Cigarette smoke exposure attenuates the induction of antigen-specific IgA in the murine upper respiratory tract.

The upper respiratory tract is highly exposed to airborne pathogens and serves as an important inductive site for protective antibody responses, including mucosal IgA and systemic IgG. However, it is currently unknown to what extent inhaled environmental toxins, such as a cigarette smoke, affect the ability to induce antibody-mediated immunity at this site. Using a murine model of intranasal lipopolysaccharide and ovalbumin (LPS/OVA) immunization, we show that cigarette smoke exposure compromises the induction of antigen-specific IgA in the upper airways and systemic circulation. Deficits in OVA-IgA were observed in conjunction with a reduced accumulation of OVA-specific IgA antibody-secreting cells (ASCs) in the nasal mucosa, inductive tissues (NALT, cervical lymph nodes, spleen) and the blood. Nasal OVA-IgA from smoke-exposed mice also demonstrated reduced avidity during the acute post-immunization period in association with an enhanced mutational burden in the cognate nasal Igha repertoire. Mechanistically, smoke exposure attenuated the ability of the nasal mucosa to upregulate VCAM-1 and pIgR, suggesting that cigarette smoke may inhibit both nasal ASC homing and IgA transepithelial transport. Overall, these findings demonstrate the immunosuppressive nature of tobacco smoke and illustrate the diversity of mechanisms through which this noxious stimulus can interfere with IgA-mediated immunity in the upper airways.

Nasal Tissue Extraction Is Essential for Characterization of the Murine Upper Respiratory Tract Microbiota.

Respiratory infections are a leading cause of morbidity and mortality worldwide. Bacterial pathogens often colonize the upper respiratory tract (nose or mouth) prior to causing lower respiratory infections or invasive disease. Interactions within the upper respiratory tract between colonizing bacteria and the resident microbiota could contribute to colonization success and subsequent transmission. Human carriage studies have identified associations between pathogens such as Streptococcus pneumoniae and members of the resident microbiota, although few mechanisms of competition and cooperation have been identified and would be aided by the use of animal models. Little is known about the composition of the murine nasal microbiota; thus, we set out to improve assessment, including tissue sampling, composition, and comparison between mouse sources. Nasal washes were efficient in sampling the nasopharyngeal space but barely disrupted the nasal turbinates. Nasal tissue extraction increased the yield of cultivable bacterial compared to nasal washes, revealing distinct community compositions. Experimental pneumococcal colonization led to dominance by the colonizing pathogen in the nasopharynx and nasal turbinates, but the composition of the microbiota, and interactions with resident microbes, differed depending on the sampling method. Importantly, vendor source has a large impact on microbial composition. Bacterial interactions, including cooperation and colonization resistance, depend on the biogeography of the nose and should be considered during research design of experimental colonization with pathogens.IMPORTANCE The nasal microbiota is composed of species that play a role in the colonization success of pathogens, including Streptococcus pneumoniae and Staphylococcus aureus Murine models provide the ability to explore disease pathogenesis, but little is known about the natural murine nasal microbiota. This study established techniques to allow the exploration of the bacterial members of the nasal microbiota. The mouse nasal microbiota included traditional respiratory bacteria, including Streptococcus, Staphylococcus, and Moraxella species. Analyses were affected by different sampling methods as well as the commercial source of the mice, which should be included in future research design of infectious disease research.

Streptococcus pneumoniae Colonization Is Required To Alter the Nasal Microbiota in Cigarette Smoke-Exposed Mice.

Smokers have nasal microbiota dysbiosis, with an increased frequency of colonizing bacterial pathogens. It is possible that cigarette smoke increases pathogen acquisition by perturbing the microbiota and decreasing colonization resistance. However, it is difficult to disentangle microbiota dysbiosis due to cigarette smoke exposure from microbiota changes caused by increased pathogen acquisition in human smokers. Using an experimental mouse model, we investigated the impact of cigarette smoke on the nasal microbiota in the absence and presence of nasal pneumococcal colonization. We observed that cigarette smoke exposure alone did not alter the nasal microbiota composition. The microbiota composition was also unchanged at 12 h following low-dose nasal pneumococcal inoculation, suggesting that the ability of the microbiota to resist initial nasal pneumococcal acquisition was not impaired in smoke-exposed mice. However, nasal microbiota dysbiosis occurred as a consequence of established high-dose nasal pneumococcal colonization at day 3 in smoke-exposed mice. Similar to clinical reports on human smokers, an enrichment of potentially pathogenic bacterial genera such as Fusobacterium, Gemella, and Neisseria was observed. Our findings suggest that cigarette smoke exposure predisposes to pneumococcal colonization independent of changes to the nasal microbiota and that microbiota dysbiosis observed in smokers may occur as a consequence of established pathogen colonization.

Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3-/- Mice.

The pathogenesis of Crohn's disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3-/- mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3-/- mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3-/- mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3-/- mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.

Gastrointestinal dysbiosis and the use of fecal microbial transplantation in Clostridium difficile infection.

