Family Of Innate Immune Receptors Research Articles

Monitoring Innate Immune Receptor Stability via Post‐Translational Modification by OGT

The human body's first line of defense against infectious disease is the innate immune system, which is tasked with distinguishing harmful pathogens from commensal and beneficial microbes. On a molecular level, this system contains different pattern recognition receptors (PRRs) that detect microbe associated molecular patterns (MAMPs) specific to particular microorganisms, and the interaction between a PRR and its respective MAMP triggers an immune response. The Nod‐like receptors (NLRs) are one major family of innate immune receptors that contain the proteins Nod1 and Nod2, which use different bacterial cell wall peptidoglycan (PGN) fragments as their designated MAMPs. Binding between the NLR and PGN fragment is mediated by the leucine rich repeat domain (LRR), which exists at the C‐termini of Nod1 and Nod2. Upon LRR‐MAMP stimulation, NF‐kB and other signaling cascades are initiated, ending with the production of proinflammatory cytokines and chemokines. Misregulation of signaling by NLRs can lead to disease, and mutations in Nod1 and Nod2 are associated with gastric cancer and Crohn's disease, respectively. These NLRs are critically important for maintaining the relationship between the human body and the trillions of microorganisms that it hosts, yet so much about their functions and regulation in the body remain unclear. Recently, Nod2 was identified as being O‐‐GlcNAcylated, which is a post‐translational modification mediated by two enzymes that affect thousands of proteins in diverse ways. An emerging cellular thermal shift assay (CETSA) that is used for identifying protein‐ligand binding was repurposed to measure the stabilizing effect of O‐GlcNAc modification on Nod1 and Nod2. When protein O‐GlcNAc levels are increased, melting temperatures of both NLRs increase as well. These data are supported by cycloheximide half‐life experiments and NF‐kB luciferase assays, which demonstrated that the modification also led to increased NLR half‐lives and NF‐kB activity. Additionally, two Nod2 Crohn's associated variants were studied, and both G908R‐Nod2 and 1007fs‐Nod2 are O‐GlcNAcylated in the cell. While the modification led to similar increases in melting temperature and NF‐kB activity for 1007fs‐Nod2, its effects on G908R‐Nod2 are less clear. These results show that CETSA is a useful tool for measuring stability changes caused by O‐GlcnAc modification and that NLRs may be commonly post‐translationally modified to regulate stability.Support or Funding InformationMITZUTANI GLYCOSCIENCE FOUNDATION, NATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967), NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Site-specific effect of polar functional group-modification in lipids of TLR2 ligands for modulating the ligand immunostimulatory activity

Toll-like receptor 2 (TLR2), a member of the TLR innate immune receptor family, recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. TLR2 has a large hydrophobic pocket that recognizes long fatty acyl groups on TLR2 ligands. However, few studies have focused on the property of the hydrophobic TLR2 pocket. Based on the X-ray crystal structure of TLR2, small polar regions were found in the hydrophobic TLR2 pocket. Interactions between the polar residues and ligands were explored here by designing and synthesizing a Pam2CSK4 derivative of the TLR2 ligands, containing an amide group within the lipid moiety. We evaluated the binding affinities and immunomodulatory activities of these ligands. Results suggested that the amide groups in the lipid chain interacted with the polar residues in the hydrophobic lipid-binding pocket of TLR2.

Deficiency of Toll-like receptors 2, 3 or 4 extends life expectancy in Huntington’s disease mice

Huntington’s disease (HD), an autosomal dominant neurodegenerative disorder characterized by progressive striatal and cortical atrophy, has been strongly linked with neuroinflammation. Toll-like receptors, a family of innate immune receptors, are a major pathway for neuroinflammation with pleiotropic effects on neuronal plasticity and neurodevelopment. We assessed whether deficiency for TLRs 2, 3 or 4 affects life expectancy in the N171-82Q mouse model of HD. Our data indicate that homozygous TLRs 2 and 3 as well as heterozygous TLR4 deficiency significantly extends the life expectancy of HD mice. Our data suggest that multiple TLR pathways may be involved in the neuroinflammatory and degenerative processes during HD.

