Epithelial Antimicrobial Peptides Research Articles

Mechanisms and Therapeutic Potential of Key Anti-inflammatory Metabiotics: Trans-Vaccenic Acid, Indole-3-Lactic Acid, Thiamine, and Butyric Acid.

Identifying metabolites produced by probiotic bacteria, also known as metabiotics, is becoming increasingly common due to their anti-inflammatory, anti-obesogenic, and immunomodulatory effects. Postbiotics alongside diet, regulate both physical and mental health, as the microbiota members can interact physically with host cells or through secretion of nutrients and metabiotics. These metabiotics also reduce the severity of certain metabolic disorders and support the proper functioning of various organs and systems. In this review, we describe the mechanisms of action of trans-vaccenic acid (TVA), indole-3-lactic acid (ILA), thiamine (vitamin B1), and butyric acid metabolites produced or induced by probiotics such as Lactobacillus and/or Bifidobacterium, among others and previously identified using analytical techniques such as mass spectrometry (LC-MS). Within their mechanisms of action, Trans-vaccenic acid exerts anti-inflammatory effects and helps alleviate complications associated with metabolic diseases. Indole metabolites promote IL-22 production and regulate epithelial cell proliferation and antimicrobial peptide production. Thiamin is essential for energy metabolism regulation, and butyric acid regulates the brain-gut axis and also regulates immune response. This review expands our understanding of the potential therapeutic useofmetabiotics.

Evidence that the loss of colonic anti-microbial peptides may promote dysbiotic Gram-negative inflammaging-associated bacteria in aging mice.

Introduction: Aging studies in humans and mice have played a key role in understanding the intestinal microbiome and an increased abundance of "inflammaging" Gram-negative (Gn) bacteria. The mechanisms underlying this inflammatory profile in the aging microbiome are unknown. We tested the hypothesis that an aging-related decrease in colonic crypt epithelial cell anti-microbial peptide (AMP) gene expression could promote colonic microbiome inflammatory Gn dysbiosis and inflammaging. Methods: As a model of aging, C57BL/6J mice fecal (colonic) microbiota (16S) and isolated colonic crypt epithelial cell gene expression (RNA-seq) were assessed at 2 months (mth) (human: 18years old; yo), 15mth (human: 50yo), and 25mth (human: 84yo). Informatics examined aging-related microbial compositions, differential colonic crypt epithelial cell gene expressions, and correlations between colonic bacteria and colonic crypt epithelial cell gene expressions. Results: Fecal microbiota exhibited significantly increased relative abundances of pro-inflammatory Gn bacteria with aging. Colonic crypt epithelial cell gene expression analysis showed significant age-related downregulation of key AMP genes that repress the growth of Gn bacteria. The aging-related decrease in AMP gene expressions is significantly correlated with an increased abundance in Gn bacteria (dysbiosis), loss of colonic barrier gene expression, and senescence- and inflammation-related gene expression. Conclusion: This study supports the proposed model that aging-related loss of colonic crypt epithelial cell AMP gene expression promotes increased relative abundances of Gn inflammaging-associated bacteria and gene expression markers of colonic inflammaging. These data may support new targets for aging-related therapies based on intestinal genes and microbiomes.

Beta-defensin index: A functional biomarker for oral cancer detection

There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

Bacterial pneumonia-induced shedding of epithelial heparan sulfate inhibits the bactericidal activity of cathelicidin in a murine model.

