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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., (© 2019 British Society for Immunology.)

Aims: NOD2 and CD1d play a key role in innate immunity by recognizing conserved molecular patterns of pathogens. While NOD2-/- and CD1d-/- mice display structural and functional alterations in Paneth cells, animal studies have reported no impact of NOD2 or CD1d deficiency on experimental colitis. NOD2 mutations increase the susceptibility to inflammatory bowel diseases and the CD1d bound to α-galactosylceramide [α-GalCer] alleviates intestinal inflammation. We evaluated the effect of CD1d modulation on experimental colitis in NOD2-/- mice., Methods: The effect of CD1d augmentation and depletion in NOD2-/- mice was assessed in a dextran sodium sulphate [DSS]-induced colitis model via administration of α-GalCer and construction of NOD2-/-CD1d-/- mice. The structural and functional changes in Paneth cells were evaluated using transmission electron microscopy and pilocarpine administration. Colitogenic taxa were analysed in the faeces of NOD2-/-CD1d-/- mice using 16S rRNA gene sequencing., Results: In NOD2-/- mice, α-GalCer alleviated and CD1d depletion [NOD2-/-CD1d-/- mice] aggravated colitis activity and histology compared with co-housed littermates NOD2-/-, CD1d-/- and wild-type mice after administration of 3% DSS. In NOD2-/-CD1d-/- mice, the ultrastructure and degranulation ability of secretary granules in Paneth cells were altered and the intestinal microbial composition differed from that of their littermates. Faecal microbiota transplantation [FMT] with NOD2-/-CD1d-/- mice faeces into wild-type mice aggravated DSS-induced colitis, while FMT with wild-type mice faeces into NOD2-/-CD1d-/- mice alleviated DSS-induced colitis. Acetatifactor muris was identified only in NOD2-/-CD1d-/- mice faeces and the oral gavage of A. muris in wild-type mice aggravated DSS-induced colitis., Conclusion: CD1d modulates colonic inflammation in NOD2-/- mice by altering the intestinal microbial composition comprising A. muris., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mannose-capped lipoarabinomannan (ManLAM), present in all members of the Mycobacterium tuberculosis complex and in other pathogenic Mycobacterium spp, is a high molecular mass amphipathic lipoglycan with a defined critical role in mycobacterial survival during infection. In particular, ManLAM is well-characterized for its importance in providing M. tuberculosis a safe portal of entry to phagocytes, regulating the intracellular trafficking network, as well as immune responses of infected host cells. These ManLAM immunological characteristics are thought to be linked to the subtle but unique and well-defined structural characteristics of this molecule, including but not limited to the degree of acylation, the length of the D-mannan and D-arabinan cores, the length of the mannose caps, as well as the presence of other acidic constituents such as succinates, lactates and/or malates, and also the presence of 5-methylthioxylosyl. The impact of all these structural features on ManLAM spatial conformation and biological functions during M. tuberculosis infection is still uncertain. In this review, we dissect the relationship between ManLAM structure and biological function addressing how this relationship determines M. tuberculosis interactions with host cells, and how it aids this exceptional pathogen during the course of infection.

The innate immune system is critical for clearing infection, and is tightly regulated to avert excessive tissue damage. Nod1/2-Rip2 signaling, which is essential for initiating the innate immune response to bacterial infection and ER stress, is subject to many regulatory mechanisms. In this study, we found that LRRK2, encoded by a gene implicated in Crohn's disease, leprosy and familial Parkinson's disease, modulates the strength of Nod1/2-Rip2 signaling by enhancing Rip2 phosphorylation. LRRK2 deficiency markedly reduces cytokine production in macrophages upon Nod2 activation by muramyl dipeptide (MDP), Nod1 activation by D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) or ER stress. Our biochemical study shows that the presence of LRRK2 is necessary for optimal phosphorylation of Rip2 upon Nod2 activation. Therefore, this study reveals that LRRK2 is a new positive regulator of Rip2 and promotes inflammatory cytokine induction through the Nod1/2-Rip2 pathway.

