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In obesity, CD11c+ innate immune cells are recruited to adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. We found that ablation of Gnas, the gene that encodes Gas, in CD11c expressing cells protects mice from obesity, glucose intolerance, and insulin resistance. Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and did not require adaptive immunity. Loss of cAMP signaling was associated with increased adipose tissue norepinephrine and cAMP signaling, and prevention of catecholamine resistance. The adipose tissue had reduced expression of catecholamine transport and degradation enzymes suggesting that the elevated norepinephrine resulted from decreased catabolism. Collectively, our results identified an important role for cAMP signaling in CD11c+ innate immune cells in whole-body metabolism by controlling norepinephrine levels in WAT, modulating catecholamine-induced lipolysis, and increasing thermogenesis, which together created a lean phenotype.

In obesity, CD11c+ innate immune cells are recruited to adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. We found that ablation of Gnas, the gene that encodes Gαs, in CD11c expressing cells protects mice from obesity, glucose intolerance, and insulin resistance. Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and did not require adaptive immunity. Loss of cAMP signaling was associated with increased adipose tissue norepinephrine and cAMP signaling, and prevention of catecholamine resistance. The adipose tissue had reduced expression of catecholamine transport and degradation enzymes suggesting that the elevated norepinephrine resulted from decreased catabolism. Collectively, our results identified an important role for cAMP signaling in CD11c+ innate immune cells in whole-body metabolism by controlling norepinephrine levels in WAT, modulating catecholamine-induced lipolysis, and increasing thermogenesis, which together created a lean phenotype.

CD11c+ immune cells are a potential therapeutic target for treatment of obesity-related insulin resistance and type 2 diabetes (T2D). In obesity, CD11c+ immune cells are recruited to white adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. In this study, we found that ablation of Gnas, the gene that encodes Gas, in CD11c expressing cells protects mice from high-fat diet-induced obesity, glucose intolerance and insulin resistance. GnasΔCD11c mice (KO) had increased oxygen consumption, energy expenditure, and beigeing of white adipose tissue (WAT). Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and the absence of T and B cells by crossing the KO to a Rag1-/- background did not alter the phenotype. Notably, we observed elevated norepinephrine and elevated cAMP signaling in the WAT of KO mice. The KO adipose tissue also had reduced expression of catecholamine transport and degradation enzymes. Collectively, our results identified an important role of Gas in CD11c+ cells in whole body metabolism regulation by controlling norepinephrine levels in WAT, modulating catecholamine-induced lipolysis and increasing thermogenesis that together created a lean phenotype.

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings, but how cells compensate for such decreases is unknown. We have used a unique system—murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein Gαs—to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in Gαs-depleted DCs. PDE4B expression is dynamic, falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations, respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations.

Cyclic AMP (cAMP) is involved in multiple biological processes. However, little is known about its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent) regulates conventional type-2 Dendritic Cells (cDC2s), but not cDC1s and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors (TFs) for Th2 induction. Genetic loss of IRF4 phenocopies the effects of cAMP signaling on Th17-induction, indicating that the cAMP effect is secondary to repression of IRF4. Moreover, signaling in cDC2s by a PRR-dependent microbial product, curdlan, represses IRF4 and KLF4, resulting in a pro-Th17 phenotype. These results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the novel cDC2 and cDC17 classification. In addition, the data reveal that cAMP signaling can alter DCs function and fate by repressing IRF4 and KLF4, a pathway that can be harnessed for immuno-regulation.

