosanoid production would impact intestinal injury by utilizing cPLA2 knockout mice. cPLA2 deletion enhanced colonic injury, reflected by increased mucosal ulceration and pro-inflammatory cytokine expression. Increased disease severity was associated with a significant reduction in the levels of several eicosanoid metabolites, including PGE2. We further assessed the precise role of PGE2 synthesis on mucosal injury and repair by utilizing mice with a genetic deletion of microsomal PGE synthase-1 (mPGES-1), the terminal synthase in the formation of inducible PGE2. DSS exposure caused more extensive acute injury as well as impaired recovery in knockout mice compared to wild-type littermates. Increased intestinal damage was associated with both reduced PGE2 levels as well as altered levels of other eicosanoids including PGD2. To determine whether this metabolic redirection impacted inflammation-associated intestinal tumorigenesis, Apc(Min/+) and Apc(Min/+):mPGES-1(-/-) mice were exposed to DSS. DSS administration caused a reduction in the number of intestinal polyps only in Apc(Min/+):mPGES-1(-/-) mice. These results demonstrate the importance of the balance of prostaglandins produced in the intestinal tract for maintaining intestinal homeostasis and impacting tumor development.

Directing traffic: IL-17 and IL-22 coordinate pulmonary immune defense.

Respiratory infections and diseases are among the leading causes of death worldwide, and effective treatments probably require manipulating the inflammatory response to pathogenic microbes or allergens. Here, we review mechanisms controlling the production and functions of interleukin-17 (IL-17) and IL-22, cytokines that direct several aspects of lung immunity. Innate lymphocytes (γδ T cells, natural killer cells, innate lymphoid cells) are the major source of IL-17 and IL-22 during acute infections, while CD4(+) T-helper 17 (Th17) cells contribute to vaccine-induced immunity. The characterization of dendritic cell (DC) subsets has revealed their central roles in T-cell activation. CD11b(+) DCs stimulated with bacteria or fungi secrete IL-1ß and IL-23, potent inducers of IL-17 and IL-22. On the other hand, recognition of viruses by plasmacytoid DCs inhibits IL-1ß and IL-23 release, increasing susceptibility to bacterial superinfections. IL-17 and IL-22 primarily act on the lung epithelium, inducing antimicrobial proteins and neutrophil chemoattractants. Recent studies found that stimulation of macrophages and DCs with IL-17 also contributes to antibacterial immunity, while IL-22 promotes epithelial proliferation and repair following injury. Chronic diseases such as asthma and chronic obstructive pulmonary disease have been associated with IL-17 and IL-22 responses directed against innocuous antigens. Future studies will evaluate the therapeutic efficacy of targeting the IL-17/IL-22 pathway in pulmonary inflammation.

Acute alcohol intoxication impairs methicillin-resistant Staphylococcus aureus clearance in the lung by impeding epithelial production of Reg3γ.

The incidence of community-associated methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in previously healthy individuals has increased in the past 5 years. Such infections are associated with bronchiectasis and high mortality rates, making them a significant public health concern. The mechanisms of host defense against this pathogen are not well characterized. However, patients diagnosed with MRSA, as opposed to methicillin-susceptible S. aureus (MSSA), are more likely to have abused alcohol in the past, and these patients are more likely to die from sepsis. In the United States, USA300 is the predominant strain that causes necrotizing pneumonia. To investigate whether acute ethanol exacerbates MRSA pneumonia, mice were intraperitoneally (i.p.) administered 2 or 4 g/kg of ethanol 30 min prior to oropharyngeal inoculation of 2 × 10(7) CFU of USA300. An increased pulmonary bacterial burden was observed in alcohol-intoxicated mice at 16 and 24 h and was associated with decreased levels of interleukin 6 (IL-6). IL-6 activates signal transducer and activator of transcription 3 (STAT3) as part of an acute-phase response of infection. Reg3γ is an antimicrobial C-type lectin that is induced by STAT3 signaling in response to Gram-positive bacteria. Previously, in situ hybridization studies showed that Reg3g is highly expressed in lung epithelium. In the present study, we found that acute ethanol exacerbated USA300 in a murine model of USA300 pneumonia. This was associated with reduced IL-6 expression in vivo as well as inhibition of IL-6 induction of STAT3 signaling and Reg3g expression in mouse lung epithelial (MLE12) cells in vitro. Furthermore, recombinant Reg3γ administration 4 h after MRSA infection in alcohol-intoxicated mice rescued USA300 clearance in vivo. Therefore, acute alcohol intoxication leads to decreased MRSA clearance in part by inhibiting IL-6/STAT3 induction of the antimicrobial protein Reg3γ in the pulmonary epithelium.

Innate Stat3-mediated induction of the antimicrobial protein Reg3γ is required for host defense against MRSA pneumonia.

Pulmonary Staphylococcus aureus (SA) infections are a public health concern and a major complication of hyper-IgE syndrome, caused by mutations in STAT3. In contrast to previous findings of skin infection, we observed that clearance of SA from the lung did not require T, B, or NK cells but did require Stat3 activation. Immunohistochemistry showed robust Stat3 phosphorylation in the lung epithelium. We identified that a critical Stat3 target gene in lung epithelium is Reg3g (regenerating islet-derived 3 γ), a gene which is highly expressed in gastrointestinal epithelium but whose role in pulmonary host defense is uncharacterized. Stat3 regulated Reg3g transcription through direct binding at the Reg3g promoter region. Recombinant Reg3γ bound to SA and had both bacteriostatic and bactericidal activity in a dose-dependent fashion. Stat3 inhibition in vivo reduced Reg3g transcripts in the lung, and more importantly, recombinant Reg3γ rescued mice from defective SA clearance. These findings reveal an antibacterial function for lung epithelium through Stat3-mediated induction of Reg3γ.

Maintaining poise: commensal microbiota calibrate interferon responses.

The mechanisms of how commensal bacteria impact systemic immunity are unclear. In this issue of Immunity, Abt et al. (2012) and Ganal et al. (2012) demonstrate that microbiota poise macrophages for induction of the type I interferon pathway after virus infection.

Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

The interleukin-17 (IL-17) family of cytokines phylogenetically predates the evolution of T cells in jawed vertebrates, suggesting that the ontogeny of the Th17 cell lineage must have arisen to confer an evolutionary advantage to the host over innate sources of IL-17. Utilizing a model of mucosal immunization with the encapsulated bacteria Klebsiella pneumoniae, we found that B cells, which largely recognized polysaccharide capsular antigens, afforded protection to only the vaccine strain. In contrast, memory Th17 cells proliferated in response to conserved outer membrane proteins and conferred protection against several serotypes of K. pneumoniae, including the recently described multidrug resistant New Dehli metallolactamase strain. Notably, this heterologous, clade-specific protection was antibody independent, demonstrating the Th17 cell lineage confers a host advantage by providing heterologous mucosal immunity independent of serotype-specific antibody.