Immunotherapeutic Role of NOD-2 and TLR-4 Signaling as an Adjunct to Antituberculosis Chemotherapy.

Tuberculosis (TB) treatment is lengthy and inflicted with severe side-effects. Here, we attempted a novel strategy to reinforce host immunity through NOD-like receptor (NOD-2) and Toll-like receptor (TLR-4) signaling in the murine model of TB. Intriguingly, we noticed that it not only bolstered the immunity but also reduced the dose and duration of rifampicin and isoniazid therapy. Further, we observed expansion in the pool of effector (CD44hi, CD62Llo, CD127hi) and central (CD44hi, CD62Lhi, CD127hi) memory CD4 T cells and CD8 T cells and increased the intracellular killing of Mycobacterium tuberculosis (Mtb) by activated dendritic cells [CD86hi, CD40hi, IL-6hi, IL-12hi, TNF-αhi, nitric oxide (NO)hi] with significant reduction in Mtb load in the lungs and spleen of infected animals. We infer that the signaling through NOD-2 and TLR-4 may be an important approach to reduce the dose and duration of the drugs to treat TB.

Are Pattern Recognition Receptors Associated with Hepatocellular Carcinoma?

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the important causes of mortality due to malignancy. Toll-like receptors (TLRs) are very important in liver pathophysiology in terms of their roles in the innate immune system, such as the regulation of inflammation, wound healing, stimulation of adaptive immune responses, promotion of epithelial regeneration, and carcinogenesis. In this study, we planned to examine the role of TLR1 (rs4833095, rs5743551) and nucleotide-binding oligomerization domain (NOD2) (rs2066844, rs2066845, rs2066847) polymorphisms in the development of HCC and their effects on the clinical presentation of HCC patients. METHODS: Our study was designed prospectively. Cirrhotic and HCC patients who were followed up in our clinic between January 2015 and September 2018 were included in the study. Sex, age, cirrhosis etiology, Child-Pugh class, and MELD scores were recorded. TLR1 and NOD2 polymorphisms were studied by the PCR method. RESULTS: HCC developed in 88 (31.4%) of the 280 patients who were followed up, either during the recruitment phase of our study or during the follow-up. The mean follow-up time of our patient group was 17.04 ± 11.72 months, and the mean follow-up time of HCC patients was 12.09 ± 10.26 months. TLR1 (rs5743551) polymorphism was associated with HCC development (P = .003). TLR1 (rs5743551) and NOD2 (rs2066844) polymorphisms were associated with the development of spontaneous bacterial peritonitis (SBP) in the HCC patient group (P = .013 and P = .021, respectively). CONCLUSION: We think that increased bacterial translocation in cirrhotic patients may contribute to HCC development by causing chronic inflammation, especially in patients with TLR 1 (rs5743551) polymorphism.

NOD1 in the interplay between microbiota and gastrointestinal immune adaptations.

Nucleotide-binding oligomerization domain 1 (NOD1), a pattern recognition receptor (PRR) that detects bacterial peptidoglycan fragments and other danger signals, has been linked to inflammatory pathologies. NOD1, which is expressed by immune and non-immune cells, is activated after recognizing microbe-associated molecular patterns (MAMPs). This recognition triggers host defense responses and both immune memory and tolerance can also be achieved during these processes. Since the gut microbiota is currently considered a master regulator of human physiology central in health and disease and the intestine metabolizes a wide range of nutrients, drugs and hormones, it is a fact that dysbiosis can alter tissues and organs homeostasis. These systemic alterations occur in response to gastrointestinal immune adaptations that are not yet fully understood. Even if previous evidence confirms the connection between the microbiota, the immune system and metabolic disorders, much remains to be discovered about the contribution of NOD1 to low-grade inflammatory pathologies such as obesity, diabetes and cardiovascular diseases. This review compiles the most recent findings in this area, while providing a dynamic and practical framework with future approaches for research and clinical applications on targeting NOD1. This knowledge can help to rate the consequences of the disease and to stratify the patients for therapeutic interventions.

Influence of NOD-like receptor 2 gene polymorphisms on muramyl dipeptide induced pro-inflammatory response in patients with active pulmonary tuberculosis and household contacts.

