Nucleotide-binding oligomerization domain 2 (Nod2) is dispensable for the innate immune responses of macrophages against Yersinia enterocolitica

Nucleotide-binding oligomerization domain 2 (Nod2) is a cytosolic sensor for muramyl dipeptide, a component of bacterial peptidoglycan. In this study, we have examined whether Nod2 mediates the immune response of macrophages against Yersinia enterocolitica. Bone-marrow-derived macrophages (BMDMs) were isolated from WT and Nod2-deficient mice and were infected with various strains of Y. enterocolitica. ELISA showed that the production of IL-6 and TNF-α in BMDMs infected with Y. enterocolitica was not affected by the Nod2 deficiency. iNOS mRNA expression was induced in both WT and Nod2-deficienct BMDMs in response to Y. enterocolitica, beginning 2 h after infection. Nitric oxide (NO) production by Y. enterocolitica did not differ between WT and Nod2-deficient BMDMs. Western blot analysis revealed that Y. enterocolitica induces activation of NF-κB, p38, and ERK MAPK through a Nod2-independent pathway. Neither LDH release by Y. enterocolitica nor the phagocytic activity of the macrophages was altered by Nod2 deficiency. An in vivo experiment showed that bacterial clearance ability and production of IL-6 and KC in serum were comparable in WT and Nod2-deficient mice infected with Y. enterocolitica. These findings suggest that Nod2 may not be critical for initiating the innate immune response of macrophages against Yersinia infection.     

Defective innate cell response and lymph node infiltration specify Yersinia pestis infection

Since its recent emergence from the enteropathogen Yersinia pseudotuberculosis, Y. pestis, the plague agent, has acquired an intradermal (id) route of entry and an extreme virulence. To identify pathophysiological events associated with the Y. pestis high degree of pathogenicity, we compared disease progression and evolution in mice after id inoculation of the two Yersinia species. Mortality studies showed that the id portal was not in itself sufficient to provide Y. pseudotuberculosis with the high virulence power of its descendant. Surprisingly, Y. pseudotuberculosis multiplied even more efficiently than Y. pestis in the dermis, and generated comparable histological lesions. Likewise, Y. pseudotuberculosis translocated to the draining lymph node (DLN) and similar numbers of the two bacterial species were found at 24 h post infection (pi) in this organ. However, on day 2 pi, bacterial loads were higher in Y. pestis-infected than in Y. pseudotuberculosis-infected DLNs. Clustering and multiple correspondence analyses showed that the DLN pathologies induced by the two species were statistically significantly different and identified the most discriminating elementary lesions. Y. pseudotuberculosis infection was accompanied by abscess-type polymorphonuclear cell infiltrates containing the infection, while Y. pestis-infected DLNs exhibited an altered tissue density and a vascular congestion, and were typified by an invasion of the tissue by free floating bacteria. Therefore, Y. pestis exceptional virulence is not due to its recently acquired portal of entry into the host, but is associated with a distinct ability to massively infiltrate the DLN, without inducing in this organ an organized polymorphonuclear cell reaction. These results shed light on pathophysiological processes that draw the line between a virulent and a hypervirulent pathogen.     

Reduced secretion of YopJ by Yersinia limits in vivo cell death but enhances bacterial virulence

Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow-derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis-infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen.     

Yersinia pseudotuberculosis effector YopJ subverts the Nod2/RICK/TAK1 pathway and activates caspase-1 to induce intestinal barrier dysfunction

Yersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y.?pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide. YopJ, but not the catalytically inactive mutant YopJ(C172A), acetylates critical sites in the activation loops of the RICK and TAK1 kinases, which are central mediators of Nod2 signaling, and decreases the affinity of Nod2 for RICK. Concomitantly, Nod2 interacts with and activates caspase-1, resulting in increased levels of IL-1β. Finally, IL-1β within PP plays an essential role in inducing intestinal barrier dysfunction. Thus, YopJ alters intestinal permeability and promotes the dissemination of Yersinia as well as commensal bacteria by exploiting the mucosal inflammatory response.     

NOD2-deficient mice have impaired resistance to Mycobacterium tuberculosis infection through defective innate and adaptive immunity

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2(-/-) mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2(-/-) mice had decreased production of type 1 cytokines and reduced recruitment of CD8(+) and CD4(+) T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2(-/-) mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2(-/-) mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.     