The impact of antibiotics on the human gut microbiota is a significant concern. Antibiotic-associated diarrhea has been on the rise for the past few decades with the increasing usage of antibiotics. Clostridium difficile infections (CDI) have become one of the most prominent types of infectious diarrheal disease, with dramatically increased incidence in both the hospital and community setting worldwide. Studies show that variability in the innate host response may in part impact upon CDI severity in patients. That being said, CDI is a disease that shows the most prominent links to alterations to the gut microbiota, in both cause and treatment. With recurrence rates still relatively high, it is important to explore alternative therapies to CDI. Fecal microbiota transplantation (FMT) and other types of bacteriotherapy have become exciting avenues of treatment for CDI. Recent clinical trials have generated excitement for the use of FMT as a therapeutic option for CDI; however, the exact components of the human gut microbiota needed for protection against CDI have remained elusive. Additional investigations on the effects of antibiotics on the human gut microbiota and subsequent CDI will help reduce the socioeconomic burden of CDI and potentially lead to new therapeutic modalities.

The Src kinase Fyn is protective in acute chemical-induced colitis and promotes recovery from disease.

Despite progress in understanding enteric inflammation, current therapies, although effective in many patients with inflammatory bowel disease (IBD), have significant side-effects, and, in many patients, it is refractory to treatment. The Src kinase Fyn mediated IFN-γ-induced increased permeability in model epithelia, and so we hypothesized that inhibition of Fyn kinase would be anti-colitic. Mice [B6.129SF2/J wild-type (WT), Fyn KO, or chimeras] received 2.5% dextran sodium sulfate (DSS) or normal water for 10 d and were necropsied immediately or 3 d later. Gut permeability was assessed by FITC-dextran flux, colitis by macroscopic and histologic parameters, and immune cell status by cytokine production and CD4(+) T cell Foxp3 expression. Fyn KO mice consistently displayed significantly worse DSS-induced disease than WT, correlating with decreased IL-10 and increased IL-17 in splenocytes and the gut; Fyn KO mice failed to thrive after removal of the DSS water. Analysis of chimeric mice indicated that the increased sensitivity to DSS was due to the lack of Fyn kinase in hematopoietic, but not stromal, cells, in accordance with Fyn(+) T cell increases in WT mice exposed to DSS and Fyn KO mice having a reduced number of CD4(+)Foxp3(+) cells in baseline or colitic conditions and a reduced capacity to induce Foxp3 expression in vitro. Other experiments suggest that the colonic microbiota in Fyn KO mice is not preferentially colitogenic. Contrary to our expectation, the absence of Fyn kinase resulted in greater DSS-induced disease, and analysis of chimeric mice indicated that leukocyte Fyn kinase is beneficial in limiting colitis.

Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain Giardia infections may attenuate PMN accumulation by decreasing the expression of the mediators responsible for their recruitment.

The P2Y6 receptor mediates Clostridium difficile toxin-induced CXCL8/IL-8 production and intestinal epithelial barrier dysfunction.

C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.

Attenuation of Clostridium difficile toxin-induced damage to epithelial barrier by ecto-5'-nucleotidase (CD73) and adenosine receptor signaling.

BACKGROUND: Clostridium difficile (Cdf) releases toxins (TcdA and TcdB) that damage the intestinal epithelial barrier. Ecto-5'-nucleotidase (CD73) is expressed on intestinal epithelial cells, and it is hypothesized to protect against toxin-induced epithelial damage through the cleavage of 5'-AMP to adenosine (Ado) and subsequent activation of adenosine receptors (AdoRs). Herein, we sought to assess the potential protective effects of CD73 and AdoR signaling on the injurious effects of Cdf toxins. METHODS: Barrier function was assessed with T84 colonocytes. Transepithelial electrical resistance (TEER), paracellular fluorescein isothiocyanate (FITC)-dextran flux, and tight junction protein (ZO-1) integrity were monitored. Intrarectal installation of Cdf toxin was used to assess epithelial damage in vivo. KEY RESULTS: TcdA/B caused reduced TEER and increased paracellular flux in vitro. Concurrent treatment with 5'-AMP attenuated these responses to Cdf toxin; an effect that was blocked with ZM241385 (AdoRA2 antagonist). APCP, a CD73 inhibitor, also suppressed the protective effects of 5'-AMP on paracellular flux. 5'-AMP reduced toxin-induced disruption of ZO-1, an effect that was abolished by APCP and ZM241385. Inhibition of CD73 with APCP during Cdf toxin exposure led to increased intestinal barrier permeability and epithelial damage in vivo. Intrarectal instillation of 5'-AMP had no effect on toxin-induced intestinal injury. CONCLUSIONS & INFERENCES: Our data suggest that CD73 has a protective role against TcdA/B-induced damage. 5'-AMP treatment attenuated the damaging effects of Cdf toxin in vitro, and inhibitors of CD73 (APCP) and AdoRs (ZM241385) revealed that the cleavage of 5'-AMP to Ado was necessary for the protective effects. Inhibition of CD73 in vivo increases colonic tissue damage and epithelial permeability during Cdf toxin exposure.

Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure.

Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated "ileal loop." This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1ß. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.

Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome.

BACKGROUND & AIMS: Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1beta production, which contributes to the pathogenesis of CDAD. METHODS: Macrophages exposed to TcdA and TcdB were assessed for IL-1beta production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist-treated mice. RESULTS: TcdA and TcdB triggered inflammasome activation and IL-1beta secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1beta release. TcdB-induced IL-1beta release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra. CONCLUSIONS: TcdA and TcdB trigger IL-1beta release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC-/- mice. These data suggest that targeting inflammasome or IL-1beta signaling may represent new therapeutic targets in the treatment of CDAD.