PURPOSE: The immune response induced by nucleotide-binding oligomerization domain-2(NOD2) is associated with the production of cytokines affected by the host's genetic background. The present study aimed to examine the effects of NOD2; 802C > T, 2105G > A polymorphisms associated with altered cytokine levels in patients with active pulmonary tuberculosis disease, Latent TB subjects (household contacts(HHC) and healthy controls(HC). METHODS: Genetic polymorphisms were analyzed by Restriction Fragment Length Polymorphism(RFLP) in 102-PTB patients, 102-HHC, and 132-HC. QuantiFERON-TB Gold In-Tube test was performed to identify latent TB infection in 60-HHC. Estimated their cytokine levels by ELISA in MDP (muramyl dipeptide) stimulated culture supernatants of all the groups. Further, we studied pre-mRNA structures by insilico analysis and relative gene expression by RT-PCR. RESULTS: Recessive genetic models of NOD2 802C > T SNP with TT genotype and AA genotype of NOD2 2105G > A SNP were significantly associated with increased TB risk in PTB patients and HHC compared with HC. In vitro stimulations were performed with NOD2 ligand MDP in PTB patients and latent TB subjects: QuantiFERON positive household contacts (QFT + ve HHC)and QuantiFERON negative household contacts(QFT-ve HHC). The results showed that reduced TNF-α and enhanced IL-12, IL-1ß indicate that these cytokines may play an essential role in the initial maintenance of cell-mediated immunity. Our study demonstrated the correlation between NOD2 polymorphism with IL-1ß, TNF-α, IL-12 levels. Insilico analysis represents the pre-mRNA secondary structures affected by NOD2 SNPs. We also observed the difference in m RNA levels in variant and wild genotypes. CONCLUSION: This finding may lead to the forthcoming development of immunotherapy and may be used as predictive markers to identify high-risk individuals for TB disease.

IL-6 effector function of group 2 innate lymphoid cells (ILC2) is NOD2 dependent.

Cutaneous group 2 innate lymphoid cells (ILC2) are spatially and epigenetically poised to respond to barrier compromise and associated immunological threats. ILC2, lacking rearranged antigen-specific receptors, are primarily activated by damage-associated cytokines and respond with type 2 cytokine production. To investigate ILC2 potential for direct sensing of skin pathogens and allergens, we performed RNA sequencing of ILC2 derived from in vivo challenged human skin or blood. We detected expression of NOD2 and TLR2 by skin and blood ILC2. Stimulation of ILC2 with TLR2 agonist alone not only induced interleukin-5 (IL-5) and IL-13 expression but also elicited IL-6 expression in combination with Staphylococcus aureus muramyl dipeptide (MDP). Heat-killed skin-resident bacteria provoked an IL-6 profile in ILC2 in vitro that was notably impaired in ILC2 derived from patients with nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutations. In addition, we show that NOD2 signaling can stimulate autophagy in ILC2, which was also impaired in patients with NOD2 mutations. Here, we have identified a role for ILC2 NOD2 signaling in the differential regulation of ILC2-derived IL-6 and have reported a previously unrecognized pathway of direct ILC2 bacterial sensing.

NOD2 drives early IL-33-dependent expansion of group 2 innate lymphoid cells during Crohn's disease-like ileitis.

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn's disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., the SAMP1/YitFc (SAMP) mouse strain, we showed that ILC2s, compared with ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP versus AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from CD patients versus healthy controls were also increased and expanded, similarly to ILC1s, in greater proportion compared with ILC3s. Importantly, we report that the intracellular bacteria-sensing protein, nucleotide-binding oligomerization domaining-containing protein 2, encoded by Nod2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP mice lacking NOD2 and in SAMP mice raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.

T cell-intrinsic role for Nod2 in protection against Th17-mediated uveitis.

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.

NOD2 receptor is crucial for protecting against the digestive form of Chagas disease.