Objective Transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-1 (TRPV1) are calcium (Ca 2+ )-permeable ion channels mostly known as pain receptors in sensory neurons. However, growing evidence suggests their crucial involvement in the pathogenesis of IBD. We explored the possible contribution of TRPA1 and TRPV1 to T-cell-mediated colitis. Design We evaluated the role of Trpa1 gene deletion in two models of experimental colitis (ie, interleukin-10 knockout and T-cell-adoptive transfer models). We performed electrophysiological and Ca 2+ imaging studies to analyse TRPA1 and TRPV1 functions in CD4+ T cells. We used genetic and pharmacological approaches to evaluate TRPV1 contribution to the phenotype of Trpa1 −/− CD4+ T cells. We also analysed TRPA1 and TRPV1 gene expression and TRPA1 + TRPV1 + T cell infiltration in colonic biopsies from patients with IBD. Results We identified a protective role for TRPA1 in T-cell-mediated colitis. We demonstrated the functional expression of TRPA1 on the plasma membrane of CD4+ T cells and identified that Trpa1 −/− CD4+ T cells have increased T-cell receptor-induced Ca 2+ influx, activation profile and differentiation into Th1-effector cells. This phenotype was abrogated upon genetic deletion or pharmacological inhibition of the TRPV1 channel in mouse and human CD4+ T cells. Finally, we found differential regulation of TRPA1 and TRPV1 gene expression as well as increased infiltration of TRPA1 + TRPV1 + T cells in the colon of patients with IBD. Conclusions Our study indicates that TRPA1 inhibits TRPV1 channel activity in CD4+ T cells, and consequently restrains CD4+ T-cell activation and colitogenic responses. These findings may therefore have therapeutic implications for human IBD.

The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC., It is unclear how the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways interact with other signals in intestinal epithelial cells. Here, the authors show that upon loss of Erk1/2, or pharmacological inhibition of MEK1/2, the ERK5 pathway is upregulated to maintain epithelial cell proliferation.

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.

Rodents are a natural host for the dimorphic pathogenic fungi Coccidioides immitis and Coccidioides posadasii , and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 in vivo is uncertain. Myeloid differentiation factor 88 (MyD88) is the adapter protein for TLR2 and -4, interleukin-1R1 (IL-1R1), and IL-18R1. MyD88/TRIF −/− and MyD88 −/− mice were equally susceptible to C. immitis infection, in contrast to C57BL/6 (B6) controls. Of the four surface receptors, only IL-1R1 was required for resistance to C. immitis , partially explaining the susceptibility of MyD88 −/− mice. We also found that FKS stimulated production of IL-1Ra by bone marrow-derived dendritic cells (BMDCs), independent of MyD88 and Dectin-1. There also was a very high concentration of IL-1Ra in the lungs of infected B6 mice, supporting the potential importance of this regulatory IL-1 family protein in the largely ineffective response of B6 mice to coccidioidomycosis. These results suggest that IL-1R1 signaling is important for defense against C. immitis infection.

The transient receptor potential (TRP) family of ion channels is widely expressed in many cell types and plays various physiological roles. Growing evidence suggests that certain TRP channels are functionally expressed in the immune system. Indeed, an increasing number of reports have demonstrated the functional expression of several TRP channels in innate and adaptive immune cells and have highlighted their critical role in the activation and function of these cells. However, very few reviews have been entirely dedicated to this subject. Here, we will summarize the recent findings with regards to TRP channel expression in T cells and discuss their emerging role as regulators of T cell activation and functions. Moreover, these studies suggest that beyond their pharmaceutical interest in pain management, certain TRP channels may represent potential novel therapeutic targets for various immune-related diseases.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

The inductive role of dendritic cells (DC) in Th2 differentiation has not been fully defined. We addressed this gap in knowledge by focusing on signaling events mediated by the heterotrimeric GTP binding proteins Gαs, and Gαi, which respectively stimulate and inhibit the activation of adenylyl cyclases and the synthesis of cAMP. We show here that deletion of Gnas, the gene that encodes Gαs in mouse CD11c(+) cells (Gnas(ΔCD11c) mice), and the accompanying decrease in cAMP provoke Th2 polarization and yields a prominent allergic phenotype, whereas increases in cAMP inhibit these responses. The effects of cAMP on DC can be demonstrated in vitro and in vivo and are mediated via PKA. Certain gene products made by Gnas(ΔCD11c) DC affect the Th2 bias. These findings imply that G protein-coupled receptors, the physiological regulators of Gαs and Gαi activation and cAMP formation, act via PKA to regulate Th bias in DC and in turn, Th2-mediated immunopathologies.