MyD88-, but Not Nod1- and/or Nod2-Deficient Mice,Show Increased Susceptibility to Polymicrobial Sepsis due to Impaired Local Inflammatory Response

Pathogen recognition and triggering of the inflammatory response following infection in mammals depend mainly on Toll-like and Nod-like receptors. Here, we evaluated the role of Nod1, Nod2 and MyD88-dependent signaling in the chemokine production and neutrophil recruitment to the infectious site during sepsis induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. We demonstrate that Nod1 and Nod2 are not involved in the release of chemokines and recruitment of neutrophils to the infectious site during CLP-induced septic peritonitis because these events were similar in wild-type, Nod1-, Nod2-, Nod1/Nod2- and Rip2-deficient mice. Consequently, the local and systemic bacterial loads were not altered. Accordingly, neither Nod1 nor Nod2 was involved in the production of the circulating cytokines and in the accumulation of leukocytes in the lungs. By contrast, we showed that MyD88-dependent signaling is crucial for the establishment of the local inflammatory response during CLP-induced sepsis. MyD88-deficient mice were susceptible to sepsis because of an impaired local production of chemokines and defective neutrophil recruitment to the infection site. Altogether, these data show that Nod1, Nod2 and Rip2 are not required for local chemokine production and neutrophil recruitment during CLP-induced sepsis, and they reinforce the importance of MyD88-dependent signaling for initiation of a protective host response.     

Acute oral toxicity of Yersinia pseudotuberculosis to fleas: implications for the evolution of vector-borne transmission of plague

Yersinia pestis diverged from Yersinia pseudotuberculosis相似文献   

Nod1 activation by bacterial iE-DAP induces maternal-fetal inflammation and preterm labor

There is a strong association between infection and prematurity; however, the underlying mechanisms remain largely unknown. Nod1 and Nod2 are intracellular pattern recognition receptors that are activated by bacterial peptides and mediate innate immunity. We previously demonstrated that human first-trimester trophoblasts express Nod1 and Nod2, which trigger inflammation upon stimulation. This study sought to determine the expression and function of Nod1 and Nod2 in third-trimester trophoblasts, and to characterize the in vivo effects of Nod1 activation on pregnancy outcome. Human term placental tissues and isolated term trophoblast expressed Nod1, but not Nod2. Activation of Nod1 by its agonist, bacterial γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), in term trophoblast cultures induced a proinflammatory cytokine profile, characterized by elevated levels of secreted IL-6, GRO-α, and MCP-1, when compared with the control. However, these cytokines were not upregulated in response to Nod2 stimulation with bacterial MDP. Administration of high-dose bacterial iE-DAP to pregnant C57BL/6J mice on embryonic day 14.5 triggered preterm delivery within 24 h. iE-DAP at a lower dose that did not induce prematurity, reduced fetal weight, altered the cytokine profile at the maternal-fetal interface, and induced fetal inflammation. Thus, functional Nod1 is expressed by trophoblast cells across gestation and may have a role in mediating infection-associated inflammation and prematurity. This study demonstrates that pattern recognition receptors, other than the TLRs, may be implicated or involved in infection-associated preterm labor.     

Viral infection augments Nod1/2 signaling to potentiate lethality associated with secondary bacterial infections

Secondary bacterial infection is a common sequela to?viral infection and is associated with increased lethality and morbidity. However, the underlying mechanisms remain poorly understood. We show that the TLR3/MDA5 agonist poly I:C or viral infection dramatically augments signaling via the NLRs Nod1 and Nod2 and enhances the production of proinflammatory cytokines. Enhanced Nod1 and Nod2 signaling by poly I:C required the TLR3/MDA5 adaptors TRIF and IPS-1 and was mediated by type I IFNs. Mechanistically, poly I:C or IFN-β induced the expression of Nod1, Nod2, and the Nod-signaling adaptor Rip2. Systemic administration of poly I:C or IFN-β or infection with murine norovirus-1 promoted inflammation and lethality in mice superinfected with E.?coli, which was independent of bacterial burden but attenuated in the absence of Nod1/Nod2 or Rip2. Thus, crosstalk between type I IFNs and Nod1/Nod2 signaling promotes bacterial recognition, but induces harmful effects in the virally infected host.     