Digestive and cardiodigestive forms of Chagas' disease are observed in 2% to 27% of the patients, depending on their geographic location, Trypanosoma cruzi strain and immunopathological responses. The aim of this work was to evaluate the role of NOD2 innate immune receptor in the pathogenesis of the digestive system in Chagas' disease. Patients with digestive form of the disease showed lower mRNA expression of NOD2, higher expression of RIP2 and α-defensin 6, compared to indeterminate form, detected by Real-time PCR in peripheral blood mononuclear cells. In addition, there was a negative correlation between the expression of NOD2 and the degree of dilation of the esophagus, sigmoid and rectum in those patients. The infection of NOD2-/- mice with T. cruzi strain isolated from the digestive patient induced a decrease in intestinal motility. Histopathological analysis of the colon and jejunum of NOD2-/- and wild type C57BL/6 animals revealed discrete inflammatory foci during the acute phase of infection. Interestingly, during the chronic phase of the infection there was inflammation and hypertrophy of the longitudinal and circular muscular layer more pronounced in the colon and jejunum from NOD2-/- animals, when compared to wild type C57BL/6 mice. Together, our results suggest that NOD2 plays a protective role against the development of digestive form of Chagas' disease.

Lessons from Bacillus Calmette-Guérin: Harnessing Trained Immunity for Vaccine Development.

Vaccine design traditionally focuses on inducing adaptive immune responses against a sole target pathogen. Considering that many microbes evade innate immune mechanisms to initiate infection, and in light of the discovery of epigenetically mediated innate immune training, the paradigm of vaccine design has the potential to change. The Bacillus Calmette-Guérin (BCG) vaccine induces some level of protection against Mycobacterium tuberculosis (Mtb) while stimulating trained immunity that correlates with lower mortality and increased protection against unrelated pathogens. This review will explore BCG-induced trained immunity, including the required pathways to establish this phenotype. Additionally, potential methods to improve or expand BCG trained immunity effects through alternative vaccine delivery and formulation methods will be discussed. Finally, advances in new anti-Mtb vaccines, other antimicrobial uses for BCG, and "innate memory-based vaccines" will be examined.

New chimeric TLR7/NOD2 agonist is a potent adjuvant to induce mucosal immune responses.

BACKGROUND: PRR (Pattern Recognition Receptor) agonists have been widely tested as potent vaccine adjuvants. TLR7 (Toll-Like Receptor 7) and NOD2 (nucleotide-binding oligomerization domain 2) are key innate receptors widely expressed at mucosal levels. METHODS: Here, we evaluated the immunostimulatory properties of a novel hybrid chemical compound designed to stimulate both TLR7 and NOD2 receptors. FINDING: The combined TLR7/NOD2 agonist showed increase efficacy than TLR7L or NOD2L agonists alone or combined in different in vitro models. Dual TLR7/NOD2 agonist efficiently stimulates TLR7 and NOD2, and promotes the maturation and reprogramming of human dendritic cells, as well as the secretion of pro-inflammatory or adaptive cytokines. This molecule also strongly induces autophagy in human cells which is a major intracellular degradation system that delivers cytoplasmic constituents to lysosomes in both MHC class I and II-restricted antigen presentation. In vivo, TLR7/NOD2L agonist is a potent adjuvant after intranasal administration with NP-p24 HIV vaccine, inducing high-quality humoral and adaptive responses both in systemic and mucosal compartments. Use of TLR7/NOD2L adjuvant improves very significantly the protection of mice against an intranasal challenge with a vaccinia virus expressing the p24. INTERPRETATION: Dual TLR7/NOD2L agonist is a very potent and versatile vaccine adjuvant and promote very efficiently both systemic and mucosal immunity. FUNDING: This work was supported by Sidaction.

Synergistic Activation of Toll-Like and NOD Receptors by Complementary Antigens as Facilitators of Autoimmune Disease: Review, Model and Novel Predictions.

Persistent activation of toll-like receptors (TLR) and nucleotide-binding oligomerization domain-containing proteins (NOD) in the innate immune system is one necessary driver of autoimmune disease (AD), but its mechanism remains obscure. This study compares and contrasts TLR and NOD activation profiles for four AD (autoimmune myocarditis, myasthenia gravis, multiple sclerosis and rheumatoid arthritis) and their animal models. The failure of current AD theories to explain the disparate TLR/NOD profiles in AD is reviewed and a novel model is presented that explains innate immune support of persistent chronic inflammation in terms of unique combinations of complementary AD-specific antigens stimulating synergistic TLRs and/or NODs. The potential explanatory power of the model is explored through testable, novel predictions concerning TLR- and NOD-related AD animal models and therapies.