Significance Current therapy for advanced colorectal cancer (CRC) is unsatisfactory and CRC remains a major cause of cancer-related deaths. Thus, novel and ubiquitously acting oncogenic mediators that are amenable to pharmacological targeting need to be identified. We found that loss of adenomatous polyposis coli ( APC ), which is mutated in the majority of human CRC, results in up-regulation of the signaling protein IL-6ST/gp130. This results in activation of Src family kinases (SFKs), YAP, Notch, and STAT3, which are simultaneously activated in 64% of human CRC. In addition to better explaining how APC loss initiates colorectal tumorigenesis, we show that combined treatment with SFK and JAK inhibitors results in regression of established colorectal tumors in mice.

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (Apc(Min/+) mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in Apc(Min/+) mice, similar to--as well as in conjunction with--a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis.

The innate immune system detects infection and tissue injury through different families of pattern-recognition receptors (PRRs), such as Toll-like receptors. Most PRR-mediated responses initiate elaborate processes of signaling, transcription, translation, and secretion of effector mediators, which together require time to achieve. Therefore, PRR-mediated processes are not active in the early phases of infection. These considerations raise the question of how the host limits microbial replication and invasion during this critical period. Here, we examine the crucial defense mechanisms, such as antimicrobial peptides or extracellular traps, typically activated within minutes of the initial infection phase, which we term the “immediate protective response”. Deficiencies in different components of the immediate protective response are often associated with severe and recurrent infectious diseases in humans, highlighting their physiologic importance.

Background & Aims Foxp3 + T-regulatory cells (Tregs) maintain intestinal homeostasis under conditions of continuous challenge with inflammatory microbes. However, plasticity of the Treg population under certain conditions has been reported; Foxp3 + Tregs can be converted to Foxp3 − CD4 + T cells. Methods We used mice with a T cell–induced colitis model to study the regulatory role of type I interferons (IFNs) in adaptive immunity. We transferred CD4 + CD45RB hi (RB hi ) T cells, with or without CD4 + CD45RB lo CD25 + T cells, from wild-type or IFN-αβR −/− mice into Rag1 −/− recipients. We analyzed induction of colitis by flow cytometry, confocal microscopy, and enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction analyses. IFN-αβR −/− Rag −/− mice were given injections of recombinant IFN-α following transfer of IFN-αβR −/− RB hi T cells and CD4 + Foxp3 + cells from Foxp3-eGFP mice. Results Signaling by type I IFNs was required for maintenance of Foxp3 expression and the suppressive activity of Tregs in mice. Transfer of CD4 + CD45RB lo CD25 + Tregs from IFN-αβR −/− mice did not prevent T-cell induction of colitis in mice. Foxp3 expression by Tregs transferred from IFN-αβR −/− mice was significantly lower than that of Tregs from wild-type mice. Administration of recombinant IFN-α reduced T cell–mediated colitis by increasing the number of Foxp3 + Tregs and their suppressive functions. Conclusions Type I IFNs regulate intestinal homeostasis by maintaining Foxp3 expression on Tregs in colons of mice under inflammatory conditions.

ATG16L1 is an essential component of the autophagasome. The T300A allele of ATG16L1 is associated with the increased susceptibility to Crohn disease. In this study, we identified a novel function of ATG16L1, which suppresses signaling of the pro-inflammatory cytokine IL-1β. Deletion of ATG16L1 in mouse embryonic fibroblasts significantly amplifies IL-1β signal transduction cascades. This amplification is due to elevated p62 levels in ATG16L1-deficient cells. We found that ATG16L1 regulates p62 levels via both autolysosomal and proteasomal pathways. For proteasomal degradation, we found that Cullin-3 (Cul-3) is a E3 ubiquitin ligase of p62 and that ATG16L1 is essential for neddylation of Cul-3, a step required for Cul-3 activation. Taken together our data indicate that loss-of-function of ATG16L1 results in a hyper-responsiveness to the IL-1β signaling because of the increased p62 level.

Interferon-α (IFNα) and IFNβ, collectively known as type I IFNs, are the major effector cytokines of the host immune response against viral infections. However, the production of type I IFNs is also induced in response to bacterial ligands of innate immune receptors and/or bacterial infections, indicating a broader physiological role for these cytokines in host defence and homeostasis than was originally assumed. The main focus of this Review is the underappreciated immunomodulatory functions of type I IFNs in health and disease. We discuss their function in the regulation of innate and adaptive immune responses, the response to bacterial ligands, inflammasome activation, intestinal homeostasis and inflammatory and autoimmune diseases.

