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1.  The NOD/RIP2 Pathway Is Essential for Host Defenses Against Chlamydophila pneumoniae Lung Infection 
PLoS Pathogens  2009;5(4):e1000379.
Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae–induced pneumonia in mice. Rip2−/− mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-γ levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2−/− mice compared to wild-type (WT) mice at day 3. Rip2−/− mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1−/− and Nod2−/− mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2−/− mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.
Author Summary
Chlamydophila pneumoniae (C. pneumoniae) is a common intracellular parasite that causes lung infections and contributes to several diseases characterized by chronic inflammation. Toll-like receptors expressed on the cell surface detect C. pneumoniae and mount a vigorous defense, but it is not known how the cell defends itself once the pathogen has taken up residence as a parasite. We reasoned that cytosolic pattern recognition receptors called Nods (nucleotide oligomerization domain) that detect microbes that gain entry into the cell might be involved. Using mice genetically deficient in Nod1 and Nod2 or their common downstream adaptor (Rip2), we show that in lung infection, Nod proteins are indeed essential in directing a defense against C. pneumoniae. Mice with defective Nod/Rip2-dependent signaling exhibited delayed recruitment of neutrophils, blunted production of pro-inflammatory cytokines and chemokines, and evidence of defective iNOS expression and NO production. These impaired responses led to delayed clearance of bacteria, intense persistent lung inflammation, and increased mortality. By performing bone marrow transplantation experiments and direct transfer of cells into the lungs of mice, we demonstrated that intact Nod-dependent signaling in bone marrow–derived cells was critical in the defense against C. pneumoniae. Our results indicate that Nod proteins also play an important role in host defense against C. pneumoniae. Coordinated and sequential activation of TLR and Nod signaling pathways may be necessary for an efficient immune response and host defense against C. pneumoniae.
doi:10.1371/journal.ppat.1000379
PMCID: PMC2660273  PMID: 19360122
2.  NOD2 Signaling Contributes to Host Defense in the Lungs against Escherichia coli Infection 
Infection and Immunity  2012;80(7):2558-2569.
Bacterial pneumonia remains a significant cause of mortality in the United States. The innate immune response is the first line of defense against invading bacteria. Neutrophil recruitment to the lungs is the first step in a multistep sequence leading to bacterial clearance. Ligand interaction with pattern-recognizing receptors (PRRs) leads to chemokine production, which drives neutrophils to the site of infection. Although we demonstrated that RIP2 is important for host defense in the lungs against Escherichia coli, the individual roles of NOD1 and NOD2 in pulmonary defense have not been addressed. Here, we explored the role of NOD2 in neutrophil-mediated host defense against an extracellular pathogen, E. coli. We found enhanced bacterial burden and reduced neutrophil and cytokine/chemokine levels in the lungs of NOD2−/− mice following E. coli infection. Furthermore, we observed reduced activation of NF-κB and mitogen-activated protein kinases (MAPKs) in the lungs of NOD2−/− mice upon E. coli challenge. Moreover, NOD2−/− neutrophils show impaired intracellular bacterial killing. Using NOD2/RIP2−/− mice, we observed bacterial burden and neutrophil accumulation in the lungs similar to those seen with NOD2−/− mice. In addition, bone marrow-derived macrophages obtained from NOD2/RIP2−/− mice demonstrate a reduction in activation of NF-κB and MAPKs similar to that seen with NOD2−/− mice in response to E. coli. These findings unveil a previously unrecognized role of the NOD2-RIP2 axis for host defense against extracellular Gram-negative bacteria. This pathway may represent a novel target for the treatment of lung infection/inflammation.
doi:10.1128/IAI.06230-11
PMCID: PMC3416478  PMID: 22547547
3.  Nonhematopoietic Cells Control the Outcome of Infection with Listeria monocytogenes in a Nucleotide Oligomerization Domain 1-Dependent Manner▿  
Infection and Immunity  2009;77(7):2908-2918.
We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1−/− mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480+ Gr1+ inflammatory monocytes and lower numbers of F480− Gr1+ neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1−/− mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1−/− BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1−/− BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.
doi:10.1128/IAI.01068-08
PMCID: PMC2708577  PMID: 19398545
4.  Cyclosporine A Impairs Nucleotide Binding Oligomerization Domain (Nod1)-Mediated Innate Antibacterial Renal Defenses in Mice and Human Transplant Recipients 
PLoS Pathogens  2013;9(1):e1003152.
Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may contribute to sensitizing kidney grafts to APN.
Author Summary
Patients who have received a kidney graft are treated with immunosuppressive drugs, such as the cyclosporine A (CsA). Transplanted patients under CsA are prone to bacterial infections. In this study, we used an experimental mouse model of kidney infection with Escherichia coli (E. coli) bacteria to study the effect of CsA. We show that CsA treatment of mice reduced their renal defense against E. coli. We found that CsA, in addition to its inhibitory action on the TLR4-mediated production of chemoattractant chemokines, also inhibited the expression of nucleotide-binding oligomerization domain 1 (Nod1), an intracellular receptor involved in the innate immune response against bacteria, in phagocytic cells. CsA acts by inhibiting the functions of the transcription factor NFAT. We show that NFAT is required for the proper expression of Nod1. Since Nod1 has already been reported to be involved in the phagocytic functions of polymorphonuclear neutrophils, we looked for and found a severe defect in neutrophil bacterial killing associated with reduced expression of Nod1 in both mice and patients treated by CsA. Interestingly, when mice treated with CsA are given synthetic molecules known to bind Nod1, this permitted the restoration of the Nod1 expression and renal defenses. This paper describes a novel mechanism which may explain, at least in part, why transplant patients receiving CsA have increased susceptibility to bacterial infection, and also provides a potential therapeutic strategy to restore renal antibacterial defenses.
doi:10.1371/journal.ppat.1003152
PMCID: PMC3561241  PMID: 23382681
5.  NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila 
European journal of immunology  2010;40(12):3519-3527.
The role of nucleotide-binding oligomerization domain-1 (NOD1) and nucleotide-binding oligomerization domain-2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat-killed Legionella and stimulate NF-κb and IFN-β promoter activity using an in vitro luciferase reporter system. We next infected NOD1- and NOD2-deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1–/– mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1–/– mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2–/– animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1–/– animals when compared to WT animals. In contrast, increased 4-h proinflammatory cytokines were seen in the Nod2–/– animals. Furthermore, the lungs of both Nod1–/– and Nod2–/– mice had significantly increased pro-inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.
doi:10.1002/eji.201040518
PMCID: PMC3063513  PMID: 21108472
Legionella pneumophila; NOD1; NOD2; Pneumonia
6.  NOD2-mediated Suppression of CD55 on Neutrophils Enhances C5a Generation During Polymicrobial Sepsis 
PLoS Pathogens  2013;9(5):e1003351.
Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that plays a defensive role in bacterial infection by sensing peptidoglycans. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, whether NOD2 regulates C5a generation during sepsis remains to be determined. To address this issue, cecal ligation & puncture (CLP)-induced sepsis was compared in wild type and Nod2−/− mice. Nod2−/− mice showed lower levels of C5a, IL-10, and IL-1β in serum and peritoneum, but higher survival rate during CLP-induced sepsis compared to wild type mice. Injection of recombinant C5a decreased survival rates of Nod2−/− mice rate during sepsis, whereas it did not alter those in wild type mice. These findings suggest a novel provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Furthermore, we found that NOD2-mediated IL-10 production by neutrophils enhanced C5a generation by suppressing CD55 expression on neutrophils in IL-1β-dependent and/or IL-1β-independent manners, thereby aggravating CLP-induced sepsis. SB203580, a receptor-interacting protein 2 (RIP2) inhibitor downstream of NOD2, reduced C5a generation by enhancing CD55 expression on neutrophils, resulting in attenuation of polymicrobial sepsis. Therefore, we propose a novel NOD2-mediated complement cascade regulatory pathway in sepsis, which may be a useful therapeutic target.
