Obesity has been associated with dysbiosis, but innate mechanisms linking intestinal epithelial cell subsets and obesity remain poorly understood. Using mice lacking Paneth cells (Sox9 ΔIEC mice), small intestinal epithelial cells specialized in the production of antimicrobial products and cytokines, we show that dysbiosis alone does not induce obesity or metabolic disorders. Loss of Paneth cells reduced ILC3 and increased ILC2 numbers in the intestinal lamina propria. High-fat diet (HFD) induced higher weight gain and more severe metabolic disorders in Sox9 ΔIEC mice. Further, HFD enhances the number of ILC1 in the intestinal lamina propria of Sox9 ΔIEC mice and increases intestinal permeability and the accumulation of immune cells (inflammatory macrophages and T cells, and B cells) in abdominal fat tissues of obese Sox9 ΔIEC . Transplantation of fecal materials from Sox9 ΔIEC mice in germ-free mice before HFD further confirmed the regulatory role of Paneth cells for gut ILC subsets and the development of obesity.

Cystic fibrosis (CF) is a genetic disorder that causes severe damage to the lungs and digestive system. CF affects the cells that produce mucus, sweat, and digestive enzymes and is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Expression and function of CFTR have been extensively studied on epithelial cells and several recent publications have shown that CFTR also regulates the functions of myeloid cells. We addressed whether CFTR is expressed by B cells and the role of CFTR in B cell functions. We found that CFTR is expressed on murine and human B cells. Compared to control wild-type (WT) mice, CFTR KO exhibited reduced frequency of B cells in lymphoid tissues and lower levels of serum immunoglobulin isotypes. Upon systemic immunization with an alum-based vaccine, CFTR KO mice developed lower antibody responses than control mice. Interestingly, vaccine supplementation with an inhibitor of the serine protease elastase (NEI) enhanced the titers of vaccine-specific serum antibody responses in CFTR KO mice and CFTR heterozygote (CFTR Het) mice. We have previously reported that NEI supplementation could promote mucosal immunity in mice immunized with the alum-based injected vaccine. In this regard, CFTR KO and CFTR Het mice immunized with NEI-supplemented alum-based injected developed higher IgA and IgG responses in saliva than control WT mice. These results provide new insights on the regulatory role of CFTR for B cell differentiation and function. They also suggest that vaccine supplementation with NEI could improve protection of individuals leaving with CF against respiratory pathogens. NIH R01 DK101323

Paneth cells are a subset of small intestinal epithelial cells specialized in the production of antimicrobial products and cytokines. Gastrointestinal dysbiosis has been linked to many health concerns and is believed to be a contributing factor in obesity. Despite the role of Paneth cells as a major source of antimicrobial products, the contribution of these cells to health or disease conditions has not been fully explored. We examine the impact loss of Paneth cells would have on the gut microbiota and whether the subsequent dysbiosis could affect metabolic functions of the host and fat accumulation in a mouse model of diet induced obesity. For this purpose, were used wild-type and Sox9ΔIEC mice, which lack Paneth cells due to a Sox9 gene deletion within the intestinal epithelium. Compared to control wild-type mice, the small intestine of Sox9ΔIEC exhibited an altered profile of ILCs characterized by increase ILC2 and decrease ILC3 numbers. After exposure to a high-fat diet for 13 weeks, the Sox9ΔIEC mice gained more weight and had higher glucose intolerance. The intestinal homeostasis was also affected, with an increase in intestinal permeability and an increase in ILC1s within the small intestine of Sox9ΔIEC mice. In the abdominal fat Sox9ΔIEC mice showed increased numbers of immune cells including inflammatory macrophages, T cells, and B cells. Finally, fecal material transplantation experiments revealed that fecal material from the Sox9ΔIEC mice transfer the phenotype in recipient germ-free mice as indicated by a trend towards an increase in weight gain and immune cell infiltration of abdominal fat. These results highlight the importance of Paneth cells as regulators of metabolic functions of the host and fat accumulation. Supported by grants from OSU and NIH.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti- SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alumbased injected vaccines for the induction of broad immunity, including mucosal immunity.

It is well established that dendritic cells and macrophages play a role in antigen presentation to B and T cells and in shaping B and T cell responses via cytokines they produce. We have previously reported that depletion of neutrophils improves the production of mucosal IgA after sublingual immunization with Bacillus anthracis edema toxin as adjuvant. These past studies also demonstrated that an inverse correlation exists between the number of neutrophils and production of IgA by B cells. Using specific inhibitors of elastase, we addressed whether the elastase activity of neutrophil could be the factor that interferes with production of IgA and possibly other immunoglobulin isotypes. We found that murine splenocytes and mesenteric lymph node cells cultured for 5 days in the presence of neutrophil elastase inhibitors secreted higher levels of IgG and IgA than cells cultured in the absence of inhibitors. The effect of the inhibitors was dose-dependent and was consistent with increased frequency of CD138+ cells expressing IgG or IgA. Finally, neutrophil elastase inhibitors increased transcription of mRNA for AID, IL-10, BAFF and APRIL, factors involved in B cell differentiation. These findings identify inhibitors of elastase as potential adjuvants for increasing production of antibodies.