Spotted Gar and the Evolution of Innate Immune Receptors.

The resolution of the gar genome affords an opportunity to examine the diversification and functional specialization of immune effector molecules at a distant and potentially informative point in phylogenetic development. Although innate immunity is effected by a particularly large number of different families of molecules, the focus here is to provide detailed characterization of several families of innate receptors that are encoded in large multigene families, for which orthologous forms can be identified in other species of bony fish but not in other vertebrate groups as well as those for which orthologs are present in other vertebrate species. The results indicate that although teleost fish and the gar, as a holostean reference species, share gene families thought previously to be restricted to the teleost fish, the manner in which the members of the multigene families of innate immune receptors have undergone diversification is different in these two major phylogenetic radiations. It appears that both the total genome duplication and different patterns of genetic selection have influenced the derivation and stabilization of innate immune genes in a substantial manner during the course of vertebrate evolution.

Induction of Toll-like receptor (TLR) 2 and Myeloid88-dependent TLR-Signaling Response to Aeromonas Hydrophila Infections in Gouramy (Osphronemus Gouramy)

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns associated with microbial pathogens (PAMP) and induce antimicrobial immune responses. Here we report the expression of TLR2, Myeloid differentiation primary response gene 88 (Myd88) and Interleukin 1beta (IL1β) from the Gouramy an economically freshwater fish in Indonesia. Upon stimulation with Aeromonas hydrophila, the TLR2 expression was obviously up-regulated in spleen and liver, Myd88 expression up-regulated in spleen and kidney, and IL1β expression was up-regulated in a spleen, kidney, and liver. The highest expression of TLR2 was in liver or hepatopancreas, MyD88 in kidney and IL1β was in the spleen. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish and indicated its important role in immune surveillance of various organs during the pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish and will be helpful in developing preventive measures against infectious diseases in fish.

Activation of the Innate Immune Receptors: Guardians of the Micro Galaxy : Activation and Functions of the Innate Immune Receptors.

The families of innate immune receptors are the frontline responders to danger. These superheroes of the host immune systems populate innate immune cells, surveying the extracellular environment and the intracellular endolysosomal compartments and cytosol for exogenous and endogenous danger signals. As a collective the innate immune receptors recognise a wide array of stimuli, and in response they initiate specific signalling pathways leading to activation of transcriptional or proteolytic pathways and the production of inflammatory molecules to destroy foreign pathogens and/or resolve tissue injury. In this review, I will give an overview of the innate immune system and the activation and effector functions of the families of receptors it comprises. Current key concepts will be described throughout, including innate immune memory, formation of innate immune receptor signalosomes, inflammasome formation and pyroptosis, methods of extrinsic cell communication and examples of receptor cooperation. Finally, several open questions and future directions in the field of innate immunity will be presented and discussed.

C-type Lectin Mincle Recognizes Glucosyl-diacylglycerol of Streptococcus pneumoniae and Plays a Protective Role in Pneumococcal Pneumonia.

Among various innate immune receptor families, the role of C-type lectin receptors (CLRs) in lung protective immunity against Streptococcus pneumoniae (S. pneumoniae) is not fully defined. We here show that Mincle gene expression was induced in alveolar macrophages and neutrophils in bronchoalveolar lavage fluids of mice and patients with pneumococcal pneumonia. Moreover, S. pneumoniae directly triggered Mincle reporter cell activation in vitro via its glycolipid glucosyl-diacylglycerol (Glc-DAG), which was identified as the ligand recognized by Mincle. Purified Glc-DAG triggered Mincle reporter cell activation and stimulated inflammatory cytokine release by human alveolar macrophages and alveolar macrophages from WT but not Mincle KO mice. Mincle deficiency led to increased bacterial loads and decreased survival together with strongly dysregulated cytokine responses in mice challenged with focal pneumonia inducing S. pneumoniae, all of which was normalized in Mincle KO mice reconstituted with a WT hematopoietic system. In conclusion, the Mincle-Glc-DAG axis is a hitherto unrecognized element of lung protective immunity against focal pneumonia induced by S. pneumoniae.