Cholera toxin (CT) elicits a mucosal immune response in mice when used as a vaccine adjuvant. The mechanisms by which CT exerts its adjuvant effects are incompletely understood. We show that protection against inhalation anthrax by an irradiated spore vaccine depends on CT-mediated induction of IL-17-producing CD4 Th17 cells. Furthermore, IL-17 is involved in the induction of serum and mucosal antibody responses by CT. Th17 cells induced by CT have a unique cytokine profile compared with those induced by IL-6 and TGF-β, and their induction by CT requires cAMP-dependent secretion of IL-1β and β-calcitonin gene-related peptide by dendritic cells. These findings demonstrate that Th17 cells mediate mucosal adjuvant effects of CT and identify previously unexplored pathways involved in Th17 induction that could be targeted for development of unique mucosal adjuvants.

Toll-like receptor (TLR) signaling is essential for intestinal tumorigenesis in Apc(min/+) mice, but the mechanisms by which Apc enhances tumor growth are unknown. Here we show that microflora-MyD88-ERK signaling in intestinal epithelial cells (IECs) promotes tumorigenesis by increasing the stability of the c-Myc oncoprotein. Activation of ERK (extracellular signal-related kinase) phosphorylates c-Myc, preventing its ubiquitination and subsequent proteasomal degradation. Accordingly, Apc(min/+)/Myd88(-/-) mice have lower phospho-ERK (p-ERK) levels and fewer and smaller IEC tumors than Apc(min/+) mice. MyD88 (myeloid differentiation primary response gene 88)-independent activation of ERK by epidermal growth factor (EGF) increased p-ERK and c-Myc and restored the multiple intestinal neoplasia (Min) phenotype in Apc(min/+)/Myd88(-/-) mice. Administration of an ERK inhibitor suppressed intestinal tumorigenesis in EGF-treated Apc(min/+)/Myd88(-/-) and Apc(min/+) mice and increased their survival. Our data reveal a new facet of oncogene-environment interaction, in which microflora-induced TLR activation regulates oncogene expression and related IEC tumor growth in a susceptible host.

We explored the physiological role of conventional dendritic cells (cDCs) in acute colitis induced by a single cycle of dextran sodium sulfate administration. Depending on their mode of activation and independently of T cells, cDCs can enhance or attenuate the severity of dextran sodium sulfate-induced colitis. The latter beneficial effect was achieved, in part, by IFN-1 induced by Toll-like receptor 9-activated cDCs. IFN-1 inhibits colonic inflammation by regulating neutrophil and monocyte trafficking to the inflamed colon and restraining the inflammatory products of tissue macrophages. These data highlight a novel role of cDCs in the regulation of other innate immune cells and position them as major players in acute colonic inflammation.

Since intestinal epithelium expresses Toll-like receptors, it was suggested that the intestinal epithelium is actively involved in the maintenance of colonic homeostasis. Here we describe our recent findings, which support an active contribution of colonic epithelium to intestinal homeostasis via a unique activation of epithelial TLR9.Recent data indicate that stimulation of Toll-like receptors by intestinal microbiota supports colonic homeostasis. Several Toll-like receptors are expressed in intestinal epithelium. TLR9, an intracellular protein in immune cells, is expressed on the cell surfaces of intestinal epithelium, both on the apical and the basolateral membrane. TLR9 signaling varies in a domain-specific manner; whereas JNK is activated by TLR9 ligand both apically or basolaterally, NF-kappaB is activated only via basolateral stimulation. In apical TLR9 stimulation, IkappaB is phosphorylated and ubiquitinated but is not degraded, and NF-kappaB-dependent inflammatory signals are not transduced. Stimulation of apical TLR9 compromises the inflammatory cascade induced basolaterally by several other Toll-like receptor ligands, suggesting that apical exposure to luminal microbial DNA restrains intestinal inflammation.These data indicate that certain luminal bacterial products support colonic homeostasis via activation of epithelial Toll-like receptors. The role of epithelial Toll-like receptor expression and activation in the pathogenesis of human inflammatory bowel disease is yet to be explored.

