Mucosal Host Defense Research Articles

Th22 Cells Are an Important Source of IL-22 for Host Protection against Enteropathogenic Bacteria

Interleukin-22 (IL-22) is central to host protection against bacterial infections at barrier sites. Both innate lymphoid cells (ILCs) and Tcells produce IL-22. However, the specific contributions of CD4(+) Tcells and their developmental origins are unclear. We found that the enteric pathogen Citrobacter rodentium induced sequential waves of IL-22-producing ILCs and CD4(+) Tcells that were each critical to host defense during a primary infection. Whereas IL-22 production by ILCs was strictly IL-23 dependent, development of IL-22-producing CD4(+) Tcells occurred via an IL-6-dependent mechanism that was augmented by, but not dependent on, IL-23 and was dependent on both transcription factors T-bet and AhR. Transfer of CD4(+) Tcells differentiated with IL-6 in the absence of TGF-β ("Th22" cells) conferred complete protection of infected IL-22-deficient mice whereas transferred Th17 cells did not. These findings establish Th22 cells as an important component of mucosal antimicrobial host defense.

Active vitamin D (1,25-dihydroxyvitamin D3) increases host susceptibility toCitrobacter rodentiumby suppressing mucosal Th17 responses

Vitamin D deficiency affects more that 1 billion people worldwide and is associated with an increased risk of developing a number of inflammatory/autoimmune diseases, including inflammatory bowel disease (IBD). At present, the basis for the impact of vitamin D on IBD and mucosal immune responses is unclear; however, IBD is known to reflect exaggerated immune responses to luminal bacteria, and vitamin D has been shown to play a role in regulating bacteria-host interactions. Therefore, to test the effect of active vitamin D on host responses to enteric bacteria, we gave 1,25(OH)(2)D(3) to mice infected with the bacterial pathogen Citrobacter rodentium, an extracellular microbe that causes acute colitis characterized by a strong Th1/Th17 immune response. 1,25(OH)(2)D(3) treatment of infected mice led to increased pathogen burdens and exaggerated tissue pathology. In association with their increased susceptibility, 1,25(OH)(2)D(3)-treated mice showed substantially reduced numbers of Th17 T cells within their infected colons, whereas only modest differences were noted in Th1 and Treg numbers. In accordance with the impaired Th17 responses, 1,25(OH)(2)D(3)-treated mice showed defects in their production of the antimicrobial peptide REG3γ. Taken together, these studies show that 1,25(OH)(2)D(3) suppresses Th17 T-cell responses in vivo and impairs mucosal host defense against an enteric bacterial pathogen.

The Amino Acid Sequence of Neisseria lactamica PorB Surface-Exposed Loops Influences Toll-Like Receptor 2-Dependent Cell Activation

Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical "TLR2-binding signature" within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro, a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis, suggesting a role for PorB in induction of host cell activation by whole bacteria.

Innate immune induction and influenza protection elicited by a response-selective agonist of human C5a.

The anaphylatoxin C5a is an especially potent mediator of both local and systemic inflammation. However, C5a also plays an essential role in mucosal host defense against bacterial, viral, and fungal infection. We have developed a response-selective agonist of human C5a, termed EP67, which retains the immunoenhancing activity of C5a at the expense of its inflammatory, anaphylagenic properties. EP67 insufflation results in the rapid induction of pulmonary cytokines and chemokines. This is followed by an influx of innate immune effector cells, including neutrophils, NK cells, and dendritic cells. EP67 exhibits both prophylactic and therapeutic protection when tested in a murine model of influenza A infection. Mice treated with EP67 within a twenty-four hour window of non-lethal infection were significantly protected from influenza-induced weight loss. Furthermore, EP67 delivered twenty-four hours after lethal infection completely blocked influenza-induced mortality (0% vs. 100% survival). Since protection based on innate immune induction is not restricted to any specific pathogen, EP67 may well prove equally efficacious against a wide variety of possible viral, bacterial, and fungal pathogens. Such a strategy could be used to stop the worldwide spread of emergent respiratory diseases, including but not limited to novel strains of influenza.