Author Summary
Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic protein that senses peptidoglycans of bacteria and exerts a defensive effect on bacterial infection. Sepsis is a complex dysregulated inflammatory response in bacterial infection, causing multiple organ dysfunction, coagulopathy, and fatal outcome. C5a, which has harmful effects in sepsis, interacts with innate proteins. However, it remains unclear the mechanism by which NOD2 affects sepsis responses in vivo by regulating C5a generation. Here, we demonstrate that NOD2 enhances C5a generation by IL-10-mediated suppression of CD55 expression on neutrophils, thereby aggravating polymicrobial sepsis. These findings suggest a provocative role for NOD2 in sepsis, in contrast to its protective role during bacterial infection. Therefore, we propose that a novel NOD2-mediated complement cascade regulatory pathway in neutrophils may be a useful therapeutic target for sepsis.
doi:10.1371/journal.ppat.1003351
PMCID: PMC3649968  PMID: 23675299
7.  Muramyl Dipeptide Induces NOD2-Dependent Ly6Chigh Monocyte Recruitment to the Lungs and Protects Against Influenza Virus Infection 
PLoS ONE  2012;7(5):e36734.
Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6Chigh “inflammatory” monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6Chigh monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6Chigh monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV.
doi:10.1371/journal.pone.0036734
PMCID: PMC3348889  PMID: 22590599
8.  Salmonella enterica serovar Typhimurium ΔmsbB Triggers Exacerbated Inflammation in Nod2 Deficient Mice 
PLoS ONE  2014;9(11):e113645.
The intracellular pathogen Salmonella enterica serovar Typhimurium causes intestinal inflammation characterized by edema, neutrophil influx and increased pro-inflammatory cytokine expression. A major bacterial factor inducing pro-inflammatory host responses is lipopolysaccharide (LPS). S. Typhimurium ΔmsbB possesses a modified lipid A, has reduced virulence in mice, and is being considered as a potential anti-cancer vaccine strain. The lack of a late myristoyl transferase, encoded by MsbB leads to attenuated TLR4 stimulation. However, whether other host receptor pathways are also altered remains unclear. Nod1 and Nod2 are cytosolic pattern recognition receptors recognizing bacterial peptidoglycan. They play important roles in the host's immune response to enteric pathogens and in immune homeostasis. Here, we investigated how deletion of msbB affects Salmonella's interaction with Nod1 and Nod2. S. Typhimurium Δ msbB-induced inflammation was significantly exacerbated in Nod2−/− mice compared to C57Bl/6 mice. In addition, S. Typhimurium ΔmsbB maintained robust intestinal colonization in Nod2−/− mice from day 2 to day 7 p.i., whereas colonization levels significantly decreased in C57Bl/6 mice during this time. Similarly, infection of Nod1−/− and Nod1/Nod2 double-knockout mice revealed that both Nod1 and Nod2 play a protective role in S. Typhimurium ΔmsbB-induced colitis. To elucidate why S. Typhimurium ΔmsbB, but not wild-type S. Typhimurium, induced an exacerbated inflammatory response in Nod2−/− mice, we used HEK293 cells which were transiently transfected with pathogen recognition receptors. Stimulation of TLR2-transfected cells with S. Typhimurium ΔmsbB resulted in increased IL-8 production compared to wild-type S. Typhimurium. Our results indicate that S. Typhimurium ΔmsbB triggers exacerbated colitis in the absence of Nod1 and/or Nod2, which is likely due to increased TLR2 stimulation. How bacteria with “genetically detoxified” LPS stimulate various innate responses has important implications for the development of safe and effective bacterial vaccines and adjuvants.
doi:10.1371/journal.pone.0113645
PMCID: PMC4244092  PMID: 25423082
9.  MyD88-, but Not Nod1- and/or Nod2-Deficient Mice, Show Increased Susceptibility to Polymicrobial Sepsis due to Impaired Local Inflammatory Response 
PLoS ONE  2014;9(8):e103734.
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.
doi:10.1371/journal.pone.0103734
PMCID: PMC4118952  PMID: 25084278
10.  Nucleotide-Binding Oligomerization Domain-1 and -2 Play No Role in Controlling Brucella abortus Infection in Mice 
Nucleotide-binding oligomerization domain proteins (NODs) are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. Further, several in vivo studies have demonstrated a role for Nod1 and Nod2 in host defense against bacterial pathogens. Here, we demonstrated that macrophages from NOD1-, NOD2-, and Rip2-deficient mice produced lower levels of TNF-α following infection with live Brucella abortus compared to wild-type mice. Similar reduction on cytokine synthesis was not observed for IL-12 and IL-6. However, NOD1, NOD2, and Rip2 knockout mice were no more susceptible to infection with virulent B. abortus than wild-type mice. Additionally, spleen cells from NOD1-, NOD2-, and Rip2-deficient mice showed unaltered production of IFN-γ compared to C57BL/6 mice. Taken together, this study demonstrates that NOD1, NOD2 and Rip2 are dispensable for the control of B. abortus during in vivo infection.
doi:10.1155/2012/861426
PMCID: PMC3235452  PMID: 22203860
11.  Nod1/Nod2-Mediated Recognition Plays a Critical Role in Induction of Adaptive Immunity to Anthrax after Aerosol Exposure▿  
Infection and Immunity  2009;77(10):4529-4537.
