Maternal seeding of the microbiome in neonates promotes a long-lasting biological footprint, but how it impacts disease susceptibility in early life remains unknown. We hypothesized that feeding butyrate to pregnant mice influences the newborn's susceptibility to biliary atresia, a severe cholangiopathy of neonates. Here, we show that butyrate administration to mothers renders newborn mice resistant to inflammation and injury of bile ducts and improves survival. The prevention of hepatic immune cell activation and survival trait is linked to fecal signatures of Bacteroidetes and Clostridia and increases glutamate/glutamine and hypoxanthine in stool metabolites of newborn mice. In human neonates with biliary atresia, the fecal microbiome signature of these bacteria is under-represented, with suppression of glutamate/glutamine and increased hypoxanthine pathways. The direct administration of butyrate or glutamine to newborn mice attenuates the disease phenotype, but only glutamine renders bile duct epithelial cells resistant to cytotoxicity by natural killer cells. Thus, maternal intake of butyrate influences the fecal microbial population and metabolites in newborn mice and the phenotypic expression of experimental biliary atresia, with glutamine promoting survival of bile duct epithelial cells.

Anthrax is caused by Bacillus anthracis, a zoonotic bacterial pathogen affecting humans and livestock worldwide. The current human anthrax vaccine, anthrax vaccine adsorbed (AVA), is an injected vaccine with a cumbersome administration schedule and fails to promote mucosal immunity. Bacterial enterotoxins, which stimulate production of the cyclic nucleotide cAMP are effective experimental mucosal vaccine adjuvants, but their inherent toxicity has precluded their use in humans. We investigated whether cyclic dinucleotides that target Stimulator of Interferon Gamma Genes (STING) in mammalian cells could represent an alternative to bacterial enterotoxins as adjuvant for sublingual immunization and promotion of mucosal immunity and secretory IgA responses in addition to systemic immunity. We found that sublingual immunization of mice with Bacillus anthracis protective antigen (PA) and the STING ligand 3'3'-cGAMP promotes PA-specific serum IgG Ab responses of the same magnitude as those induced after immunization with PA and the experimental adjuvant cholera toxin (CT). Interestingly, this STING ligand also promoted serum anti-PA IgA and IgA-producing cells in the bone marrow. Furthermore, the saliva of mice immunized with the STING ligand exhibited similar levels of PA-specific IgA Abs as groups immunized with CT as adjuvant. The adjuvant activity of 3'3'-cGAMP was associated with mixed Th1, Th2, and Th17 responses. This STING ligand also induced rapid IFN-β and IL-10 responses in sublingual tissues and cervical lymph nodes, and TGF-β responses in the cervical lymph nodes, which could contribute to promoting IgA responses after sublingual immunization.

Biliary atresia is progressive fibro-inflammatory cholangiopathy of young children. Central to pathogenic mechanisms of injury is the tissue targeting by the innate and adaptive immune cells. Among these cells, neutrophils and the IL-8/Cxcl-8 signaling via its Cxcr2 receptor have been linked to bile duct injury. Here, we aimed to investigate whether the intestinal microbiome modulates Cxcr2-dependent bile duct injury and obstruction. Adult wild-type (WT) and Cxcr2-/- mice were fed a diet supplemented with sulfamethoxazole/trimethoprim (SMZ/TMP) during pregnancy and lactation, and their pups were injected intraperitoneally with rhesus rotavirus (RRV) within 24 hours of life to induce experimental biliary atresia. The maternal exposure to SMZ/TMP significantly lowered the incidence of jaundice and bile duct obstruction and resulted in improved survival, especially in Cxcr2-/- mice. Analyses of the microbiome by deep sequencing of 16S rRNA of the neonatal colon showed a delay in bacterial colonization of WT mice induced by SMZ/TMP, with a notable switch from Proteobacteria to Firmicutes. Interestingly, the genetic inactivation of Cxcr2 alone produced a similar bacterial shift. When treated with SMZ/TMP, Cxcr2-/- mice infected with RRV to induce experimental biliary atresia showed further enrichment of Corynebacterium, Anaerococcus and Streptococcus. Among these, Anaerococcus lactolyticus was significantly associated with a suppression of biliary injury, cholestasis, and survivability. These results suggest that the postnatal development of the intestinal microbiota is an important susceptibility factor for experimental biliary atresia.