Bacterial pneumonia is a common clinical syndrome leading to significant morbidity and mortality worldwide. In the current study, we investigate a novel, multidirectional relationship between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Using an in vivo pneumonia model, we demonstrate that highly sulfated heparan sulfate (HS) oligosaccharides are shed into the airspaces in response to MRSA pneumonia. In vitro, these HS oligosaccharides do not directly alter MRSA growth or gene transcription. However, in the presence of an antimicrobial peptide (cathelicidin), increasing concentrations of HS inhibit the bactericidal activity of cathelicidin against MRSA as well as other nosocomial pneumonia pathogens (Klebsiella pneumoniae and Pseudomonas aeruginosa) in a dose-dependent manner. Surface plasmon resonance shows avid binding between HS and cathelicidin with a dissociation constant of 0.13 μM. These findings highlight a complex relationship in which shedding of airspace HS may hamper host defenses against nosocomial infection via neutralization of antimicrobial peptides. These findings may inform future investigation into novel therapeutic targets designed to restore local innate immune function in patients suffering from primary bacterial pneumonia.NEW & NOTEWORTHY Primary Staphylococcus aureus pneumonia causes pulmonary epithelial heparan sulfate (HS) shedding into the airspace. These highly sulfated HS fragments do not alter bacterial growth or transcription, but directly bind with host antimicrobial peptides and inhibit the bactericidal activity of these cationic polypeptides. These findings highlight a complex local interaction between the pulmonary epithelial glycocalyx and antimicrobial peptides in the setting of bacterial pneumonia.

Sympathetic activity regulates epithelial proliferation and wound healing via adrenergic receptor α2A

Innervation of the intestinal mucosa by the sympathetic nervous system is well described but the effects of adrenergic receptor stimulation on the intestinal epithelium remain equivocal. We therefore investigated the effect of sympathetic neuronal activation on intestinal cells in mouse models and organoid cultures, to identify the molecular routes involved. Using publicly available single-cell RNA sequencing datasets we show that the α2A isoform is the most abundant adrenergic receptor in small intestinal epithelial cells. Stimulation of this receptor with norepinephrine or a synthetic specific α2A receptor agonist promotes epithelial proliferation and stem cell function, while reducing differentiation in vivo and in intestinal organoids. In an anastomotic healing mouse model, adrenergic receptor α2A stimulation resulted in improved anastomotic healing, while surgical sympathectomy augmented anastomotic leak. Furthermore, stimulation of this receptor led to profound changes in the microbial composition, likely because of altered epithelial antimicrobial peptide secretion. Thus, we established that adrenergic receptor α2A is the molecular delegate of intestinal epithelial sympathetic activity controlling epithelial proliferation, differentiation, and host defense. Therefore, this receptor could serve as a newly identified molecular target to improve mucosal healing in intestinal inflammation and wounding.

Investigating the Correlation Between Clostridioides difficile Infection and Vitamin D Deficiency.

Clostridioides difficile infection (CDI) is the most common healthcare-associated infection in the US. Symptoms include watery diarrhea, nausea, and anorexia and it can present with leukocytosis on laboratory evaluation. Treatment is based on disease severity and recurrence. Despite antibioticusage being the highest risk factor for infection, they are also the first-line treatment for initial CDI. Prevention of CDI mostly involves good hand hygiene, antibiotic stewardship, and appropriate precautions when interacting with infected individuals. Vitamin D deficiency (VDD) has been linked to CDI, however, there is limited insight into the correlation between both states. Our aim was to further investigate the potential link between VDD and CDI. Data were obtained from the National Inpatient Sample (NIS) from 2016 to 2019. Patients with CDI were identified and stratified based on a diagnosis of VDD. Primary outcomes were mortality, CDI recurrence, ileus, toxic megacolon, perforation, and colectomy. Chi-squared and independent t-tests were performed to assess categorical and continuous data, respectively. Multiple logistic regression was used to control for confounders. Patients with VDD had higher rates of CDI recurrence (17.4% versus 14.7%,p<0.05), but lower rates of mortality (3.1% versus 6.1%, p<0.05). Differences in rates of ileus, toxic megacolon, perforation, and colectomy were statistically insignificant. Length of stay was higher in the VDD group (10.38 days versus 9.83 days). Total charges were lower in the VDD group ($93,935.85 versus $102,527.9). CDI patients with comorbid VDD are at higher risk for the recurrence of CDI. This is likely due to the role of vitamin D in the expression of intestinal epithelial antimicrobial peptides, macrophage activation, and maintenance of tight junctions between gut epithelial cells. Furthermore, vitamin D plays a role in maintaining a healthy gut microbiome. Alternatively, deficiency results in poor gut health and detrimental changes to the gut microbiome. In effect, VDD promotes the proliferation of C. difficile within the large colon, resulting in an increased predisposition for CDI.