Prostaglandin E2 Suppresses Antifungal Immunity by Inhibiting Interferon Regulatory Factor 4 Function and Interleukin-17 Expression in T Cells

T helper 17 (Th17) cells play an important role in mucosal host defense through production of the signature cytokines IL-17 and IL-22. Prostaglandin E2 (PGE2) has been shown to enhance IL-17 production by mature Th17 cells. However, when present during Th17 cell differentiation, we found that PGE2 inhibited the transcription factor IRF4 and suppressed production of IL-17 but not IL-22. We show that IRF4 was required for IL-17 expression but inhibited IL-22 expression, highlighting the potential for discordant regulation of these two cytokines in Th17 cells. The pathogenic fungus Cryptococcus neoformans produces PGE2, and we found that it uses PGE2- and IRF4-dependent mechanisms to specifically inhibit induction of IL-17 during Th17 cell differentiation. Blockade of host PGE2 during infection led to increased IL-17 production from CD4(+) T cells and increased survival of mice. These findings suggest that host- or pathogen-derived PGE2 can act directly on Th17 cells during differentiation to inhibit IL-17-dependent antimicrobial responses.

Suppression of Adenosine-Activated Chloride Transport by Ethanol in Airway Epithelia

Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (ISC) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A2B adenosine receptor (A2BAR), largely abolished the adenosine-stimulated chloride transport, suggesting that A2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

Differential Regional Expression Of Innate Immune Antimicrobial Proteins In Sinonasal Mucosa

Nasal mucosal innate host defense is essential for suppressing growth of pathogenic organisms and proper functioning of the upper airways. It has been recognized for long that sinonasal tissues express antimicrobial proteins important in host defense; however the regional distribution of these antimicrobial proteins in upper airways has not been studied in detail. In this study we analyzed the expression of selected antimicrobial proteins in sinonasal tissues at the front (inferior turbinate-IT) and rear (uncinate tissue - UT) of the anterior chamber of the nose in control subjects.

Immunology in prevention of dental caries

Dental caries is one of the most common infectious diseases that exist on the face of this world today. About 25% of the population (in the United States) carries about 80% of the disease burden. Cariogenic micro-organisms enter the dental biofilm early in life and can subsequently emerge, under favorable environmental conditions, to cause disease. Adaptive host defenses aroused by these infections are expressed in the saliva and gingival crevicular fluid in the oral cavity. Thanks to numerous scientific advances, tooth decay is not as rampant as it once was, but it is still five times more common in children than asthma and seven times more common than hay fever. So it is still a serious problem, especially for those populations who are very young, very old, economically disadvantaged, chronically ill, or institutionalized. Contemporary research is aimed at evolving a potent and effective caries vaccine to prevent dental caries. By means of this review we would focus on methods by which mucosal host defenses can be induced by immunization to interfere with dental caries caused by mutans streptococci and describe the development of mucosal adjuvants and viable and nonviable delivery systems for dental caries vaccines.

Regulation and function of innate and adaptive interleukin‐17‐producing cells

Interleukin-17 (IL-17)-mediated immune responses play a crucial role in the mucosal host defence against microbial and fungal pathogens. However, the chronic activation of IL-17-producing T helper cells can cause autoimmune disease. In addition, recent studies have highlighted key roles of innate cell-mediated IL-17 responses in various inflammatory settings. Besides inflammation, there have also been intriguing findings regarding the involvement of IL-17 responses in the pathogenesis of cardiovascular diseases and tumour formation. Here, we discuss the latest discoveries in regulation and function of innate and adaptive IL-17-producing cells.