Toll-like receptors and Nod-like receptors (NLR) play an important role in sensing invading microorganisms for pathogen clearance and eliciting adaptive immunity for protection against rechallenge. Nod1 and Nod2, members of the NLR family, are capable of detecting bacterial peptidoglycan motifs in the host cytosol for triggering proinflammatory cytokine production. In the current study, we sought to determine if Nod1/Nod2 are involved in sensing Bacillus anthracis infection and eliciting protective immune responses. Using mice deficient in both Nod1 and Nod2 proteins, we showed that Nod1/Nod2 are involved in detecting B. anthracis for production of tumor necrosis factor alpha, interleukin-1α (IL-1α), IL-1β, CCL5, IL-6, and KC. Proinflammatory responses were higher when cells were exposed to viable spores than when they were exposed to irradiated spores, indicating that recognition of vegetative bacilli through Nod1/Nod2 is significant. We also identify a critical role for Nod1/Nod2 in priming responses after B. anthracis aerosol exposure, as mice deficient in Nod1/Nod2 were impaired in their ability to mount an anamnestic antibody response and were more susceptible to secondary lethal challenge than wild-type mice.
doi:10.1128/IAI.00563-09
PMCID: PMC2747953  PMID: 19620350
12.  Role of Nod1 in Mucosal Dendritic Cells during Salmonella Pathogenicity Island 1-Independent Salmonella enterica Serovar Typhimurium Infection ▿  
Infection and Immunity  2009;77(10):4480-4486.
Recent advances in immunology have highlighted the critical function of pattern-recognition molecules (PRMs) in generating the innate immune response to effectively target pathogens. Nod1 and Nod2 are intracellular PRMs that detect peptidoglycan motifs from the cell walls of bacteria once they gain access to the cytosol. Salmonella enterica serovar Typhimurium is an enteric intracellular pathogen that causes a severe disease in the mouse model. This pathogen resides within vacuoles inside the cell, but the question of whether cytosolic PRMs such as Nod1 and Nod2 could have an impact on the course of S. Typhimurium infection in vivo has not been addressed. Here, we show that deficiency in the PRM Nod1, but not Nod2, resulted in increased susceptibility toward a mutant strain of S. Typhimurium that targets directly lamina propria dendritic cells (DCs) for its entry into the host. Using this bacterium and bone marrow chimeras, we uncovered a surprising role for Nod1 in myeloid cells controlling bacterial infection at the level of the intestinal lamina propria. Indeed, DCs deficient for Nod1 exhibited impaired clearance of the bacteria, both in vitro and in vivo, leading to increased organ colonization and decreased host survival after oral infection. Taken together, these findings demonstrate a key role for Nod1 in the host response to an enteric bacterial pathogen through the modulation of intestinal lamina propria DCs.
doi:10.1128/IAI.00519-09
PMCID: PMC2747964  PMID: 19620349
13.  Inflammasome-dependent Pyroptosis and IL-18 Protect against Burkholderia pseudomallei Lung Infection while IL-1β Is Deleterious 
PLoS Pathogens  2011;7(12):e1002452.
Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3-/- mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4-/- mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4-/- mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β.