A Specific Strain of Lactic Acid Bacteria, Lactobacillus paracasei, Inhibits Inflammasome Activation In Vitro and Prevents Inflammation-Related Disorders.

Some strains of lactic acid bacteria (LAB) have anti-inflammatory effects, but the mechanism underlying the alleviation of inflammation by LAB is not fully understood. In this study, we examined the inhibitory effect of a certain strain of LAB, Lactobacillus paracasei, on inflammasome activation, which is associated with various inflammatory disorders. Using bone marrow-derived macrophages from BALB/c mice, we found that L. paracasei, but not L. rhamnosus, suppressed NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1ß secretion. L. paracasei also had inhibitory effects on AIM2 and NLRC4 inflammasome activation as well as the NLRP3 inflammasome. These inhibitory effects of L. paracasei on inflammasome activation were dependent on autocrine IL-10 induced by L. paracasei-stimulated macrophages. Furthermore, IL-10 production by L. paracasei-stimulated macrophages was involved with phagocytosis and the NOD2 signaling pathway in macrophages. In addition to in vitro studies, oral administration of L. paracasei in C57BL/6 mice reduced monosodium urate crystal-induced peritoneal inflammation in vivo. Moreover, continuous intake of L. paracasei in C57BL/6 mice alleviated high fat diet-induced insulin resistance and aging-induced expression of biomarkers for T cell senescence. Taken together, we demonstrated that L. paracasei inhibits inflammasome activation in vitro and exhibits an anti-inflammatory function in vivo. These results indicate that LAB that have inhibitory effects on inflammasome activation might contribute to the alleviation of inflammation-related disorders.

Collagen Peptides Isolated from Salmo salar and Tilapia nilotica Skin Accelerate Wound Healing by Altering Cutaneous Microbiome Colonization via Upregulated NOD2 and BD14.

Collagen peptides can promote wound healing and are closely related to microbiome colonization. We investigated the relationship among collagen peptides, wound healing, and wound microflora colonization by administering the murine wound model with Salmo salar skin collagen peptides (Ss-SCPs) and Tilapia nilotica skin collagen peptides (Tn-SCPs). We analyzed the vascular endothelial growth factor (VEGF), fibroblast growth factors (ß-FGF), pattern recognition receptor (NOD2), antimicrobial peptides (ß-defence14, BD14), proinflammatory (TNF-α, IL-6, and IL-8) and anti-inflammatory (IL-10) cytokines, macrophages, neutrophil infiltration levels, and microbial communities in the rat wound. The healing rates of the Ss-SCP- and Tn-SCP-treated groups were significantly accelerated, associated with decreased TNF-α, IL-6, and IL-8 and upregulated BD14, NOD2, IL-10, VEGF, and ß-FGF. Accelerated healing in the collagen peptide group shows that the wound microflora such as Leuconostoc, Enterococcus, and Bacillus have a positive effect on wound healing (P < 0.01). Other microbiome species such as Stenotrophomonas, Bradyrhizobium, Sphingomonas, and Phyllobacterium had a negative influence and decreased colonization (P < 0.01). Altogether, these studies show that collagen peptide could upregulate wound NOD2 and BD14, which were implicated in microflora colonization regulation in the wound tissue and promoted wound healing by controlling the inflammatory reaction and increasing wound angiogenesis and collagen deposition.

First-in-human phase I clinical trial of the NY-ESO-1 protein cancer vaccine with NOD2 and TLR9 stimulants in patients with NY-ESO-1-expressing refractory solid tumors.