Giardia lamblia is a leading protozoan cause of diarrheal disease worldwide. It colonizes the lumen and epithelial surface of the small intestine, but does not invade the mucosa. Acute infection causes only minimal mucosal inflammation. Effective immune defenses exist, yet their identity and mechanisms remain incompletely understood. Interleukin (IL)-17A has emerged as an important cytokine involved in inflammation and antimicrobial defense against bacterial pathogens at mucosal surfaces. In this study, we demonstrate that IL-17A has a crucial function in host defense against Giardia infection. Using murine infection models with Giardia muris and G. lamblia, we observed marked and selective induction of intestinal IL-17A with peak expression after two weeks. Th17 cells in the lamina propria and innate immune cells in the epithelial compartment of the small intestine were responsible for the IL-17A response. Experiments in gene-targeted mice revealed that the cytokine, and its cognate receptor IL-17RA, were required for eradication of the parasite. The actions of the cytokine were mediated by hematopoietic cells, and were required for the transport of IgA into the intestinal lumen, since IL-17A deficiency led to marked reduction of fecal IgA levels, as well as for increased intestinal expression of several other potential effectors, including β-defensin 1 and resistin-like molecule β. In contrast, intestinal hypermotility, another major antigiardial defense mechanism, was not impacted by IL-17A loss. Taken together, these findings demonstrate that IL-17A and IL-17 receptor signaling are essential for intestinal defense against the important lumen-dwelling intestinal parasite Giardia.

Transient receptor potential vanilloid 1 (TRPV1), which has been identified as a molecular target for the activation of sensory neurons by various painful stimuli, was reported to regulate the signaling and activation of CD4+ T cells. However, the role of TRPV1 in CD4+ T cell in allergic rhinitis remains poorly understood. In this study, TRPV1 expression was localized in CD4+ T cells. Both knockout and chemical inhibition of TRPV1 suppressed Th2/Th17 cytokine production in CD4 T cells and Jurkat T cells, respectively, and can suppress T cell receptor signaling pathways including NF-κB, MAP kinase, and NFAT. In TRPV1 knockout allergic rhinitis (AR) mice, eosinophil infiltration, Th2/Th17 cytokines in the nasal mucosa, and total and ova-specific IgE levels in serum decreased, compared with wild-type AR mice. The TRPV1 antagonists, BCTC or theobromine, showed similar inhibitory immunologic effects on AR mice models. In addition, the number of TRPV1+/CD4+ inflammatory cells increased in the nasal mucosa of patients with AR, compared with that of control subjects. Thus, TRPV1 activation on CD4+ T cells is involved in T cell receptor signaling, and it could be a novel therapeutic target in AR.

Atg16L1 (autophagy-related 16-like 1), an essential component of autophagy, interacts with the Atg12-Atg5 conjugate for formation of autophagosome. A single nucleotide polymorphism (SNP) in Atg16L1 was identified by genome-wide association studies (GWASs) as a risk factor in Crohn’s disease (CD), along with other autophagy-related genes including LRRK2, NOD2, and IRGM. These findings suggest that Atg16L1 exerts anti-inflammatory activities in the gastrointestinal (GI) tract. Atg16L1 in macrophages suppresses post-transcriptional maturation of IL-1β in response to TLR4 stimulation, and it regulates the granule exocytosis pathway in Paneth cells specialized in secreting antimicrobial peptides in the GI tract. In addition, it promotes clearance of phagocytosed bacteria in epithelial cells. We investigated how autophagy regulates the innate response to IL-1β. We found that the IL-1β signal transduction pathways are significantly amplified in Atg16L1- or Atg5-deficient mouse embryonic fibroblasts (MEF). Accumulated p62 in autophagy-deficient cells was solely responsible for the amplified IL-1β signaling. We demonstrated that in addition to the autolysosomal degradative pathway, p62 is also degraded by the ubiquitination proteasomal system (UPS). Our genetic and biochemical analyses revealed that Cullin-3 is the E3 ubiquitin ligase of p62, and that Atg16L1 mediates neddylation of Cullin-3, a critical prerequisite for its activation. Taken together, we provided a novel mechanism by which Atg16L1 suppresses a proinflammatory signal.