BACKGROUND:The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.METHODS:Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts.RESULTS:We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells.CONCLUSIONS:CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD.

Secretory IgA antibody plays an important role in the protection of mucosal surfaces against microbial pathogens. Induction of the IgA antibody has been known to be largely dependent upon the cytokine milieu. However, the importance of innate immune cell subsets for IgA responses has not been fully appreciated yet. We investigated the role of myeloid cell-subsets on IgA responses to sublingual immunization. In contrast with cholera toxin as an adjuvant which promotes IgA responses, sublingual application of the Bacillus anthracis edema toxin (EdTx) to C57BL/6 mice transiently increased the frequencies of neutrophils in sublingual tissue and cervical lymph nodes (CLNs), and promoted antigen-specific serum IgG, but not serum or mucosal IgA responses. However, EdTx as an adjuvant failed to increase frequencies of neutrophils in sublingual tissue and CLNs of mice lacking IKKβ in myeloid cells (IKKβΔMye), and these mice developed antigen-specific mucosal IgA responses. Interestingly, depletion of neutrophils in C57BL/6 mice promoted antigen-specific fecal and serum IgA responses, similar to those of IKKβΔMye mice, and this effect was associated with increased frequencies of surface IgA expressing plasma cells, α4β7+CCR9+ B cells in lamina propria and number of Peyer’s patch. Our results show that the presence of neutrophils at mucosal inductive sites negatively influences induction of IgA responses and underlying mechanisms are under investigations.

Gender influences the incidence and/or the severity of several diseases and evidence suggests a higher rate of allergy and asthma among women. Most experimental models of allergy use mice sensitized via the parenteral route despite the fact that the mucosal tissues of the gastrointestinal and respiratory tracts are major sites of allergic sensitization and/or allergic responses. We analyzed allergen-specific Ab responses in mice sensitized either by gavage or intraperitoneal injection of ovalbumin together with cholera toxin as adjuvant, as well as allergic inflammation and lung functions following subsequent nasal challenge with the allergen. Female mice sensitized intraperitoneally exhibited higher levels of serum IgE than their male counterparts. After nasal allergen challenge, these female mice expressed higher Th2 responses and associated inflammation in the lung than males. On the other hand, male and female mice sensitized orally developed the same levels of allergen-specific Ab responses and similar levels of lung inflammation after allergen challenge. Interestingly, the difference in allergen-specific Ab responses between male and female mice sensitized by the intraperitoneal route was abolished in IKKβΔMye mice, which lack IKKβ in myeloid cells. In summary, the oral or systemic route of allergic sensitization and IKKβ signaling in myeloid cells regulate how the gender influences allergen-specific responses and lung allergic inflammation.

Abstract Cadmium is a toxic heavy metal that can be ingested due to its presence in contaminated water and its accumulation in leafy vegetables, fish and grains. Since cadmium can compete with iron for intestinal absorption, long-term ingestion of cadmium can change composition of gut-commensal bacteria by inhibiting the growth of selected bacteria. Gut-commensal bacteria are critical to maintain intestinal homeostasis since they limit attachment of infectious agents, regulate pro-and anti-inflammatory milieu and secretory IgA levels. However, consequences of chronic exposure to cadmium for gut immune homeostasis, and mucosal innate immunity are poorly understood. To investigate the effect of cadmium on gut-homeostasis, C57BL/6 mice were treated with cadmium-contaminated water for 28 days. Mice exposed to cadmium exhibited a significant reduction of secretory IgA levels, which correlated with reduced expression of polymeric Ig receptor mRNA. These mice also showed increased intestinal permeability to macromolecules when compared to control untreated mice. The chronic exposure to cadmium also render these mice more responsive to oral administration of cholera toxin since the frequency of Paneth and goblet cells, as well as the levels of mucus production and anti-microbial mRNA responses seen 16 hours after ingestion of cholera toxin were higher than those of control mice. In summary, environmental pollutant cadmium is a major regulator of gut immune homeostasis and innate immunity.