Loss of a biofilm-inhibiting glycosyl hydrolase during the emergence of Yersinia pestis

Yersinia pestis, the bacterial agent of plague, forms a biofilm in the foregut of its flea vector to produce a transmissible infection. The closely related Yersinia pseudotuberculosis, from which Y. pestis recently evolved, can colonize the flea midgut but does not form a biofilm in the foregut. Y. pestis biofilm in the flea and in vitro is dependent on an extracellular matrix synthesized by products of the hms genes; identical genes are present in Y. pseudotuberculosis. The Yersinia Hms proteins contain functional domains present in Escherichia coli and Staphylococcus proteins known to synthesize a poly-beta-1,6-N-acetyl-D-glucosamine biofilm matrix. In this study, we show that the extracellular matrices (ECM) of Y. pestis and staphylococcal biofilms are antigenically related, indicating a similar biochemical structure. We also characterized a glycosyl hydrolase (NghA) of Y. pseudotuberculosis that cleaved beta-linked N-acetylglucosamine residues and reduced biofilm formation by staphylococci and Y. pestis in vitro. The Y. pestis nghA ortholog is a pseudogene, and overexpression of functional nghA reduced ECM surface accumulation and inhibited the ability of Y. pestis to produce biofilm in the flea foregut. Mutational loss of this glycosidase activity in Y. pestis may have contributed to the recent evolution of flea-borne transmission.     

The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected with Legionella pneumophila

The intracellular bacterium Legionella pneumophila induces a severe form of pneumonia called Legionnaires diseases, which is characterized by a strong neutrophil (NE) infiltrate to the lungs of infected individuals. Although the participation of pattern recognition receptors, such as Toll-like receptors, was recently demonstrated, there is no information on the role of nod-like receptors (NLRs) for bacterial recognition in vivo and for NE recruitment to the lungs. Here, we employed a murine model of Legionnaires disease to evaluate host and bacterial factors involved in NE recruitment to the mice lungs. We found that L. pneumophila type four secretion system, known as Dot/Icm, was required for NE recruitment as dot/icm mutants fail to trigger NE recruitment in a process independent of bacterial multiplication. By using mice deficient for Nod1, Nod2, and Rip2, we found that these receptors accounted for NE recruitment to the lungs of infected mice. In addition, Rip2-dependent responses were important for cytokine production and bacterial clearance. Collectively, these studies show that Nod1, Nod2, and Rip2 account for generation of innate immune responses in vivo, which are important for NE recruitment and bacterial clearance in a murine model of Legionnaires diseases.     

Yersinia pseudotuberculosis plasmids and their significance in the realization of the epidemic process in pseudotuberculosis

The results obtained in the electrophoretic study of the plasmid spectra of 190 Y. pseudotuberculosis strains, isolated from different sources, in 0.6% agarose gel are presented. 11 types of plasmids differing in their molecular weight have been detected. Plasmids with a molecular weight of 45 MD determine Ca2+ dependence, bacterial virulence for white mice and autoagglutination. The presence of differences in Y. pseudotuberculosis strains of serovars I, III and IV has been established, which is manifested by their differing plasmid spectra. The relationship between the presence of plasmids with a molecular weight of 75 and 45 DM in the strains and the character of pseudotuberculosis morbidity in the population has been demonstrated. The epidemic course of infection correlates with the presence of both these plasmids and the sporadic course of infection, with the presence of the plasmid with a molecular weight of 45 MD only.     

Nod2 suppresses Borrelia burgdorferi mediated murine Lyme arthritis and carditis through the induction of tolerance

The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.     

Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1β in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.     

Nod proteins link bacterial sensing and autophagy

Autophagy is one of the main cellular degradation systems in eukaryotes, being responsible for the elimination of long-lived proteins and damaged organelles. Besides its well-documented role as a housekeeping mechanism, autophagy has recently caught the attention of groups working in the fields of microbiology and immunology, especially those working in innate immunity. In particular, the highly specific segregation and degradation of intracellular bacteria by the autophagic machinery was a matter of great interest. However, it was still unclear how the autophagy machinery could target intracellular bacteria with such specificity. We have recently analyzed the role of the intracellular peptidoglycan (PG) receptors Nod1 and Nod2 as a link between intracellular bacterial sensing and the induction of autophagy. Our results demonstrated that Nod2 recruits the critical autophagy protein ATG16L1 to the plasma membrane during bacterial invasion and that cells expressing mutations in these proteins, two of the most important associated with Crohn disease, autophagy is defective upon infection or stimulation with the bacterial peptidoglycan fragment MDP. Thus, our findings put together two genes previously reported as independent risk factors for the development of Crohn disease and open a venue in the study of new therapies to cure the disease.     

RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs

RICK is a kinase that has been implicated in Nod1 and Nod2 signaling. In addition, RICK has been proposed to mediate TLR signaling in that its absence confers reduced responses to certain bacterial products such as LPS. We show here that macrophages and mice lacking RICK are defective in their responses to Nod1 and Nod2 agonists but exhibit unimpaired responses to synthetic and highly purified TLR agonists. Furthermore, production of chemokines induced by the bacterial dipeptide gamma-d-glutamyl-meso-diaminopimelic acid was intact in MyD88 deficient mice but abolished in RICK-null mice. Stimulation of macrophages with muramyl dipeptide, the Nod2 activator, enhanced immune responses induced by LPS, IFN-gamma, and heat-killed Listeria in wild-type but not in RICK- or Nod2-deficient macrophages. Finally, we show that the absence of RICK or double deficiency of Nod1 and Nod2 was associated with reduced cytokine production in Listeria-infected macrophages. These results demonstrate that RICK functions in innate immunity by mediating Nod1 and Nod2 signaling but not TLR-mediated immune responses.     

Varying dependency of periplasmic peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity

Periplasmic PPIases (peptidylprolyl cis-trans isomerases) catalyse the cis-trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.     

The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis

Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2-/- mice subcutaneously with Y. pestis GB. While TLR2-/- mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.     

Nod1 participates in the innate immune response to Pseudomonas aeruginosa

The mammalian innate immune system recognizes pathogen-associated molecular patterns through pathogen recognition receptors. Nod1 has been described recently as a cytosolic receptor that detects specifically diaminopimelate-containing muropeptides from Gram-negative bacteria peptidoglycan. In the present study we investigated the potential role of Nod1 in the innate immune response against the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that Nod1 detects the P. aeruginosa peptidoglycan leading to NF-kappaB activation and that this activity is diminished in epithelial cells expressing a dominant-negative Nod1 construct or in mouse embryonic fibroblasts from Nod1 knock-out mice infected with P. aeruginosa. Finally, we demonstrate that the cytokine secretion kinetics and bacterial killing are altered in Nod1-deficient cells infected with P. aeruginosa in the early stages of infection.     

Cross-tolerization between Nod1 and Nod2 signaling results in reduced refractoriness to bacterial infection in Nod2-deficient macrophages

Nod2 is an intracellular innate immune receptor that plays a role in host defense and susceptibility to inflammatory disease. We show in this study that macrophages rendered refractory to TLR4 and Nod2 signaling by exposure to LPS and muramyl dipeptide (MDP) exhibit impaired TNF-alpha and IL-6 production in response to pathogenic Listeria monocytogenes and Yersinia pseudotuberculosis as well as commensal bacteria including Escherichia coli and Bacteroides fragilis. Surprisingly, Nod2 deficiency was associated with impaired tolerization in response to pathogenic and commensal bacteria. Mechanistically, reduced tolerization of Nod2-null macrophages was mediated by recognition of bacteria through Nod1 because it was abolished in macrophages deficient in Nod1 and Nod2. Consistently, Nod2-null macrophages tolerant to LPS and MDP showed enhanced production of TNF-alpha and IL-6 as well as increased NF-kappaB and MAPK activation in response to the dipeptide KF1B, the Nod1 agonist. Furthermore, reduced tolerization of Nod2-deficient macrophages in response to bacteria was abolished when mutant macrophages were also rendered tolerant to the Nod1 ligand. Finally, MDP stimulation induced refractoriness not only to MDP, but also to iE-DAP stimulation, providing a mechanism to explain the reduced tolerization of Nod2-deficient macrophages infected with bacteria. These results demonstrate that cross-tolerization between Nod1 and Nod2 leads to increase recognition of both pathogenic and commensal bacteria in Nod2-deficient macrophages pre-exposed to microbial ligands.