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-kappaB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IkappaBalpha degradation and activation of the NF-kappaB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IkappaB accumulates in the cytoplasm preventing NF-kappaB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-kappaB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

We evaluated gamma-irradiated Listeria monocytogenes as a killed bacterial vaccine, testing the hypothesis that irradiation preserves antigenic and adjuvant structures destroyed by traditional heat or chemical inactivation. Irradiated Listeria monocytogenes (LM), unlike heat-killed LM, efficiently activated dendritic cells via Toll-like receptors and induced protective T cell responses in mice. Like live LM, irradiated LM induced Toll-like-receptor-independent T cell priming. Cross-presentation of irradiated listerial antigens to CD8(+) T cells involved TAP- and proteasome-dependent cytosolic antigen processing. These results establish that killed LM can induce protective T cell responses, previously thought to require live infection. gamma-irradiation may be potentially applied to numerous bacterial vaccine candidates, and irradiated bacteria could serve as a vaccine platform for recombinant antigens derived from other pathogens, allergens, or tumors.

Background Airway remodeling in asthma is associated with angiogenesis. Objective We have examined whether immunostimulatory sequences of DNA (ISSs) inhibit allergen-induced airway angiogenesis and expression of angiogenic cytokines in a mouse model of airway remodeling. Methods Mice sensitized to ovalbumin were challenged repetitively with ovalbumin for three months to develop airway remodeling and angiogenesis. Levels of angiogenesis were compared in ISS-treated and control mice. Results Mice challenged with ovalbumin developed significantly increased levels of peribronchial angiogenesis (increase in the number of CD31+ peribronchial small blood vessels) and an increase in the peribronchial vascular area as assessed by image analysis. Ovalbumin-induced peribronchial angiogenesis was associated with increased bronchoalveolar lavage levels of vascular endothelial growth factor (VEGF) and an increase in the number of peribronchial cells expressing VEGF. Treatment of mice with ISS before repetitive ovalbumin challenge significantly reduced the levels of peribronchial angiogenesis as well as the levels of bronchoalveolar lavage VEGF and the number of peribronchial cells expressing VEGF. ISS is unlikely to act directly on endothelial cells to inhibit angiogenesis because lung endothelial cells did not express Toll receptor 9, the receptor for ISS as assessed by RT-PCR. In vitro studies demonstrated that ISS inhibited macrophage expression of VEGF. Conclusion The ability of ISS to inhibit angiogenesis in vivo is likely to be mediated by several mechanisms, including ISS reducing the number of peribronchial inflammatory cells that express VEGF, ISS inhibiting expression of TH2 cytokines such as IL-13 that promote VEGF expression, and direct effects of ISS on macrophages to inhibit VEGF expression.

Background Matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor of metalloproteinase 1 (TIMP-1) are hypothesized to play a role in the pathogenesis of airway remodeling in asthma. Objective We have used a mouse model of airway remodeling to determine the pattern of expression of MMP-9 and TIMP-1 in airway epithelium and peribronchial cells, and assess whether TIMP-1, an inhibitor of MMP-9, is expressed at the same sites in the airway. In addition, we have investigated whether immunostimulatory sequences (ISSs) of DNA modulate levels of expression of MMP-9, TIMP-1, and peribronchial fibrosis. Methods Levels of lung MMP-9 and TIMP-1 were assessed by zymography, ELISA, and immunohistochemistry. Results Repetitive ovalbumin challenge induced a significant increase in levels of MMP-9, TIMP-1, and peribronchial collagen deposition. The pattern of expression of MMP-9 and TIMP-1 in the remodeled airway was significantly different. MMP-9 but not TIMP-1 was expressed in airway epithelium, whereas both MMP-9 and TIMP-1 were expressed in peribronchial inflammatory cells. ISS significantly reduced expression of MMP-9 in airway epithelium (which immunostained positive for Toll receptor 9), as well as in peribronchial inflammatory cells. In vitro studies demonstrated that ISS inhibited bone marrow macrophage generation of MMP-9. Conclusion Allergen-induced peribronchial fibrosis is associated with expression of MMP-9 and TIMP-1 at different anatomical sites in the remodeled airway. The ability of ISS to inhibit the expression of MMP-9 in airway epithelium (a site where its inhibitor TIMP-1 is not induced by allergen challenge) may be important in determining whether ISS contributes to reductions in airway remodeling by reducing levels of MMP-9. Clinical implications Immunostimulatory sequences of DNA, which are being investigated as novel therapeutics in asthma, inhibit airway remodeling in mice as well as epithelial expression of MMP-9, an enzyme that degrades the extracellular matrix proteins surrounding the airway.