Cathelicidin LL-37 Ignites Primed NLRP3 Inflammasomes in Rosacea

Microbes and commensal mites contribute to the development of inflammation and neurovascular dysregulation in rosacea. Cathelicidin family proteins are epithelial antimicrobial peptides expressed in higher-order mammals. In humans, mature LL-37 is cleaved from its precursor in response to microbial infection, UV light, and injury. In their new article in the Journal of Investigative Dermatology, Yoon etal. expand on existing evidence supporting LL-37 proinflammatory activity in lipopolysaccharide (LPS)- and UV-primed models of rosacea. They show invitro that LL-37 promotes NLRP3-mediated inflammasome activation through lysosomal destabilization in the presence of LPS and that the injection of LL-37 invivo leads to skin inflammation that is abrogated by direct NLRP3 inhibition and homozygous knockout in a murine model.

Neutrophil-Specific Defensin Receptors Prevent Skin Dysbiosis and Bacterial Infection

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that epithelial cell-derived antimicrobial peptides defensins activate orphan GPCRs Mrgpra2a/b on neutrophils. This signaling axis is required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Furthermore, we found that defensins and Mrgpra2 are critical for combatting S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggers neutrophils to release the pro-inflammatory cytokine IL-1β, which is vital for proper amplification and propagation of the antibacterial immune response. This study demonstrates the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Retinal Pigment Epithelial Cells Express Antimicrobial Peptide Lysozyme - A Novel Mechanism of Innate Immune Defense of the Blood-Retina Barrier.

PurposeFor this study we aimed to understand if retinal pigment epithelial (RPE) cells express antimicrobial peptide lysozyme as a mechanism to protect the neuroretina from blood-borne pathogens.MethodsThe expression of lysozyme in human and mouse RPE cells was examined by RT-PCR or immune (cyto)histochemistry in cell cultures or retinal sections. RPE cultures were treated with different concentrations of Pam3CSK4, lipopolysaccharides (LPS), staphylococcus aureus-derived peptidoglycan (PGN-SA), Poly(I:C), and Poly(dA:dT). The mRNA expression of lysozyme was examined by qPCR and protein expression by ELISA. Poly(I:C) was injected into the subretinal space of C57BL/6J mice and eyes were collected 24 hours later and processed for the evaluation of lysozyme expression by confocal microscopy. Bactericidal activity was measured in ARPE19 cells following LYZ gene deletion using Crispr/Cas9 technology.ResultsThe mRNA and protein of lysozyme were detected in mouse and human RPE cells under normal conditions, although the expression levels were lower than mouse microglia BV2 or human monocytes THP-1 cells, respectively. Immunohistochemistry showed punctate lysozyme expression inside RPE cells. Lysozyme was detected by ELISA in normal RPE lysates, and in live bacteria-treated RPE supernatants. Treatment of RPE cells with Pam3CSK4, LPS, PGN-SA, and Poly(I:C) enhanced lysozyme expression. CRISPR/Cas9 deletion of lysozyme impaired bactericidal activity of ARPE19 cells and reduced their response to LPS and Poly(I:C) stimulation.ConclusionsRPE cells constitutively express antimicrobial peptide lysozyme and the expression is modulated by pathogenic challenges. RPE cells may protect the neuroretina from blood-borne pathogens by producing antimicrobial peptides, such as lysozyme.

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis.

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.

Mechanisms and regulation of IL-22-mediated intestinal epithelial homeostasis and repair

Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.

Hyaluronan-induced alterations of the gut microbiome protects mice against Citrobacter rodentium infection and intestinal inflammation