Resurrecting ancestral structural dynamics of an antiviral immune receptor: adaptive binding pocket reorganization repeatedly shifts RNA preference.

BackgroundAlthough resurrecting ancestral proteins is a powerful tool for understanding the molecular-functional evolution of gene families, nearly all studies have examined proteins functioning in relatively stable biological processes. The extent to which more dynamic systems obey the same ‘rules’ governing stable processes is unclear. Here we present the first detailed investigation of the functional evolution of the RIG-like receptors (RLRs), a family of innate immune receptors that detect viral RNA in the cytoplasm.ResultsUsing kinetic binding assays and molecular dynamics simulations of ancestral proteins, we demonstrate how a small number of adaptive protein-coding changes repeatedly shifted the RNA preference of RLRs throughout animal evolution by reorganizing the shape and electrostatic distribution across the RNA binding pocket, altering the hydrogen bond network between the RLR and its RNA target. In contrast to observations of proteins involved in metabolism and development, we find that RLR-RNA preference ‘flip flopped’ between two functional states, and shifts in RNA preference were not always coupled to gene duplications or speciation events. We demonstrate at least one reversion of RLR-RNA preference from a derived to an ancestral function through a novel structural mechanism, indicating multiple structural implementations of similar functions.ConclusionsOur results suggest a model in which frequent shifts in selection pressures imposed by an evolutionary arms race preclude the long-term functional optimization observed in stable biological systems. As a result, the evolutionary dynamics of immune receptors may be less constrained by structural epistasis and historical contingency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0818-6) contains supplementary material, which is available to authorized users.

Toll-like receptor agonists partially restore the production of pro-inflammatory cytokines and type I interferon in Sézary syndrome.

Sézary syndrome (SS) carries a poor prognosis, and infections represent the most frequent cause of death in SS patients. Toll-like receptors (TLRs) are a family of innate immune receptors that induce protective immune responses against infections. We sought to evaluate the ability of TLR agonists to induce inflammatory cytokine, Th2 cytokine, and type I interferon (IFN-I) production by peripheral blood mononuclear cells (PBMC) of untreated SS patients. We detected impaired IL-6, IL-10 and IL-13 secretion by PBMC induced by the agonists for TLR5, TLR3, TLR7 and TLR9 in SS patients, while it was partially recovered by TLR2/TLR4 and TLR7/8 agonists TNF secretion was restored following stimulation with TLR2/TLR4 agonists. IFN-γ was scarcely produced upon TLR activation in SS cells, albeit TLR 7/8 (CL097) enhanced their secretion at lower levels than the control group. TLR9 agonist efficiently induced IFN-I in SS patients, although this positive regulation was not observed for other cytokines, in direct contrast to the broad activity of CL097. Among the TLR agonists, TLR4 was able to induce pro-inflammatory, IL-10 and Th2 secretion, while TLR7-8 agonist induced the inflammatory cytokines, IFN-I and IFN-γ. These findings reveal a dysfunctional cytokine response upon both extracellular and intracellular TLR activation in SS patients, which was partially restored by TLRs agonists.