Coccidioides immitis is endemic in the soil of the desert Southwest. It causes a respiratory infection that is usually mild, but can last months and may disseminate beyond the lung. Disseminated infections can be fatal or require life-long therapy. Development of an effective vaccine may be a successful method of preventing serious disease. In this paper, we show that immunostimulatory-oligodeoxynucleotides (ISS-ODN) are an effective adjuvant for a recombinant coccidioidal protein known as antigen 2/proline rich antigen. Protective immunity induced by this ISS-ODN-based vaccine requires IL-12, interferon-gamma and MHC Class II-restricted T-cells. Cytotoxic CD8 T-cells are not required. This study elucidates the mechanisms needed to elicit successful immunity against coccidioidomycosis, and holds promise for development of an effective coccidioidal vaccine against coccidioidomycosis.

The interactions between genetic and environmental factors play a major role in the development of childhood asthma. We hypothesized that a pre-existing Th2/asthmatic response can promote Th2 responses to newly encountered Ags (i.e., phenotype spread). To test this hypothesis, we developed a mouse model in which the requirements for the induction and inhibition of phenotype spread to a clinically relevant neo-allergen (i.e., ragweed) were investigated. Our results indicate that 1) phenotype spread to the neo-allergen can be induced only within the first 8 h after a bronchial challenge with the first Ag (OVA); 2) Th2 differentiation of naive CD4+ T cells occurs in bronchial lymph nodes; 3) trafficking of naive CD4+ T cells to local lymph nodes and IL-4 produced by OVA-activated Th2 cells play essential roles in the differentiation of naive CD4+ T cells to Th2 cells; and 4) suppression of the production of chemokines involved in the homing of naive CD4+ T and Th2 cells to bronchial lymph nodes by a TLR9 agonist inhibited phenotype spread and abrogated the consequent development of experimental asthma. These findings provide a mechanistic insight into Th2 phenotype spread and offer an animal model for testing relevant immunomodulatory interventions.

While effective for the prevention and treatment of allergic rhinitis (AR) symptoms, currently available medications do not reverse allergen specific hypersensitivities. Therefore, pharmacotherapeutics are not curative and their daily use is often required for years. These investigations were conducted to determine whether immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) delivery protects previously sensitized mice from AR hypersensitivity responses and modulates their allergen specific immune profiles. Mice were first sensitized with ovalbumin (OVA) and alum, twenty-four hr before beginning a series of seven daily intranasal (i.n.) allergen challenges, subsets of mice received a single i.n. or intradermal (i.d.) dose of ISS-ODN or control oligodeoxynucleotide (C-ODN), a single intraperitoneal (i.p.) injection of dexamethasone (DXM), or no intervention. Mice receiving i.d. or i.n. ISS-ODN were found to have attenuated immediate and late phase effector cell responses to i.n. OVA challenge. Specifically, ISS-ODN treated mice had less histamine and cysteinyl leukotriene release and eosinophilic inflammation in their nasal passages than mice treated with C-ODN. In addition, splenocytes from ISS-ODN but not C-ODN treated mice displayed attenuated OVA-specific interleukin (IL)-4, IL-5, and IL-13 but increased interferon-gamma responses. Finally, ISS-ODN was generally a more effective treatment than DXM, both in blunting AR hypersensitivity responses and in shifting T helper 2 Th2-biased immune parameters towards Th1 dominance. As ISS-ODN delivery rapidly attenuated effector cell responses in this AR model in an allergen independent manner, the present results suggest that therapy with ISS-ODN alone may be an effective alternative to corticosteroid medications for the clinical management of AR.