Salmonellosis or Salmonella, one of the most common food-borne diseases, remains a major public health problem worldwide. Intestinal epithelial cells (IECs) play an essential role in the mucosal innate immunity of the host to defend against the invasion of Salmonella by interleukin (IL)-8 and human β-defensin-2 (hBD-2). Accumulated research has unravelled important roles of vitamin D in the regulation of innate immunity. Therefore, we investigated the effects of 1,25-dihydroxyvitamin D3 (1,25D3) on Salmonella-induced innate immunity in IECs. We demonstrate that pretreatment of 1,25D3 results in suppression of Salmonella-induced IL-8 but enhancement of hBD-2, either protein secretion and mRNA expression, in IECs. Furthermore, 1,25D3 enhanced Salmonella-induced membranous recruitment of nucleotide oligomerization domain (NOD2) and its mRNA expression and activation of protein kinase B (Akt), a downstream effector of phosphoinositide 3-kinase (PI3K). Inhibition of the PI3K/Akt signal counteracted the suppressive effect of 1,25D3 on Salmonella-induced IL-8 expression, while knock-down of NOD2 by siRNA diminished the enhanced hBD-2 expression. These data suggest differential regulation of 1,25D3 on Salmonella-induced IL-8 and hBD-2 expression in IECs via PI3K/Akt signal and NOD2 protein expression, respectively. Active vitamin D-enhanced anti-microbial peptide in Salmonella-infected IECs protected the host against infection, while modulation of proinflammatory responses by active vitamin D prevented the host from the detrimental effects of overwhelming inflammation. Thus, active vitamin D-induced innate immunity in IECs enhances the host's protective mechanism, which may provide an alternative therapy for invasive Salmonella infection., (© 2016 British Society for Immunology.)

BCG vaccination is associated with a reduced mortality from nonmycobacterial infections. This is likely to be mediated by a combination of innate-immune memory ("trained immunity") and heterologous effects on adaptive immunity. As such, BCG could be used to boost host immunity but not in immunocompromised hosts, as it is a live, attenuated vaccine. Therefore, we assessed whether killed γBCG has similar potentiating effects. In an in vitro model of trained immunity, human monocytes were incubated with γBCG for 24 h and restimulated after 6 d. Cytokine production and the role of pattern recognition receptors and histone methylation markers were assessed. The in vivo effects of γBCG vaccination were studied in a proof-of-principle trial in 15 healthy volunteers. γBCG induced trained immunity in vitro via the NOD2 receptor pathway and up-regulation of H3K4me3 histone methylation. However, these effects were less strong than those induced by live BCG. γBCG vaccination in volunteers had only minimal effects on innate immunity, whereas a significant increase in heterologous Th1/Th17 immunity was observed. Our results indicate that γBCG induces long-term training of innate immunity in vitro. In vivo, γBCG induces mainly heterologous effects on the adaptive-immune system, whereas effects on innate cytokine production are limited., (© Society for Leukocyte Biology.)

M.tb, which causes TB, is a host-adapted intracellular pathogen of macrophages. Macrophage intracellular PRRs, such as NOD proteins, regulate proinflammatory cytokine production in response to various pathogenic organisms. We demonstrated previously that NOD2 plays an important role in controlling the inflammatory response and viability of M.tb and Mycobacterium bovis BCG in human macrophages. Various inflammatory mediators, such as cytokines, ROS, and RNS, such as NO, can mediate this control. iNOS (or NOS2) is a key enzyme for NO production and M.tb control during infection of mouse macrophages; however, the role of NO during infection of human macrophages remains unclear, in part, as a result of the low amounts of NO produced in these cells. Here, we tested the hypothesis that activation of NOD2 by its ligands (MDP and GMDP, the latter from M.tb) plays an important role in the expression and activity of iNOS and NO production in human macrophages. We demonstrate that M.tb or M. bovis BCG infection enhances iNOS expression in human macrophages. The M.tb-induced iNOS expression and NO production are dependent on NOD2 expression during M.tb infection. Finally, NF-κB activation is required for NOD2-dependent expression of iNOS in human macrophages. Our data provide evidence for a new molecular pathway that links activation of NOD2, an important intracellular PRR, and iNOS expression and activity during M.tb infection of human macrophages., (© Society for Leukocyte Biology.)