IL-17RE is the functional receptor for IL-17C and mediates mucosal immunity to infection with intestinal pathogens

Interleukin 17 receptor E (IL-17RE) is an orphan receptor of the IL-17 receptor family. Here we show that IL-17RE is a receptor specific to IL-17C and has an essential role in host mucosal defense against infection. IL-17C activated downstream signaling through IL-17RE-IL-17RA complex for the induction of genes encoding antibacterial peptides as well as proinflammatory molecules. IL-17C was upregulated in colon epithelial cells during infection with Citrobacter rodentium and acted in synergy with IL-22 to induce the expression of antibacterial peptides in colon epithelial cells. Loss of IL-17C-mediated signaling in IL-17RE-deficient mice led to lower expression of genes encoding antibacterial molecules, greater bacterial burden and early mortality during infection. Together our data identify IL-17RE as a receptor of IL-17C that regulates early innate immunity to intestinal pathogens.

GM-CSF modulates pulmonary resistance to influenza A infection

Alveolar type II epithelial or other pulmonary cells secrete GM-CSF that regulates surfactant catabolism and mucosal host defense through its capacity to modulate the maturation and activation of alveolar macrophages. GM-CSF enhances expression of scavenger receptors MARCO and SR-A. The alveolar macrophage SP-R210 receptor binds the surfactant collectin SP-A mediating clearance of respiratory pathogens. The current study determined the effects of epithelial-derived GM-CSF in host resistance to influenza A pneumonia. The results demonstrate that GM-CSF enhanced resistance to infection with 1.9 × 10 4 ffc of the mouse-adapted influenza A/Puerto Rico/8/34 (PR8) H1N1 strain, as indicated by significant differences in mortality and mean survival of GM-CSF-deficient ( GM −/−) mice compared to GM −/− mice in which GM-CSF is expressed at increased levels. Protective effects of GM-CSF were observed both in mice with constitutive and inducible GM-CSF expression under the control of the pulmonary-specific SFTPC or SCGB1A1 promoters, respectively. Mice that continuously secrete high levels of GM-CSF developed desquamative interstitial pneumonia that impaired long-term recovery from influenza. Conditional expression of optimal GM-CSF levels at the time of infection, however, resulted in alveolar macrophage proliferation and focal lymphocytic inflammation of distal airways. GM-CSF enhanced alveolar macrophage activity as indicated by increased expression of SP-R210 and CD11c. Infection of mice lacking the GM-CSF-regulated SR-A and MARCO receptors revealed that MARCO decreases resistance to influenza in association with increased levels of SP-R210 in MARCO −/− alveolar macrophages. In conclusion, GM-CSF enhances early host resistance to influenza. Targeting of MARCO may reinforce GM-CSF-mediated host defense against pathogenic influenza.

Morphine Inhibits Murine Dendritic Cell IL-23 Production by Modulating Toll-like Receptor 2 and Nod2 Signaling

IL-23, produced by dendritic cells (DCs) and macrophages, plays a critical role in innate immunity against bacterial infection. Our previous studies show that morphine disrupts the IL-23/IL-17 mediated pulmonary mucosal host defense and increases susceptibility to Streptococcus pneumoniae lung infection. To determine the mechanism by which morphine modulates IL-23 production, mouse bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were treated with morphine, and infected with S. pneumoniae or stimulated with Toll-like receptor (TLR) and Nod2 ligands. We found that a significant increase in IL-23 protein production was observed in S. pneumoniae, TLR2 ligand lipoteichoic acid (LTA), and TLR4 ligand pneumolysin (PLY) stimulated BMDCs and BMDMs. Interestingly, although Nod2 ligand muramyldipeptide (MDP) alone had no effect on IL-23 production, it potentiated LTA induced IL-23 production to the same level as that observed following S. pneumoniae infection, suggesting that S. pneumoniae induced IL-23 production in DCs involves activation of both TLR2 and Nod2 signaling mechanisms. Furthermore, pretreatment of DCs with MyD88 (myeloid differentiation primary response gene 88) and IL-1 receptor-associated kinase (IRAK) 1/4 inhibitors, or TLR2 antibody diminished the S. pneumoniae induced IL-23 and abolished the inhibitory effects of morphine, indicating that S. pneumoniae induced IL-23 production depends on activation of the TLR2-MyD88-IRAK1/4 signaling pathway. Moreover, morphine decreased S. pneumoniae induced phosphorylation of interferon regulatory factor 3 (IRF3) and activating transcription factor 2 in DCs. Taken together, our study shows that morphine impairs S. pneumoniae induced IL-23 production through MyD88-IRAK1/4-dependent TLR2 and Nod2 signaling in DCs.