Epidemiological evidence points to the inverse relationship between microbial exposure and the prevalence of allergic asthma and autoimmune diseases in Westernized countries. The molecular basis for this observation has not yet been completely delineated. Here we report that the administration of certain toll-like receptor (TLR) ligands, via the activation of innate immunity, induces high levels of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism in various organs. TLR9 ligand-induced pulmonary IDO activity inhibits Th2-driven experimental asthma. IDO activity expressed by resident lung cells rather than by pulmonary DCs suppressed lung inflammation and airway hyperreactivity. Our results provide a mechanistic insight into the various formulations of the hygiene hypothesis and underscore the notion that activation of innate immunity can inhibit adaptive Th cell responses.

Recognition of microbial components by APCs and their activation through Toll-like receptors (TLR) leads to the induction of adaptive immune responses. In this study, we show that activation of TLR2 by its synthetic ligand Pam3Cys, in contrast to activation of TLR9 by immunostimulatory DNA (ISS-ODN), induces a prominent Th2-biased immune response. Activation of APCs by Pam3Cys resulted in the induction of Th2-associated effector molecules like IL-13, and IL-1β, GM-CSF and up-regulation of B7RP-1, but low levels of Th1-associated cytokines (IL-12, IFNα, IL-18, IL-27). Accordingly, TLR2 ligands aggravated experimental asthma. These data indicate that the type of TLR stimulation during the initial phase of immune activation determines the polarization of the adaptive immune response and may play a role in the initiation of Th2-mediated immune disorders, such as asthma.

Conjugation of protein antigen with immunostimulatory oligonucleotides creates a potent immunogen that elicits antigen-specific antibody responses, a Th1-biased cytokine profile, and CD8 cytotoxic T lymphocyte activity. The wide range of humoral and cellular immune responses induced by these conjugates suggests they can be used to create effective vaccines against infectious pathogens and tumors and to beneficially modulate the immune responses seen in allergic diseases. This review summarizes the available data on the use of antigen-oligonucleotide conjugates and discusses their potential use for the treatment of infectious, oncologic, and allergic diseases in humans.

The prevalence and severity of allergic diseases and other diseases of immune dysregulation are increasing in industrialized countries. One explanation for these trends is that decreased exposure to microbes, due to modern public health practices, has resulted in the loss of a main source of immune provocation, and a consequent increase in pathogenic immune responses and their associated diseases. It is now clear that molecular interactions between immunocytes and microbes are mediated largely by Toll-like receptors (TLRs) on host cells and a diversity of ligands produced by viruses, bacteria and fungi. Physiological exposures to TLR ligands are also likely to have an important yet complex role in host immune homeostasis and predisposition towards atopy.

Few peribronchial mast cells are noted either in the lungs of naive mice or in the lungs of OVA-sensitized mice challenged acutely with OVA by inhalation. In this study, we demonstrate that OVA-sensitized mice exposed to repetitive OVA inhalation for 1–6 mo have a significant accumulation of peribronchial mast cells. This accumulation of peribronchial mast cells is associated with increased expression of the Th2 cell-derived mast cell growth factors, including IL-4 and IL-9, but not with the non-Th2 cell-derived mast cell growth factor, stem cell factor. Pretreating mice with immunostimulatory sequences (ISS) of DNA containing a CpG motif significantly inhibited the accumulation of peribronchial mast cells and the expression of IL-4 and IL-9. To determine whether mast cells express Toll-like receptor-9 (TLR-9; the receptor for ISS), TLR-9 expression by mouse bone marrow-derived mast cells (MBMMCs) was assessed by RT-PCR. MBMMCs strongly expressed TLR-9 and bound rhodamine-labeled ISS. However, incubation of MBMMCs with ISS in vitro neither inhibited MBMMC proliferation nor inhibited Ag/IgE-mediated MBMMC degranulation, but they did induce IL-6. Overall these studies demonstrate that mice exposed to repetitive OVA challenge, but not acute OVA challenge, have an accumulation of peribronchial mast cells and express increased levels of mast cell growth factors in the lung. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro, suggesting that ISS inhibits accumulation of peribronchial mast cells in vivo by indirect mechanism(s), which include inhibiting the lung expression of Th2 cell-derived mast cell growth factors.