Single Nucleotide Polymorphisms of Toll-Like Receptor 7 in Hepatitis C Virus Infection and Hepatocellular Carcinoma Patients

Background: Hepatitis C virus (HCV) infection is a major health problem. Chronic HCV is the main cause of liver cirrhosis and liver cancer in Egypt and one of the top five leading causes of death. Toll-like receptors (TLRs) are a family of innate immune receptors known as pattern recognition receptors. There is an associations between single nucleo­tide polymorphisms (SNP) in TLRs and increased risk of infection. Objective: The aim of this study was to determine the association between TLR7 (rs179008 gene) polymorphism and Chronic hepatitis C infection with and without Hepatocellular Carcinoma (HCC). Methodology: Fifty nine patients suffering from HCV and HCC and twenty one sex and age matched healthy controls were included in the study. SNP of TLR 7 (rs179008 gene) was detected by genotyping assay using Real Time PCR. Results: We found no significant difference between males HCV patients and control group. However, significant differences were found for the distribution of TLR7 (rs179009) in females (χ²=5.98, p=0.01) with HCC in comparison to controls. Conclusion: The site of TLR7 (rs179009) may be a factor for susceptibility of chronic HCV in the female population to HCC. The (AT) genotype is significantly higher in female HCC group compared to control group. These patients could then be subjected to a more careful or earlier routine screening for HCC.

The confounding complexity of innate immune receptors within and between teleost species.

Teleost genomes encode multiple multigene families of immunoglobulin domain-containing innate immune receptors (IIIRs) with unknown function and no clear mammalian orthologs. However, the genomic organization of IIIR gene clusters and the structure and signaling motifs of the proteins they encode are similar to those of mammalian innate immune receptor families such as the killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LILRs), Fc receptors, triggering receptors expressed on myeloid cells (TREMs) and CD300s. Teleost IIIRs include novel immune-type receptors (NITRs); diverse immunoglobulin domain containing proteins (DICPs); polymeric immunoglobulin receptor-like proteins (PIGRLs); novel immunoglobulin-like transcripts (NILTs) and leukocyte immune-type receptors (LITRs). The accumulation of genomic sequence data has revealed that IIIR gene clusters in zebrafish display haplotypic and gene content variation. This intraspecific genetic variation, as well as significant interspecific variation, frequently confounds the identification of definitive orthologous IIIR sequences between teleost species. Nevertheless, by defining which teleost lineages encode (and do not encode) different IIIR families, predictions can be made about the presence (or absence) of specific IIIR families in each teleost lineage. It is anticipated that further investigations into available genomic resources and the sequencing of a variety of multiple teleost genomes will identify additional IIIR families and permit the modeling of the evolutionary origins of IIIRs.

Comparative analysis of peptidoglycan recognition proteins in endoparasitoid wasp Microplitis mediator

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune receptors that specifically recognize peptidoglycans (PGNs) on the surface of a number of pathogens. Here, we have identified and characterized six PGRPs from endoparasitoid wasp, Microplitis mediator (MmePGRPs). To understand the roles of PGRPs in parasitoid wasps, we analyzed their evolutionary relationship and orthology, expression profiles during different developmental stages, and transcriptional expression following infection with Gram-positive and -negative bacteria and a fungus. MmePGRP-S1 was significantly induced in response to pathogenic infection. This prompted us to evaluate the effects of RNA interference mediated gene specific knockdown of MmePGRP-S1. The knockdown of MmePGRP-S1 (iMmePGRP-S1) dramatically affected wasps' survival following challenge by Micrococcus luteus, indicating the involvement of this particular PGRP in immune responses against Gram-positive bacteria. This action is likely to be mediated by the Toll pathway, but the mechanism remains to be determined. MmePGRP-S1 does not play a significant role in anti-fungal immunity as indicated by the survival rate of iMmePGRP-S1 wasps. This study provides a comprehensive characterization of PGRPs in the economically important hymenopteran species M. mediator.

Modulation of TLR3/TLR4 inflammatory signaling by the GABAB receptor agonist baclofen in glia and immune cells: relevance to therapeutic effects in multiple sclerosis.