ABSTRACT AIM IκBζ is a transcriptional factor induced in immune cells upon Toll-like receptor (TLR) activation. Recent studies demonstrate unconventional, constitutive expression of IκBζ in epithelium of mouse skin and eyes, possibly reflecting continuous activation of TLRs by pathogen-associated molecular patterns (PAMPs). In this context, the lung epithelium which constitutes another important barrier also expresses IκBζ but may not be as actively exposed to pathogens as skin and eyes. Our aim was to determine if IκBζ expression in the lungs is constitutive or induced. SIGNIFICANCE IκBζ is linked to lung disorders due to its role in regulating protective cytokines and antimicrobial peptides in airway epithelium and can therefore be a potential biomarker and a key therapeutic target. METHODS We evaluated IκBζ expression in airway epithelia of healthy humans and three kinds of mice: normal, gnotobiotic and Nfkbiz −/− knockout, using immunostaining and immunoblotting. RESULTS Immunohistochemistry of ciliated airway epithelial cells in healthy humans and normal mice was positive for IκBζ. The pathogen free airway cells from gnotobiotic mice also stained positive, suggesting that lung epithelial IκBζ expression does not require induction by PAMPs. Although lung epithelia from Nfkbiz −/− knockout mice also stained positive, this knockout may not have eliminated exons 3-4 and 9-14, and so did not provide the specificity control for the IκBζ antiserum. Importantly, immunoblotting tissue homogenates from gnotobiotic mouse lungs and primary human airway epithelial cells demonstrated constitutive IκBζ expression at its correct 86 kDa size. CONCLUSIONS Our data demonstrates constitutive expression of IκBζ protein in airway epithelium, indicating a potential role for this molecule in lung homeostasis.

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.

Allergic sensitization to food allergens has subsequent potential to developing allergic responses in the gastrointestinal tract, but also to skin or lung. Our previous study showed that lack of IKKβ in intestinal epithelial cells regulates favors IgA responses to ingested allergen, which in turn limits the severity of allergic responses in the airway. In this study we investigated whether intestinal epithelial IKKβ also regulated allergic responses to oral antigens. Wild-type C57BL/6 and IKKβΔIEC mice, which lack IKKβ in intestinal epithelial cells, were orally sensitized to a food antigen in the presence of cholera toxin. Allergen-specific serum IgE responses and fecal IgA responses were similar between the groups. However, after oral allergen-challenge, IKKβΔIEC mice only developed minimal clinical and histological signs of allergy, including drop in body temperature and mucus in small intestinal villi and crypts. Interestingly, IKKβΔIEC mice expressed lower levels of CCL11 (eotaxin) and eosinophils than control wild-type mice and their levels were only weakly increased after oral allergen sensitization and challenge. In summary, this study reveals a new role of intestinal epithelial cells in the regulation of allergy in the GI tract through a NF-κB-CCL11 axis.

Abstract Low doses of cadmium can be ingested due to its presence in contaminated water and its accumulation in leafy vegetables, fish and grains. Pollutants are believe to contribute to the increased incidence of allergy diseases. We addressed whether chronic ingestion of low doses of cadmium could impact allergic sensitization and thus, favor the development of allergic diseases. Conventional C57BL/6 mice given low doses of cadmium in the drinking water for 28 days exhibited a significant reduction of bacterial diversity, and an alteration of the Firmicutes to Bacteriodetes ratio. This treatment also activated both the canonical and the non-canonical NF-κB pathway and promoted proinflammatory cytokine and antimicrobial responses in the gut. The effects of cadmium were at least partially independent of the gut microbiome since germ-free C57BL/6 mice subjected to the same treatment developed the same profile of responses although at a lower degree. Finally, conventional mice chronically treated with low doses of cadmium developed higher antigen-specific IgE responses upon oral sensitization with OVA and cholera toxin as adjuvant. Furthermore, upon nasal antigen, cadmium-treated mice developed higher airway allergic response, which were characterized by the increased levels of IL-17 and Th1 responses control mice. In summary, the environmental pollutant cadmium can be a major regulator of gut immune homeostasis and a cause for increased allergic responses at distant mucosal sites.

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. 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.

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.

It has been demonstrated that nuclear factor κB (NF-κB) activation by IκB kinase (Ikk)β deletion in macrophages increases pro-inflammatory cytokines responses and enhances pathogen clearance. To address whether this pathway also regulates the mucosal adjuvant activity of edema toxin (EdTx), LysMcre x Ikkβf/f (IkkβΔMø) mice with Ikkβ-deleted macrophages were sublingually immunized with 50µg of Yersinia pestis F1V fusion protein with or without 15µg of EdTx as an adjuvant. Both control and IkkβΔMø mice immunized with F1V+EdTx developed higher levels of serum Abs, i.e., IgG, IgG1, IgG2a and IgA, when compared to mice immunized with F1V alone. The IkkβΔMø mice exhibited higher serum IgG2a levels (and IgG2a:IgG1 ratio) than control counterpart immunized with F1V+EdTx, suggesting that immune responses to F1V are biased toward Th1-type. These mice also showed elevated levels of F1V-specific serum IgA Abs than control littermates. The effect of Ikkβ deletion was more pronounced in secretory Abs since EdTx as adjuvant promoted higher fecal IgA Abs, as well as IgG and IgA Ab titers in saliva and vaginal washes of IkkβΔMø mice. These studies reveal a new role for macrophages and NF-κB in shaping mucosal immune responses induced by EdTx derivative adjuvants via the sublingual mucosa.