Background: PEPT1 was proposed to be expressed only in inflamed colonic tissues in which it could contribute to inflammatory bowel disease (IBD) development by transporting bacterial peptides, such as muramyl dipeptide (MDP), that activate intracellular pattern recognition receptors, such as the nucleotide-binding and oligomerization domain 2. To better define the pathological relevance of this transporter, we analyzed PEPT1 expression during intestinal inflammation and studied the susceptibility of Pept1-deficient (Pept1) mice to experimental colitis., Methods: Wild-type and Pept1 mice were treated with dextran sulfate sodium and 2,4,6-trinitrobenzene sulfonic acid to induce colitis, and MDP-induced cytokine expression was studied in colonic tissue cultures. PEPT1 expression was characterized in mouse models of Crohn's disease-like ileitis (Tnf) or colitis (Il-10, Il-10XTlr2) and endoscopic tissue samples from descending colon of patients with IBD (n = 11) and controls (n = 17). Moreover, the prevalence of the PEPT1 single-nucleotide polymorphism rs2297322 was tested in German patients with IBD (n = 458) and controls (n = 452)., Results: PEPT1 expression was consistently reduced under condition of acute or chronic experimental inflammation. Wild-type and Pept1 mice revealed comparable susceptibility to dextran sulfate sodium-induced and 2,4,6-trinitrobenzene sulfonic acid-induced colitis, and MDP-induced cytokine expression was PEPT1-independent. PEPT1 expression levels were also decreased in descending colon of patients with IBD during acute inflammation, but the rs2297322 single-nucleotide polymorphism was not associated with IBD susceptibility in the German cohort., Conclusions: PEPT1 expression is reduced during intestinal inflammation and PEPT1 is neither required for MDP-induced immune response nor is the PEPT1 rs2297322 single-nucleotide polymorphism associated with IBD susceptibility in our German cohort. These data strongly argue against a primary role of PEPT1 in the initiation or progression of IBD.

Respiratory tract bacterial infection can amplify and sustain airway inflammation. Intracytosolic nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is one member of the nucleotide binding and oligomerization domain (NOD)-like receptor (NLR) family, which senses the conserved structural peptidoglycan component muramyl dipeptide (MDP) in almost all bacteria. In the present study, activation of the NOD2 ligand MDP on primary human bronchial epithelial cells (HBE) co-cultured with human basophils was investigated. Cytokines, NOD2, adhesion molecules and intracellular signalling molecules were assayed by enzyme-linked immunosorbent assay or flow cytometry. The protein expression of NOD2 was confirmed in basophils/KU812 cells and HBE/human bronchial epithelial cell line (BEAS-2B) cells. MDP was found to up-regulate significantly the cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 on basophils and HBE in the co-culture system with or without basophil priming by interleukin (IL)-33 (all P < 0·05). MDP could further enhance the release of inflammatory cytokine IL-6 and chemokine CXCL8, and epithelium-derived anti-microbial peptide β-defensin 2 in the co-culture. HBE cells were the major source for the release of IL-6, CXCL8 and β-defensin2 upon stimulation by MDP in the co-culture system. The expression of ICAM-1 and VCAM-1 and release of IL-6 and CXCL8 were suppressed by various signalling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be regulated differentially by the mitogen-activated protein kinase pathways and nuclear transcription factors. The results therefore provide a new insight into the functional role of basophils in innate immunity, and the link between respiratory bacteria-mediated innate immunity and subsequent amplification of allergic inflammation in the airway., (© 2012 British Society for Immunology.)

Retinochoroiditis manifests in patients infected with Toxoplasma gondii. Here, we assessed 30 sibships and 89 parent/case trios of presumed ocular toxoplasmosis (POT) to evaluate associations with polymorphisms in the NOD2 gene. Three haplotype-tagging single-nucleotide polymorphisms (tag-SNPs) within the NOD2 gene were genotyped. The family-based association test showed that the tag-SNP rs3135499 is associated with retinochoroiditis (P = .039). We then characterized the cellular immune response of 59 cases of POT and 4 cases of active ocular toxoplasmosis (AOT). We found no differences in levels of interferon γ (IFN-γ) and interleukin 2 produced by T-helper 1 cells when comparing patients with AOT or POT to asymptomatic individuals. Unexpectedly, we found an increased interleukin 17A (IL-17A) production in patients with POT or OAT. In patients with POT or AOT, the main cellular source of IL-17A was CD4(+)CD45RO(+)T-bet(-)IFN-γ(-) T-helper 17 cells. Altogether, our results suggest that NOD2 influences the production of IL-17A by CD4(+) T lymphocytes and might contribute to the development of ocular toxoplasmosis.