Cholesteryl pullulan (CHP) is a novel antigen delivery system. CHP and New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen complexes (CHP-NY-ESO-1) present multiple epitope peptides to the MHC class I and II pathways. Adjuvants are essential for cancer vaccines. MIS416 is a non-toxic microparticle that activates immunity via the nucleotide-binding oligomerization domain 2 (NOD2) and TLR9 pathways. However, no reports have explored MIS416 as a cancer vaccine adjuvant. We conducted a first-in-human clinical trial of CHP-NY-ESO-1 with MIS416 in patients with NY-ESO-1-expressing refractory solid tumors. CHP-NY-ESO-1/MIS416 (µg/µg) was administered at 100/200, 200/200, 200/400 or 200/600 (cohorts 1, 2, 3 and 4, respectively) every 2 weeks for a total of 6 doses (treatment phase) followed by one vaccination every 4 weeks until disease progression or unacceptable toxicity (maintenance phase). The primary endpoints were safety and tolerability, and the secondary endpoint was the immune response. In total, 26 patients were enrolled. Seven patients (38%) continued vaccination in the maintenance phase. Grade 3 drug-related adverse events (AEs) were observed in six patients (23%): anorexia and hypertension were observed in one and five patients, respectively. No grade 4-5 drug-related AEs were observed. Eight patients (31%) had stable disease (SD). Neither augmentation of the NY-ESO-1-specific IFN-γ-secreting CD8+ T cell response nor an increase in the level of anti-NY-ESO-1 IgG1 was observed as the dose of MIS416 was increased. In a preclinical study, adding anti-PD-1 monoclonal antibody to CHP-NY-ESO-1 and MIS416 induced significant tumor suppression. This combination therapy is a promising next step.

Pattern Recognition Receptor-reactivity Screening of Liver Transplant Patients: Potential for Personalized and Precise Organ Matching to Reduce Risks of Ischemia-reperfusion Injury.

OBJECTIVE AND BACKGROUND: Pattern recognition receptors (PRRs) on immune and parenchymal cells can detect danger-associated molecular patterns (DAMPs) released from cells damaged during ischemia-reperfusion injury (IRI), in heart attack or stroke settings, but also as an unavoidable consequence of solid organ transplantation. Despite IRI being a significant clinical problem across all solid organ transplants, there are limited therapeutics and patient-specific diagnostics currently available. METHODS: We screened portal blood samples obtained from 67 human liver transplant recipients both pre- [portal vein (PV) sample] and post-(liver flush; LF) reperfusion for their ability to activate a panel of PRRs, and analyzed this reactivity in relation to biopsy-proven IRI. RESULTS: PV samples from IRI+ orthotopic liver transplantation (OLT) patients (n = 35) decreased activation of hTLR4- and hTLR9-transfected cells, whereas PV from IRI- patients (n = 32) primarily increased hTLR7 and hNOD2 activation. LF samples from OLT-IRI patients significantly increased activation of hTLR4 and hTLR9 over IRI- LF. In addition, the change from baseline reactivity to hTLR4/9/NOD2 was significantly higher in IRI+ than IRI- OLT patients. CONCLUSIONS: These results demonstrate that TLR4/7/9 and NOD2 are involved in either promoting or attenuating hepatic IRI, and suggest a diagnostic screening of portal blood for reactivity to these PRRs might prove useful for prediction and/or therapeutic intervention in OLT patients before transplantation.

Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide.

Peptide transporters 1 and 2 (PEPT1 and PEPT2) are proton-coupled oligopeptide transporter members of the solute carrier 15 family and play a role in the cellular uptake of di/tri-peptides and peptidomimetics. Our previous work showed that PEPT2 is predominantly expressed within undifferentiated keratinocytes. Here we show that PEPT2 expression decreases as keratinocyte differentiation progresses and that PEPT1 alternately is expressed at later stages. Absolute quantification using quantitative polymerase chain reaction revealed that the expression level of PEPT1 is about 17 times greater than that of PEPT2. Immunohistochemical study of human skin provided evidence of PEPT1 in the epidermis. The uptake of glycylsarcosine into keratinocytes was significantly blocked by PEPT inhibitors, including nateglinide and glibenclamide. Moreover, we found that PEPT1 knockdown in differentiated keratinocytes significantly suppressed the influence of a bacterial-derived peptide, muramyl dipeptide (MDP), on the production of proinflammatory cytokine interleukin-8, implying that bacteria-derived oligopeptides can be transported by PEPT1 in advanced differentiated keratinocytes. Taken together, PEPT1 and PEPT2 may concertedly play an important role in MDP-NOD2 signaling in the epidermis, which provides new insight into the mechanisms of skin homeostasis against microbial pathogens.