ABSTRACT Hyaluronan is a glycosaminoglycan polymer that has been shown to play an important role in homeostasis of the gastrointestinal tract. However, its mechanistic significance in gastrointestinal epithelial barrier elements remain unexplored. Here, our results revealed that hyaluronan treatment resulted in significant changes in the gut microbiota in mice. To demonstrate the functional consequences of hyaluronan-treatment and hyaluronan-induced microbiota alterations, Citrobacter rodentium- and DSS-induced colitis models and microbiota transplantation approaches were utilized. We showed that hyaluronan alleviated intestinal inflammation in both pathogen and chemically induced intestinal mucosal damage. The protection in bacterial colitis was associated with enhanced C. rodentium clearance and alleviation of pathogen-induced gut dysbiosis. Microbiota transplantation experiments showed that the hyaluronan-altered microbiota is sufficient to confer protection against C. rodentium infection. Colonization with Akkermansia muciniphila, a commensal bacterium that is greatly enriched by hyaluronan treatment, alleviated C. rodentium-induced bacterial colitis in mice. Additionally, Akkermansia-induced protection was found to be associated with the induction of goblet cells and the production of mucins and epithelial antimicrobial peptides. Collectively, these results provide novel insights into the regulatory role of hyaluronan in modulating the gut microbiota and immunity in enteric infection and inflammation, with therapeutic potential for gut microbiome-targeted immunotherapy.

Feeding-dependent VIP neuron-ILC3 circuit regulates the intestinal barrier.

The intestinal mucosa serves as both a conduit for uptake of food-derived nutrients and microbiome-derived metabolites and as a barrier that prevents tissue invasion by microbes and tempers inflammatory responses to the myriad contents of the lumen. How the intestine coordinates physiological and immune responses to food consumption to optimize nutrient uptake while maintaining barrier functions remains unclear. Here we show in mice how a gut neuronal signal triggered by food intake is integrated with intestinal antimicrobial and metabolic responses controlled by type 3 innate lymphoid cells (ILC3)1–3. Food consumption rapidly activates a population of enteric neurons that express vasoactive intestinal peptide (VIP)4. Projections of VIP-producing neurons (VIPergic neurons) in the lamina propria are in close proximity to clusters of ILC3 that selectively express VIP receptor type 2 (VIPR2; also known as VPAC2). Production of interleukin (IL)-22 by ILC3, which is up-regulated by commensal microbes such as segmented filamentous bacteria (SFB)5–7, is inhibited upon engagement of VIPR2. As a consequence, there is a reduction in epithelial cell-derived antimicrobial peptide, but enhanced expression of lipid-binding proteins and transporters8. During food consumption, activation of VIPergic neurons thus enhances growth of epithelial-associated SFB and increases lipid absorption. Our results reveal a feeding- and circadian-regulated dynamic intestinal neuro-immune circuit that promotes a trade-off between IL-22-mediated innate immune protection and efficiency of nutrient absorption. Modulation of this pathway may hence be effective for enhancing resistance to enteropathogen2,3,9 and for treatment of metabolic diseases.

Effect of tetramethylpyrazine on growth performance, Campylobacter jejuni carriage and endogenous antimicrobial peptides in rabbits

The present study aimed to investigate the effect of tetramethylpyrazine (TMP) supplementation on growth performance, Campylobacter jejuni (C. jejuni) carriage and antimicrobial peptides in the epithelial tissue of caecum and skin in rabbits. Five treatments included control and C. jejuni challenge with the addition of TMP at 0, 50, 100 or 150 mg/kg of diet. The trial lasted for 35 days and C. jejuni challenge occurred on first day of feeding trial. The results showed that C. jejuni challenge worsened (P &amp;lt; 0.05) feed intake, body weight gain and feed efficiency, whereas TMP supplementation partially compensated (P &amp;lt; 0.05) growth performance. C. jejuni populations in the caecal content and on the skin were decreased (P &amp;lt; 0.05) in the treatments containing TMP. The mRNA levels of antimicrobial peptides, including defensin neutrophil peptide 4, macrophage cationic peptide 2, galectin 3 and cathelicidin were also decreased (P &amp;lt; 0.05) by C. jejuni challenge while they were increased (P &amp;lt; 0.05) with supplemental TMP. Linear and quadratic trends (P ≤ 0.012) of the three doses of TMP were found in growth performance, linear trends (P ≤ 0.049) in C. jejuni carriage, and linear and quadratic trends (P ≤ 0.012) in galectin 3. The results suggest that TMP can partially protect from C. jejuni infection by decreasing C. jejuni carriage and activating epithelial antimicrobial peptides.