Background: Genetic variants of nucleotide-binding oligomerization domain 2 (NOD2) lead to aberrant microbial recognition and can cause chronic inflammatory diseases in patients with Crohn's disease (CD)., Methods: We utilized gene-specific siRNA mediated knockdown and expression of guanine nucleotide exchange factor H1 (GEF-H1) in wildtype, Rip2-, and Nod2-deficient macrophages, HCT-116 and HEK 293 cells to determine the role of GEF-H1 in NOD2 and Rip2-mediated NF-κB-dependent induction of proinflammatory cytokine expression. Confocal microscopy was used to determine subcellular distribution of GEF-H1, Rip2, and NOD2., Results: We identified GEF-H1 as an unexpected component of innate immune regulation during microbial pattern recognition by NOD2. Surprisingly, GEF-H1-mediated the activation of Rip2 during signaling by NOD2, but not in the presence of the 3020 insC variant of NOD2 associated with CD. GEF-H1 functioned downstream of NOD2 as part of Rip2-containing signaling complexes and was responsible for phosphorylation of Rip2 by Src tyrosine kinase. Rip2 variants lacking the tyrosine target of GEF-H1-mediated phosphorylation were unable to mediate NF-κB activation in Rip2-deficient macrophages and failed to transduce NOD2 signaling. GEF-H1 is required downstream of NOD2 as part of Rip2-containing signaling complexes for the activation of innate immune responses., Conclusions: GEF-H1 connects tyrosine kinase function to NOD-like receptor signaling and is fundamental to the regulation of microbial recognition by ubiquitous innate immune mechanisms mediated by Rip2 kinase., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

In about 70% of patients Crohn's disease (CD) affects the small intestine. This disease location is stable over time and associated with a genetic background different from isolated colonic disease. A characteristic feature of small intestinal host defense is the presence of Paneth cells at the bottom of the crypts of Lieberkühn. These cells produce different broad spectrum antimicrobial peptides (AMPs) most abundantly the α-defensins HD-5 and -6 (DEFA5 und DEFA6). In small intestinal Crohn's disease both these PC products are specifically reduced. As a functional consequence, ileal extracts from Crohn's disease patients are compromised in clearing bacteria and enteroadherent E. coli colonize the mucosa. Mechanisms for defective antimicrobial Paneth cell function are complex and include an association with a NOD2 loss of function mutation, a disturbance of the Wnt pathway transcription factor TCF7L2 (also known as TCF4), the autophagy factor ATG16L1, the endosomal stress protein XBP1, the toll-like receptor TLR9, the calcium mediated potassium channel KCNN4 as well as mutations or inactivation of HD5. Thus we conclude that small intestinal Crohn's disease is most likely a complex disease of the Paneth cell: Paneth's disease., (Copyright © 2010 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.)

Toll-like receptor 2 (TLR2) plays an important role in the recognition of Borrelia bacteria, the causative agent of Lyme disease, but the existence and importance of additional receptors in this process has been hypothesized. In the present study, we confirmed the role played by TLR2 in the recognition of Borrelia bacteria but also demonstrated a crucial role for the intracellular peptidoglycan receptor NOD2 for sensing the spirochete. Cells from individuals who were homozygous for the loss-of-function mutation 3020insC in the NOD2 gene were defective with respect to cytokine release after stimulation with Borrelia species, and this was confirmed in peritoneal macrophages from mice lacking RICK, the adaptor molecule used by NOD2. In contrast, NOD1 played no major role in the recognition of Borrelia spirochetes. This raises the intriguing possibility that recognition of Borrelia spirochetes is exerted by TLR2 in combination with NOD2 and that both receptors are necessary for an effective induction of cytokines by Borrelia species. The interplay between TLR2 and NOD2 might not only be necessary for the induction of a proper immune response but may also contribute to inflammatory-induced pathology.

Background: Mutations in the gene encoding the nucleotide-binding oligomerization domain 2 (NOD2) protein are associated with Crohn's disease (CD), but the mechanism underlying this is not completely understood. To study the mechanism of CD resulting from NOD2 mutations, we analyzed NOD2-dependent whole-genome expression profiles of patient-derived antigen-presenting cells., Patients and Methods: Monocyte-derived dendritic cells (DCs) from CD carriers of double-dose NOD2 mutations, wild-type CD patients, and wild-type healthy volunteers were stimulated with the NOD2 ligand muramyl dipeptide. Whole-genome microarrays were used to assess the differential gene expression. The clustering of significantly changed genes was analyzed by online gene ontology mapping software., Results: In the DCs from the wild-type CD patient group, 683 genes were significantly changed, with most of the genes clustering in the pathways of inflammatory response. In addition, a significant number of genes clustered in the apoptosis regulation-related pathway. In the DCs from the healthy volunteer group, only 50 genes were significantly changed, predominantly those belonging to the response to pathogen pathway. Analysis of differentially expressed gene ontology pathways in the DCs from the NOD2 mutant CD patient group showed that the transcription of pathogen response genes was absent. In this group, 298 genes were significantly changed, predominantly clustering in the negative apoptosis regulation and cell organization and biogenesis pathways., Conclusions: Our results suggest that NOD2 mutations may result in perpetuation of mucosal inflammation through insufficient pathogen elimination. Further, these observations implicate a possible role of defective regulation of dendritic cell apoptosis in CD pathogenesis.