Author Summary
The disease melioidosis is caused by the intracellular bacterium Burkholderia pseudomallei, a potential bioterrorism agent. Here we examined the interaction of B. pseudomallei with the inflammasome, an important innate immune pathway that regulates at least two host responses protective against infections: 1) secretion of the proinflammatory cytokines IL-1β and IL-18 and 2) induction of pyroptosis, a form of cell death that restricts intracellular bacteria growth. Using a mouse model of melioidosis we show that two distinct inflammasomes are activated by B. pseudomallei infection. One, containing the Nod-like receptor (NLR) NLRP3, mediates IL-1β and IL-18 induction. The other contains a different NLR called NLRC4 and mediates pyroptosis. Pyroptosis and IL-18 production were equally important for resistance to B. pseudomallei. Surprisingly, IL-1β was found to be deleterious in melioidosis. The detrimental role of IL-1β during melioidosis was due, in part, to excessive recruitment of neutrophils to the lung. We show that neutrophils do not express NLRC4, fail to undergo pyroptosis, and, therefore, may be permissive to B. pseudomallei intracellular replication leading to increased bacterial burden and morbidity/mortality. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β.
doi:10.1371/journal.ppat.1002452
PMCID: PMC3248555  PMID: 22241982
14.  Nod1 and Nod2 signaling does not alter the composition of intestinal bacterial communities at homeostasis 
Gut Microbes  2013;4(3):222-231.
Patients with inflammatory bowel diseases (IBD) harbour intestinal bacterial communities with altered composition compared with healthy counterparts; however, it is unknown whether changes in the microbiota are associated with genetic susceptibility of individuals for developing disease or instead reflect other changes in the intestinal environment related to the disease itself. Since deficiencies in the innate immune receptors Nod1 and Nod2 are linked to IBD, we tested the hypothesis that Nod-signaling alters intestinal immune profiles and subsequently alters bacterial community structure. We used qPCR to analyze expression patterns of selected immune mediators in the ileum and cecum of Nod-deficient mice compared with their Nod-sufficient littermates and assessed the relative abundance of major bacterial groups sampled from the ileum, cecum and colon. The Nod1-deficient ileum exhibited significantly lower expression of Nod2, Muc2, α- and β-defensins and keratinocyte-derived chemokine (KC), suggesting a weakened epithelial barrier compared with WT littermates; however, there were no significant differences in the relative abundance of targeted bacterial groups, indicating that Nod1-associated immune differences alone do not promote dysbiosis. Furthermore, Nod2-deficient mice did not display any changes in the expression of immune markers or bacterial communities. Shifts in bacterial communities that were observed in this study correlated with housing conditions and were independent of genotype. These findings emphasize the importance of using F2 littermate controls to minimize environmental sources of variation in microbial analyses, to establish baseline conditions for host-microbe homeostasis in Nod-deficient mice and to strengthen models for testing factors contributing to microbial dysbiosis associated with IBD.
doi:10.4161/gmic.24373
PMCID: PMC3669167  PMID: 23549220
Nod-like receptors; gut bacterial communities; homeostasis; intestinal environment; inflammation
15.  Defects in Innate Immunity Predispose C57BL/6J-Leprdb/Leprdb Mice to Infection by Staphylococcus aureus▿  
Infection and Immunity  2008;77(3):1008-1014.
Foot and ankle infections are the most common cause of hospitalization among diabetic patients, and Staphylococcus aureus is a major pathogen implicated in these infections. Patients with insulin-resistant (type 2) diabetes are more susceptible to bacterial infections than nondiabetic subjects, but the pathogenesis of these infections is poorly understood. C57BL/6J-Leprdb/Leprdb (hereafter, db/db) mice develop type 2 diabetes due to a recessive, autosomal mutation in the leptin receptor. We established a S. aureus hind paw infection in diabetic db/db and nondiabetic Lepr+/+ (+/+) mice to investigate host factors that predispose diabetic mice to infection. Nondiabetic +/+ mice resolved the S. aureus hind paw infection within 10 days, whereas db/db mice with persistent hyperglycemia developed a chronic infection associated with a high bacterial burden. Diabetic db/db mice showed a more robust neutrophil infiltration to the infection site and higher levels of chemokines in the infected tissue than +/+ mice. Blood from +/+ mice killed S. aureus in vitro, whereas db/db blood was defective in bacterial killing. Compared with peripheral blood neutrophils from +/+ mice, db/db neutrophils demonstrated a diminished respiratory burst when stimulated with S. aureus. However, bone marrow-derived neutrophils from +/+ and db/db mice showed comparable phagocytosis and bactericidal activity. Our results indicate that diabetic db/db mice are more susceptible to staphylococcal infection than their nondiabetic littermates and that persistent hyperglycemia modulates innate immunity in the diabetic host.