The GABAB receptor agonist, baclofen, is used to treat muscle tightness and cramping caused by spasticity in a number of disorders including multiple sclerosis (MS), but its precise mechanism of action is unknown. Neuroinflammation drives the central pathology in MS and is mediated by both immunoreactive glial cells and invading lymphocytes. Furthermore, a body of data indicates that the Toll-like receptor (TLR) family of innate immune receptors is implicated in MS progression. In the present study we investigated whether modulation of GABAB receptors using baclofen can exert anti-inflammatory effects by targeting TLR3 and(or) TLR4-induced inflammatory signaling in murine glial cells and human peripheral blood mononuclear cells (PBMCs) isolated from healthy control individuals and patients with the relapse-remitting (RR) form of MS. TLR3 and TLR4 stimulation promoted the nuclear sequestration of NF-κB and pro-inflammatory cytokine expression in murine glia, while TLR4, but not TLR3, promoted pro-inflammatory cytokine expression in PBMCs isolated from both healthy donors and RR-MS patients. Importantly, this effect was exacerbated in RR-MS patient immune cells. We present further evidence that baclofen dose-dependently attenuated TLR3- and TLR4-induced inflammatory signaling in primary glial cells. Pre-exposure of PBMCs isolated from healthy donors to baclofen attenuated TLR4-induced TNF-α expression, but did not affect TLR4-induced TNF-α expression in RR-MS patient PBMCs. Interestingly, mRNA expression of the GABAB receptor was reduced in PBMCs from RR-MS donors when compared to healthy controls, an effect that might contribute to the differential sensitivity to baclofen seen in healthy and RR-MS patient cells. Overall these findings indicate that baclofen differentially regulates TLR3 and TLR4 signaling in glia and immune cells, and offers insight on the role of baclofen in the treatment of neuroinflammatory disease states including MS.

Triggering Receptor Expressed on Myeloid Cells in Cutaneous Melanoma.

The tumor microenvironment plays an important role in the progression of melanoma, the prototypical immunologic cutaneous malignancy. The triggering receptor expressed on myeloid cells (TREM) family of innate immune receptors modulates inflammatory and innate immune signaling. It has been investigated in various neoplastic diseases, but not in melanoma. This study examines the expression of TREM-1 (a proinflammatory amplifier) and TREM-2 (an anti-inflammatory modulator and phagocytic promoter) in human cutaneous melanoma and surrounding tissue. Indirect immunofluorescence staining was performed on skin biopsies from 10 melanoma patients and staining intensity was semiquantitatively scored. Expression of TREM-1 and TREM-2 was higher in keratinocytes than melanoma tissue (TREM-1: p < 0.01; TREM-2: p < 0.01). Whereas TREM-2 was the dominant isoform expressed in normal keratinocytes, TREM-1 expression predominated in melanoma tissue (TREM-1 to TREM-2 ratio: keratinocytes = 0.78; melanoma = 2.08; p < 0.01). The increased TREM ratio in melanoma tissue could give rise to a proinflammatory and protumor state of the microenvironment. This evidence may be suggestive of a TREM-1/TREM-2 paradigm in which relative levels dictate inflammatory and immune states, rather than absolute expression of one or the other. Further investigation regarding this paradigm is warranted and could carry prognostic or therapeutic value in treatment for melanoma.

Less functional variants of TLR-1/-6/-10 genes are associated with age.

BackgroundDetermining the prerequisites for healthy aging is a major task in the modern world characterized by a longer lifespan of the individuals. Besides lifestyle and environmental influences genetic factors are involved as shown by several genome-wide association studies. Older individuals are known to have an impaired immune response, a condition recently termed “inflamm-aging”. We hypothesize that the induction of this condition in the elderly is influenced by the sensitivity of the innate immune system. Therefore, we investigated genetic variants of the Toll-like receptor (TLR) family, one of the major family of innate immune receptors, for association with age in two cohorts of healthy, disease-free subjects.ResultsAccording to sex we found a positive association of loss-of-function variants of TLR-1 and −6 with healthy aging with odds ratios of 1.54 in males for TLR-6 (249 S/S), and 1.41, 1.66, and 1.64 in females for TLR-1 prom., TLR-1 (248 S/S), and TLR-1 (602 S/S), respectively. Thus, the presence of these variants increases the probability of achieving healthy old age and indicates that a reduced TLR activity may be beneficial in the elderly.ConclusionsThis is the first report showing an association of TLR variants with age. While a loss of function of an important immune receptor may be a risk factor for acute infections as has been shown previously, in the setting of healthy ageing it appears to be protective, which may relate to “inflamm-aging”. These first results should be reproduced in larger trials to confirm this hypothesis.