Mycobacterium paratuberculosis has been suggested to be involved in the pathogenesis of Crohn's disease (CD). The importance of microorganisms in CD is supported by the association of CD with mutations in the intracellular pathogen recognition receptor (PRR) nucleotide-binding oligomerization domain 2 (NOD2). The aim of this study is to investigate the PRR involved in the recognition of M. paratuberculosis. Methods used include in vitro stimulation of transfected cell lines, murine macrophages, and human PBMC. M. paratuberculosis stimulated human TLR2 (hTLR2)-Chinese hamster ovary (CHO) cells predominantly and hTLR4-CHO cells modestly. Macrophages from TLR2 and TLR4 knockout mice produced less cytokines compared with controls after stimulation with M. paratuberculosis. TLR4 inhibition in human PBMC reduced cytokine production only after stimulation with live M. paratuberculosis. TLR-induced TNF-alpha, IL-1beta, and IL-10 production is mediated through MyD88, whereas Toll-IL-1R domain-containing adaptor inducing IFN-beta (TRIF) promoted the release of IL-1beta. hNOD2-human embryo kidney (HEK) cells, but not hNOD1-HEK cells, responded to stimulation with M. paratuberculosis. PBMC of individuals homozygous for the 3020insC NOD2 mutation showed a 70% defective cytokine response after stimulation with M. paratuberculosis. These results demonstrate that TLR2, TLR4, and NOD2 are involved in the recognition of M. paratuberculosis by the innate immune system.

Abnormal response to enteric microflora is a critical factor driving bowel inflammation in patients with inflammatory bowel disease (IBD). Mice with genetically engineered mutations have played a central role in both formulating this hypothesis and elucidating the mechanism that normally protect the host from excessive inflammation within the bowel. One emerging theme is the importance of regulation within the innate immune system in protecting from microflora-driven pathology. In this review, I describe how genetically engineered mice have played a crucial role in shaping our conceptual understanding of pathways that regulate the development of chronic bowel inflammation, and furthermore, explore data derived from the study of genetically engineered mice that implicates the fundamental importance of regulation within the innate immune system in the control of this process.

Inflammation is part of the non-specific immune response that occurs in reaction to any type of bodily injury. In some disorders, the inflammatory process - which under normal conditions is self-limiting - becomes continuous and chronic inflammatory diseases might develop subsequently. Pattern recognition molecules (PRMs) represent a diverse collection of molecules responsible for sensing danger signals, and together with other immune components they are involved in the first line of defence. NALP3 and NOD2, which belong to a cytosolic subgroup of PRMs, dubbed Nod-like-receptors (NLRs), have been associated recently with inflammatory diseases, specifically Crohn's disease and Blau syndrome (NOD2) and familial cold autoinflammatory syndrome, Muckle-Wells syndrome and chronic infantile neurological cutaneous and articular syndrome (NALP3). The exact effects of the defective proteins are not fully understood, but activation of nuclear factor (NF)-kappaB, transcription, production and secretion of interleukin (IL)-1beta and activation of the inflammasome are some of the processes that might hold clues, and the present review will provide a thorough update in this area.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of a new family of myeloid receptors, encoded by a gene cluster linked to the MHC. Engagement of TREM-1 stimulates intracellular signals, resulting in activation of phagocytosis, neutrophil degranulation, and amplification of cytokine production induced by TLRs. In the present study, a novel property following engagement of TREM-1 is described, namely the amplification of cytokine production induced by the second major class of pattern recognition receptors, the NAIP, CIITA, HET-E, TP-1-leucine-rich repeat (NACHT-LRR; NLR) receptors, which recognize intracellular microorganisms through sensing their muropeptide components of peptidoglycan. The TREM-1/NLR synergism was observed for the production of TNF-alpha, IL-1beta, and IL-6, leading to an increase in cytokine production up to tenfold greater than the additive value of TREM-1 or muropeptide stimulation alone. Several putative mechanisms are proposed to be involved in the synergism between NLRs and TREM-1, including the increase in TREM-1 expression by NLR ligands, and of the expression of nucleotide oligomerization domain-2 receptor by TREM-1 engagement. In contrast, although caspase-1 modulates IL-1beta and IL-6 production after stimulation with anti-TREM-1 antibodies or NLR ligands, it does not appear to be responsible for the synergism between these two pathways. These findings demonstrate that TREM-1 acts on both major recognition pathways of bacterial structures: the extracellular TLR receptors, and the intracellular NLR molecules. This latter finding supports the concept that TREM-1 provides optimal amplification of cytokine-induced inflammation during the initiation of host defense.