Secretory IgA (SIgA) Abs play an important role in the protection of mucosal surfaces including the respiratory tract. Several bacterial toxin and derivatives were shown to act as adjuvants and promote SIgA responses to co-administered antigens, but little is known about potential of plant toxins and derivatives to induce SIgA Abs. We found that chemically inactivated ricin toxoid (RT) increases the frequencies of CD11b+phospho-p65 NF-κB+ cells in nasopharyngeal-associated lymphoid tissues (NALT), an effect associated with increased expression of CD40 molecules on CD11b+ macrophages and enhanced pro-inflammatory cytokine responses. Three hours after intranasal administration of RT in vivo, NALT tissues showed a significant increase in the percentage of B220+ cells, which appeared to undergo rapid proliferation. When nasally co-administered with an unrelated antigen, RT induced broad antigen-specific T helper cell-cytokine responses and promoted antigen-specific SIgA Abs. This adjuvant effect of RT was altered in NALP3 KO mice, which exhibited lower number of B cells in NALT after intranasal administration of RT. Furthermore, antigen-specific T cell and B cell responses, as well as antigen-specific SIgA Ab responses were diminished significantly in NALP3 KO mice. Our results show that the plant toxin derivative RT is a mucosal adjuvant, which promotes SIgA via B cell-recruitment and activation in NALT tissues, and that NALP3 is a key regulator of this activity.

The sites where immune responses are initiated are believed to play a key role at shaping subsequent responses at distant mucosal sites. We have previously shown that IKKβ in intestinal epithelial cells (IECs) shapes the immune responses to ingested antigens and influences airway responses to subsequent antigen exposure via regulation of IgA and Th17. People with food allergy often suffer from allergic airway and/or skin diseases like asthma or atopic dermatitis but it is unknown whether T cells sensitized in the gut promote inflammation at different mucosal surfaces through the same mechanism. Using a model of oral antigen sensitization and mice with cell-specific deletion of IKKβ, we addressed the contribution of IECs to regulatory crosstalk between the gut and skin. Control C57BL/6 mice and mice deficient in IKKβ in IEC (IKKβΔIEC) were orally sensitized by administration of ovalbumin and cholera toxin as adjuvant. All groups developed similar levels of antigen-specific IgE and IgG1 responses, but IgG2b responses were enhanced in IKKβΔIEC. Subsequent epicutaneous challenges of control mice with ovalbumin induced skin inflammation characterized by increased epidermal thickness and dermal infiltration of eosinophils. Interestingly, both the epidermal thickness and dermal infiltration of eosinophils were reduced in IKKβΔIEC. Ongoing studies will determine mechanisms underlying the protective effect of IKKβ in intestinal epithelial cells against allergic responses in the skin.

Abstract The nature of immune cells recruited at the sites of antigen exposure and the cytokine microenvironment play a key role at shaping immune responses. Using a model sublingual vaccine with Bacillus anthracis edema toxin as an adjuvant, we show that alteration of IKKβ signaling in myeloid cells (IKKβΔMye) enhanced the magnitude of Ab and T cell responses, compared to control mice. While this model vaccine failed to induce S-IgA Abs in control mice, significant S-IgA Ab responses were induced in mucosal secretions of IKKβΔMye mice. We also established that the sublingual vaccine recruited lower numbers of CD11b+F4/80+Gr1high and CD11b+F4/80-Gr1high, but higher number of CD11b+F4/80+Gr1- cells in the sublingual tissues of IKKβΔMye mice. In contrast, higher numbers of CD11b+F4/80+Gr1high and CD11b+F4/80-Gr1high, but lower number of CD11b+F4/80+Gr1- cells were recruited in the sublingual tissues of control and IL-17A KO mice. Lack of IL-17A signaling did not affect the magnitude of Ab responses to the sublingual vaccine. However, the profile of B cell epitope responses was affected in IL-17A KO mice. Furthermore, CD11b+ cells isolated from IKKβΔMye mice assisted B cell-differentiation and induced production of IgA Abs by IL-17Rhigh B lymphocytes in vitro. Our results support the existence of an IKKβ-IL-17 axis for regulation of mucosal immunity and identify IKKβ signaling as a potential target for improving induction of mucosal immunity by sublingual vaccines.