IL-22 and Reg3γ overexpression affects Experimental Autoimmune Encephalomyelitis Severity

Abstract IL-22 belongs to IL-10 family cytokines and is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ, iNKT, Th17, and Th22 cells. IL-22 receptor, however, is expressed primarily by non-hematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knock out mice were previously shown to develop experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis (MS), how IL-22 manipulation in adult mice would affect EAE course has not been studied. In this study, we over-expressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to assess the impact of IL-22 modulation on EAE course. IL-22 overexpression significantly but marginally decreased EAE scores, demyelination and reduced infiltration of IFNγ+IL-17A+ Th17 cells into central nervous system (CNS). In contrast, neutralization of IL-22 exacerbated EAE scores. Surprisingly, overexpression of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22, significantly exacerbated EAE scores and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+ Th17 cells to CNS. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptides in the pathogenesis of CNS inflammation in a murine model of MS.

Adhesion requirements of methicillin-resistant Staphylococcus aureus in bacterial pneumonia and influenza super-infection

Abstract In the respiratory tract, Staphylococcus aureus is associated with infections ranging from asymptomatic colonization to severe necrotizing pneumonia. While staphylococcal pneumonia has always been highly associated with influenza infections in young children and the elderly, it has emerged as the major cause of pneumonia associated with influenza since the 2009 H1N1 pandemic. Despite the clinical relevance of methicillin-resistant S. aureus (MRSA), few studies have examined bacterial factors of MRSA in pneumonia and how preceding influenza may alter the behavior of MRSA in the lung. Successful establishment of infection by bacterial pathogens requires adhesion to host cells. Staphylococcal species express a broad range of surface proteins that are involved in adhesion to host cells, known as the MSCRAMM family. MSCRAMM proteins have known roles in upper respiratory tract colonization, however their role in the lung is unknown. Preliminary data suggest that the MSCRAMM family members are important within the lung as MRSA lacking all MSCRAMMs (ΔsrtA) has decreased immune infiltrate in influenza super-infection. Additionally, the MSCRAMM family member SdrD has a role in both bacterial adherence as well as epithelial integrity and immunity in MRSA pneumonia and super-infection. Mice infected with MRSA lacking SdrD (ΔsdrD) have lower bacterial burden and increased expression of epithelial antimicrobial peptides cathelicidin and Reg3g with no difference in overall inflammation measured by immune infiltrate and pro-inflammatory cytokine levels. This suggests that MSCRAMM family members play a role in infection within the lung in both MRSA pneumonia and super-infection and can alter the immune response.

Immunohistochemical Localization of RNase 7 in Normal and Inflamed Oral Epithelia and Salivary Glands.

RNase 7 is a skin-derived antimicrobial peptide expressed in various epithelial tissues. It is upregulated by stimulation with microbes and pro-inflammatory cytokines. Herein, we examined the expression levels of RNase 7 in tissues from normal and inflamed oral epithelia and salivary glands via immunohistochemistry. RNase 7 was expressed mainly in the surface layers of the parakeratinized and orthokeratinized oral epithelium. In addition, it was strongly and weakly expressed in oral lichen planus and radicular cysts, respectively. RNase 7 was constitutively expressed in salivary glands, particularly in the serous and duct cells. In the case of Sjogren’s syndrome, RNase 7 was strongly expressed in serous, ductal, and mucous cells in areas with lymphocytic infiltration. The localization patterns of RNase 7 were similar to those of other epithelial antimicrobial peptides, including beta-defensins. Thus, epithelial antimicrobial peptides may act against microbial infections in a coordinated manner in oral epithelia and salivary glands.

Glucocorticoids Inhibit Group 3 Innate Lymphocyte IL-22 Production.