Nicotine modulates molecules of the innate immune response in epithelial cells and macrophages during infection with M. tuberculosis.

Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll-like receptors (TLR)-2, TLR-4 and NOD-2 in all three cell types, as well as the production of the SP-D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)-6 and C-C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL-8, IL-6, tumor necrosis factor (TNF)-α, IL-10, CCL2, C-X-C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP-D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.

LACC1 Required for NOD2-Induced, ER Stress-Mediated Innate Immune Outcomes in Human Macrophages and LACC1 Risk Variants Modulate These Outcomes.

LACC1 genetic variants are associated with multiple immune-mediated diseases. However, laccase domain containing-1 (LACC1) functions are incompletely defined. We find that upon stimulation of the pattern-recognition receptor (PRR) NOD2, LACC1 localizes to the endoplasmic reticulum (ER) and forms a complex with ER-stress sensors. All three ER-stress branches, PERK, IRE1α, and ATF6, are required for NOD2-induced signaling, cytokines, and antimicrobial pathways in human macrophages. LACC1, and its localization to the ER, is required for these outcomes. Relative to wild-type (WT) LACC1, transfection of the common Val254 and rare Arg284 immune-mediated disease-risk LACC1 variants into HeLa cells and macrophages, as well as macrophages from LACC1 Val254 carriers, shows reduced NOD2-induced ER stress-associated outcomes; these downstream outcomes are restored by rescuing ER stress. Therefore, we identify ER stress to be essential in PRR-induced outcomes in macrophages, define a critical role for LACC1 in these ER stress-dependent events, and elucidate how LACC1 disease-risk variants mediate these outcomes.

Recognition of the microbiota by Nod2 contributes to the oral adjuvant activity of cholera toxin through the induction of interleukin-1ß.

The role of symbiotic bacteria in the development of antigen-specific immunity remains poorly understood. Previous studies showed that sensing of symbiotic bacteria by nucleotide-binding oligomerization domain-containing protein 2 (Nod2) regulates antibody responses in response to nasal immunization with antigen and cholera toxin (CT). In this study, we examined the role of the microbiota in the adjuvant activity of CT induced after oral immunization with antigen. Germ-free (GF) mice showed impaired production of antibody responses and T-cell-specific cytokines after oral immunization when compared with that observed in conventionally raised mice. Similar to GF mice, Nod2-deficient mice showed reduced humoral responses upon oral immunization with antigen and CT. Treatment with CT enhanced the production of interleukin-1ß (IL-1ß), but not tumor necrosis factor-α or IL-12p40, induced by stimulation of dendritic cells with muramyl dipeptide, the Nod2 ligand. Mechanistically, the enhanced production of IL-1ß induced by muramyl dipeptide and CT stimulation required Nod2 and was mediated by both increased synthesis of pro-IL-1ß and caspase-1 activation. Furthermore, antigen-specific antibody and cytokine responses induced by CT were impaired in orally immunized IL-1ß-deficient mice. Collectively, our results indicate that Nod2 stimulation by symbiotic bacteria contributes to optimal CT-mediated antigen-specific oral vaccination through the induction of IL-1ß production.

Ataxin-3 Links NOD2 and TLR2 Mediated Innate Immune Sensing and Metabolism in Myeloid Cells.

The interplay between NOD2 and TLR2 following recognition of components of the bacterial cell wall peptidoglycan is well-established, however their role in redirecting metabolic pathways in myeloid cells to degrade pathogens and mount antigen presentation remains unclear. We show NOD2 and TLR2 mediate phosphorylation of the deubiquitinase ataxin-3 via RIPK2 and TBK1. In myeloid cells ataxin-3 associates with the mitochondrial cristae protein MIC60, and is required for oxidative phosphorylation. Depletion of ataxin-3 leads to impaired induction of mitochondrial reactive oxygen species (mROS) and defective bacterial killing. A mass spectrometry analysis of NOD2/TLR2 triggered ataxin-3 deubiquitination targets revealed immunometabolic regulators, including HIF-1α and LAMTOR1 that may contribute to these effects. Thus, we define how ataxin-3 plays an essential role in NOD2 and TLR2 sensing and effector functions in myeloid cells.