Abstract IKKβ-NF-κB signaling controls a large number of biological processes via tissue-specific regulation of inflammatory and anti-inflammatory responses. Using a model of oral antigen sensitization and mice with cell-specific deletion of IKKβ, we addressed the contribution of intestinal epithelial cells (IECs) to regulatory crosstalk between distant mucosal sites of the gastrointestinal tract and the airways. Mice with localized alteration of IKKβ-NF-κB signaling in IECs (IKKβΔIEC) exhibited unaltered IgE, but enhanced Th1-associated IgG2a Abs and IgA responses after oral antigen sensitization in the presence of cholera toxin. Interestingly, these mice showed reduced lung inflammation and mucus production after nasal allergen challenge. This protection required oral sensitization and was associated with the presence of IgA in bronchoalveolar fluids and increased IL-17A in the lungs. When orally sensitized with the same regimen, mice with a generalized IKKβ deficiency in cells of the myeloid lineage (IKKβΔMye) also were protected against nasal allergen challenge. Distinct mechanisms contributed to protection in IKKβΔIEC and IKKβΔMye mice since airway hyper-responsiveness and lung inflammation were completely abolished in IKKβΔMye mice, which also showed no IgA in bronchoalveolar fluid and lower IL-17A in the lung than IKKβΔIEC mice. In summary, IKKβ in IECs is sufficient for shaping immune responses to ingested antigens, and subsequent responses to antigen exposure via the airways.

The sites where immune responses are initiated are believed to play a key role at shaping subsequent responses at distant mucosal sites. However, little is known about the relative contribution of intestinal epithelial cells (IEC) to lung reactivity to foreign antigens and the development of allergic and non-allergic asthma. It has been demonstrated that nuclear factor κB (NF-κB) activation by IκB kinase (IKK)β deletion in macrophages increases pro-inflammatory cytokines responses. To address the potential regulatory effect of IEC for lung reactivity, control C57BL/6 mice and mice deficient in IKKβ in IEC (IKKβΔIEC) or in macrophage (IKKβΔMac) were orally sensitized by administration of ovalbumin and cholera toxin as adjuvant. All groups developed similar levels of antigen-specific IgE responses, but IgG2a responses were enhanced in IKKβΔIEC and, even more in IKKβΔMac. Subsequent nasal challenges of C57BL/6 mice with ovalbumin induced lung inflammation and increased lung resistance. Interestingly, both lung inflammation and lung resistance were lower in IKKβΔIEC mice and further reduced in IKKβΔMac mice. Taken together, these results show that localized alteration of NF-κB signaling in IECs have broader effects on immune responses to ingested antigen, which were similar, although of lower magnitude, than those seen when NF-κB signaling is altered on macrophages. They also reveal that IECs can shape immune responses at distant mucosal sites of the airways.

We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.

remains to be investigated.Here we show that ATP-mediated activation of mast cells through P2X7 purinergic receptors plays a pivotal role in the induction of inflammatory responses in the colon.Using trinitrobenzenesulfonic acid (TNBS)-induced colitis model, we found that colonic inflammatory responses associated were reduced in the mast cell-deficient Wsh/sh mice.To identify key molecules in the mast cell-mediated inflammatory responses in the colon, we established monoclonal anti-mast cell antibody (1F11 mAb) inhibiting the activation of colonic mast cells in TNBS-colitis model.Biochemical and proteomics analyses revealed that 1F11 mAb recognized P2X7 purinoceptor expressed predominantly on mast cells.The 1F11 mAb inhibited ATP-dependent mast cell activation, such as degranulation and production of leukotriene and IL-1beta.Consistent with these findings, Wsh/sh mice re-constituted with bone marrow-derived mast cells (BMMCs) from P2X7-deficient mice showed less severity than that of BMMCs from wild-type mice.Taken together, current findings indicated critical roles of ATP-P2X7-dependent pathway of mast cells for the induction of acute inflammation phase of colitis, and provided possible novel strategy for the development of effective therapy against the mast cell mediated inflammatory diseases in the gut.