Glucocorticoids (GCs) are commonly prescribed to patients with a variety of inflammatory disorders, including inflammatory bowel disease (IBD). GCs mediate their immunomodulatory effects through many different mechanisms and target multiple signaling pathways. The GC dexamethasone downmodulates innate and adaptive immune cell activation. IBD is the manifestation of a dysregulated immune response involving many different immune cells. Group 3 innate lymphocytes (ILC3s) have critical roles in mucosal inflammation. ILC3s secrete high levels of the cytokine IL-22, promoting epithelial proliferation, antimicrobial peptides, and mucins. In this study, we examined the effects of dexamethasone on IL-22 production by ILC3s. We found that dexamethasone suppressed IL-23-mediated IL-22 production in human and mouse ILC3s. This was mediated in part through dexamethasone modulation of the NF-κB pathway. Inhibition of NF-κB signaling with a small molecule inhibitor also downmodulated IL-23- and IL-1β-mediated IL-22 production in ILC3s. These findings implicate NF-κB as a regulator of IL-22 in ILC3s and likely have repercussions on GC treatment of IBD patients.

Glucocorticoids inhibit group 3 innate lymphocyte IL-22 production

Abstract Glucocorticoids (GCs) are commonly prescribed to patients with a variety of inflammatory disorders, including inflammatory bowel disease (IBD). IBD is the manifestation of a dysregulated mucosal immune response, usually against commensal bacteria, involving many different immune cells. GCs mediate their immunomodulatory effects through many different mechanisms and target multiple signaling pathways. The GC dexamethasone down-modulates both innate and adaptive immune cell activation. Group 3 innate lymphocytes (ILC3s) have critical roles in mucosal inflammation. ILC3s secrete high levels of the cytokine IL-22, promoting epithelial proliferation, antimicrobial peptides and mucins. The effects of dexamethasone on T cell function are well described, however, it is not known if dexamethasone modulates the critical function of ILC3s or if dexamethasone regulates the cytokine IL-22. In this study, we examined the effects of dexamethasone on IL-22 production by ILC3s. We found that dexamethasone suppressed IL-23-mediated IL-22 production in homeostatic and activated human and mouse ILC3s. Dexamethasone did not modulate the IL-23 canonical signaling pathway of JAK2/STAT3, but did suppress phosphorylation of IkBa, an important regulator in NF-kB activation. These findings implicate NF-kB as a regulator of IL-22 production in ILC3s and have likely repercussions on GC treatment of IBD patients.

Abstract 181: Stroke Induces a Differential Colonic Epithelial Response and Leads to Impaired Gut Integrity and Increased Post-Stroke Infections in Aged Mice

Introduction: Infection is an important complication in the acute phase after stroke which effects up to 65% of stroke patients. These infections can in turn cause sepsis, which is a major contributor to hospital mortality. Emerging evidence indicates that these infections originate from the host commensal microbiota. We have found that stroke leads to increased gut permeability and bacterial translocation and that aged mice are more susceptible to post-stroke infections compared to young. We further investigated the possibility of how the response to infection differs between young and aged mice. Hypothesis: Impaired gut epithelial-specific response after stroke in aged mice leads to increased risk of infections and impaired recovery. Methods: Young (8-10 wks) and aged (18-20 mos) male mice (n=4-5/group) were subjected to either sham or 60-minute middle cerebral artery occlusion (MCAO) followed by reperfusion. Mice were sacrificed at day 8 after stroke. One cohort of mice (n=3/group) was evaluated for the histology and hypoxic barrier integrity. Finally, colonic epithelial cells (cECs) were isolated and changes of the gene expression in cECs were examined (n=4-5/group). Results: Epithelial dysfunction was observed in aged mice, including a decrease in goblet cells and mucus layer ( P &lt;0.05). Furthermore, stroke induced the loss of hypoxic barrier in the gut of aged mice. Intriguingly, multiple epithelial antimicrobial peptides of the regenerating islet-derived protein (Reg) family genes, which are produced by epithelial cells including deep secretory cells, were highly up-regulated following stroke only in young mice [~20 fold change in Reg3β and Reg3γ ( P &lt;0.05); ~2 fold change in Reg4 ( P &lt;0.05)]. In addition, aged mice showed decreased expression levels of goblet cell-secreting mucin (Muc) (~1.4 fold change in Muc2, P &lt;0.05), whereas young mice retained expression after stroke. Conclusions: Our data indicates that 1) both aging and stroke impairs gut integrity and possibly leads to increase of infections and 2) epithelial cells could differentially respond to external stimuli after stroke, in an age-dependent manner. The current study provides the first insight into how intestinal epithelium is involved in the brain-gut axis in stroke.