Postnatal TLR2 activation impairs learning and memory in adulthood.

Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth.

An Immune Effector System in the Protochordate Gut Sheds Light on Fundamental Aspects of Vertebrate Immunity.

A variety of germline and somatic immune mechanisms have evolved in vertebrate and invertebrate species to detect a wide array of pathogenic invaders. The gut is a particularly significant site in terms of distinguishing pathogens from potentially beneficial microbes. Ciona intestinalis, a filter-feeding marine protochordate that is ancestral to the vertebrate form, possesses variable region-containing chitin-binding proteins (VCBPs), a family of innate immune receptors, which recognize bacteria through an immunoglobulin-type variable region. The manner in which VCBPs mediate immune recognition appears to be related to the development and bacterial colonization of the gut, and it is likely that these molecules are critical elements in achieving overall immune and physiological homeostasis.

Novel Types of Small RNA Exhibit Sequence- and Target-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 in an Age-related Macular Degeneration (AMD) Mouse Model.

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.

Innate sensing of malaria parasites.

Innate immune receptors have a key role in immune surveillance by sensing microorganisms and initiating protective immune responses. However, the innate immune system is a classic 'double-edged sword' that can overreact to pathogens, which can have deleterious effects and lead to clinical manifestations. Recent studies have unveiled the complexity of innate immune receptors that function as sensors of Plasmodium spp. in the vertebrate host. This Review highlights the cellular and molecular mechanisms by which Plasmodium infection is sensed by different families of innate immune receptors. We also discuss how these events mediate both host resistance to infection and the pathogenesis of malaria.

143: Nod2 and Rip2 contribute to innate immune responses in mouse neutrophils

Nod-like receptors (NLRs) are a family of innate immune receptors that link cytosolic sensing of microbial and danger stimuli to the activation of immune responses. Two NLR family members, Nod1 and Nod2, recognize bacterial peptidoglycan and activate immune responses via NF-κB and MAPK. The function of Nod1 and Nod2 has been largely studied in macrophages, but the role of these receptors in other innate immune cells remains unclear. In this study, we examined the function of Nod1 and Nod2 in innate immune responses of neutrophils. Mice were injected i.p with thioglycollate, and then peritoneal neutrophils were isolated 4 h after injection. Tri-DAP and muramyl-dipeptide (MDP) were used as Nod1 and Nod2 agonist, respectively. The level of cytokines (IL-6 and TNF-α) and chemokines (CXCL1 and CCL2) was increased by MDP, but not Tri-DAP in WT neutrophils. Such production of cytokines and chemokines by MDP was abolished in Nod2- and Rip2-deficient neutrophils. MDP also induced the activation of NF-κB and MAPK in WT neutrophils, but not Nod2- and Rip2-deficient cells. Flow cytometry analysis showed that L-selectin shedding was induced by MDP in WT neutrophils, but not in Nod2- and Rip2-deficient cells. MDP and TLR agonists (Pam3CSK4 and LPS) exerted synergistic effect on the production of IL-6 and CXCL1 in neutrophils. Moreover, Nod2 and TLR4 cooperated to produce IL-6, TNF-α, CXCL1, and CCL2 in neutrophils in response to gram-negative bacteria. Our findings suggest that Nod2-Rip2 axis may contribute to innate immune response of neutrophils against bacterial infection.