Functional Specialization of Interleukin-17 Family Members

Interleukin-17A (IL-17A) is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17 has six family members (IL-17A to IL-17F). Although IL-17A and IL-17F share the highest amino acid sequence homology, they perform distinct functions; IL-17A is involved in the development of autoimmunity, inflammation, and tumors, and also plays important roles in the host defenses against bacterial and fungalinfections, whereas IL-17F is mainly involved in mucosal host defense mechanisms. IL-17E (IL-25) is an amplifier of Th2 immune responses. The functions of IL-17B, IL-17C, and IL-17D remain largely elusive. In this review, we describe the identified functions of each IL-17 family member and discuss the potential of these molecules as therapeutic targets.

Activation of type I IFN signaling by NOD1 mediates mucosal host defense against Helicobacter pylori infection

Infection of gastric epithelial cells with Helicobacter pylori (H. pylori) induces a complex array of host protective immune responses. The best known are the adaptive T helper type 1 and type 17 responses that are induced in the gastric lamina propria by antigen-presenting cells via presentation of H. pylori antigens to CD4+ T cells. Recently, it has become apparent that innate immune responses are also induced by H.pylori infection, both in epithelial cells and in underlying antigen-presenting cells. One important component of these innate responses involves the activity of NOD1, an intra-cellular sensor of peptides derived from the peptidoglycan component of the bacterial cell wall. In this review, we discuss our recent work showing that the signaling pathway utilized by NOD1 results in the generation of type I interferon and that this cytokine mediates both chemokine and cytokine responses that regulate the severity of gastric H. pylori infection.

Paneth cell defensins and the regulation of the microbiome

Recently, our laboratory demonstrated that Paneth cell defensins, innate antimicrobial peptides that contribute to mucosal host defense, are able to regulate the composition of the intestinal bacterial microbiome. Using complementary mouse models of defensin deficiency (MMP7-/-) and surplus (HD5+/+), we noted defensin-dependent reciprocal shifts in the dominant bacterial species of the small intestine, without changes in total bacterial numbers. In addition, mice that expressed HD5 showed a significant loss of segemented filamentous bacteria (SFB), resulting in reduced numbers of Th17 cells in the lamina propria. This data showed a novel role for PC defensins in intestinal homeostasis, by regulation of the small intestinal microbiome. The microbiome plays an essential role in mediating host physiology, metabolism, and immune response. The ability of PC defensins to regulate the composition of the biome suggests a much broader importance of these innate immune effectors than previously considered. In this addendum, the role of PC defensins in the regulation of the intestinal microbiome is reviewed, and discussed in the context of recent evidence that highlights the important role of PCs and defensins in the pathophysiology of inflammatory bowel disease.

Pathogenesis of invasive candidiasis

Disseminated candidiasis remains a life-threatening disease in the ICU. The development of invasive disease with Candida albicans is dependent on multiple factors, such as colonization and efficient host defense at the mucosa. In the present review, we describe the host defense mechanisms against Candida that are responsible for counteracting mucosal invasion, and eliminating the pathogen once invasion has taken place. The newly described T-helper subset Th17 is critical for mucosal anti-Candida host defense and plays a major role in controlling C. albicans colonization, whereas the Th1 response and monocyte-dependent cytokines such as IL-1 and TNF are predominantly responsible for activation of neutrophils and macrophages during disseminated candidiasis. This knowledge provides the basis of exploring new treatment options in the fight against invasive candidiasis. Reports of beneficial effects of recombinant cytokine therapy in fungal infections, renders them prime candidates for adjuvant immunotherapy in Candida sepsis.

T-cell Subsets and Antifungal Host Defenses.

It has been long appreciated that protective immunity against fungal pathogens is dependent on activation of cellular adaptive immune responses represented by T lymphocytes. The T-helper (Th)1/Th2 paradigm has proven to be essential for the understanding of protective adaptive host responses. Studies that have examined the significance of regulatory T cells in fungal infection, and the recent discovery of a new T-helper subset called Th17 have provided crucial information for understanding the complementary roles played by the various T-helper lymphocytes in systemic versus mucosal antifungal host defense. This review provides an overview of the role of the various T-cell subsets during fungal infections and the reciprocal regulation between the T-cell subsets contributing to the tailored host response against fungal pathogens.

A Biphasic Innate Immune MAPK Response Discriminates between the Yeast and Hyphal Forms of Candida albicans in Epithelial Cells

SummaryDiscriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-κB and a biphasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a “danger response” pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.

T2067 Mechanosensitivity of Na V1.5, a Voltage-Gated Sodium Selective Ion Channel Found in the Human Gastrointestinal Tract

Stress induced immunomodulation has evolved to confer maximum biological advantage to an organism exposed to varying environmental stressors. Stress is also implicated in the aetiology of Irritable Bowel Syndrome (IBS). Herein, we examined the immunophenotype of IBS patients for evidence of stress related immunomodulation. Methods: 24-IBS patients and 9-matched controls subjects were studied. Comprehensive clinical, psychological (e.g. somatisation) and symptomatic (e.g. Visceral Sensitivity Index (VSI)) evaluations were performed. Peripheral bloodmononuclear cells (PBMC's) were stimulated using lipopolysacharide (5-concentrations) in the presence or absence of exogenous corticotrophin releasing hormone (CRH). A broad panel of inflammatory markers were measured in the supernatant. A one-way ANOVA was performed and data reported as point estimates of difference, ±95%CI (IBS-Controls). Results: Levels of IL2 (-44.5 (-66,-22) p<0.001), IL4 (-32.5 (-54,11) p=0.0004), IL5 (-15.3 (-21, 8.9) p<0.001), IL10 (-1088 (685, 1491) p<0.001), IL13 (60.3 (-85, -36) p<0.001), TNFα (-5452 (-3577, -7327) p<0.001), and IFNγ (-1353 (-471, -2235) p=0.002) were lower in IBS compared to controls and negatively correlated with measures of symptom severity (e.g. IL-5 and Verbal Descriptor Visual Analogue Scale for symptom intensity (VDVAS-I) r=-0.65, p<0.001) and psychological co-morbidity (e.g. IL-5 and somatisation, r=-0.41, p=0.02). In contrast, IL6 (8431 (13160, 3703) p<0.001) and IL8 (14186 (17934, 10437) p<0.001) levels were higher in IBS and positively correlated with measures of symptom severity (e.g. IL-8 and VDVAS-I, r= 0.61, p<0.001) and psychological co-morbidity (e.g. IL-8 and somatisation, r=0.37, p=0.03). Co-incubation with CRH suppressed IFNγ levels in control subjects to the same level as IBS patients (254.1 (-491, 999) p=0.05). Conclusion: As seen in chronic stress, IBS patients suppress Th1 and Th2 cytokine release from stimulated PBMC's and this suppression is negatively correlated with increasing symptom severity and psychological co-morbidity. Elevation of IL-6, and suppression of IFN-γ, IL-2 and IL-4 may indicate a shift towards a TH17 immune profile. This immunophenotype would convey a biological advantage in terms of enhanced mucosal host defence but may also establish a pro-inflammatory mucosal cytokine profile which would promote gastrointestinal symptoms. The potential interaction between stress and TH17 immune function in IBS could represent a new insight in IBS pathophysiology.