doi:10.1128/IAI.00976-08
PMCID: PMC2643648  PMID: 19103772
16.  Immunomodulatory Function of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Autoimmune Type 1 Diabetes1 
Human clinical trials in type 1 diabetes (T1D) patients using mesenchymal stem cells (MSC) are presently underway without prior validation in a mouse model for the disease. In response to this void, we characterized bone marrow-derived murine MSC for their ability to modulate immune responses in the context of T1D, as represented in NOD mice. In comparison to NOD mice, BALB/ c-MSC mice were found to express higher levels of the negative costimulatory molecule PD-L1 and to promote a shift toward Th2-like responses in treated NOD mice. In addition, transfer of MSC from resistant strains (i.e., nonobese resistant mice or BALB/c), but not from NOD mice, delayed the onset of diabetes when administered to prediabetic NOD mice. The number of BALB/c-MSC trafficking to the pancreatic lymph nodes of NOD mice was higher than in NOD mice provided autologous NOD-MSC. Administration of BALB/c-MSC temporarily resulted in reversal of hyperglycemia in 90% of NOD mice (p = 0.002). Transfer of autologous NOD-MSC imparted no such therapeutic benefit. We also noted soft tissue and visceral tumors in NOD-MSC-treated mice, which were uniquely observed in this setting (i.e., no tumors were present with BALB/c- or nonobese resistant mice-MSC transfer). The importance of this observation remains to be explored in humans, as inbred mice such as NOD may be more susceptible to tumor formation. These data provide important preclinical data supporting the basis for further development of allogeneic MSC-based therapies for T1D and, potentially, for other autoimmune disorders.
doi:10.4049/jimmunol.0900803
PMCID: PMC3895445  PMID: 19561093
17.  The PepT1–NOD2 Signaling Pathway Aggravates Induced Colitis in Mice 
Gastroenterology  2011;141(4):1334-1345.
Background & Aims
The human di/tripeptide transporter hPepT1 is abnormally expressed in colons of patients with inflammatory bowel disease, although its exact role in pathogenesis is unclear. We investigated the contribution of PepT1 to intestinal inflammation in mouse models of colitis and the involvement of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway in the pathogenic activity of colonic epithelial hPepT1.
Methods
Transgenic mice were generated in which hPepT1 expression was regulated by the β-actin or villin promoters; colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) and the inflammatory responses were assessed. The effects of NOD2 deletion in the hPepT1 transgenic mice was also studied, to determine the involvement of the PepT1–NOD2 signaling pathway.
Results
TNBS and DSS induced more severe levels of inflammation in β-actin–hPepT1 transgenic mice than wild-type littermates. Intestinal epithelial cell (IEC)-specific hPepT1 overexpression in villin-hPepT1 transgenic mice increased the severity of inflammation induced by DSS, but not TNBS. Bone marrow transplantation studies demonstrated that hPepT1 expression in IECs and immune cells has an important role in the proinflammatory response. Antibiotics abolished the effect of hPepT1 overexpression on the inflammatory response in DSS-induced colitis in β-actinh–PepT1 and villin-hPepT1 transgenic mice, indicating that commensal bacteria are required to aggravate intestinal inflammation. Nod2−/−, β-actin–hPepT1 transgenic/Nod2−/−, and villinhPepT1 transgenic/Nod2−/− littermates had similar levels of susceptibility to DSS-induced colitis, indicating that hPepT1 overexpression increased intestinal inflammation in a NOD2-dependent manner.
Conclusions
The PepT1–NOD2 signaling pathway is involved in aggravation of DSS-induced colitis in mice.
doi:10.1053/j.gastro.2011.06.080
PMCID: PMC3186842  PMID: 21762661
IBD; mouse model; immune response; bacteria-derived peptides
18.  The role of NOD2 in murine and human melioidosis 
Journal of immunology (Baltimore, Md. : 1950)  2013;192(1):10.4049/jimmunol.1301436.
NOD2 is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments, and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-κB activation induced by B. pseudomallei stimulation of HEK293 cells. After low dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared to wild type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1,562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole blood stimulation with the NOD2 ligand, MDP, or B. pseudomallei. These findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.
doi:10.4049/jimmunol.1301436
PMCID: PMC3872087  PMID: 24298015
Burkholderia pseudomallei; melioidosis; NOD2; innate immunity; genetic variation; animal model; pneumonia; sepsis
19.  The role of NOD2 in murine and human melioidosis 
NOD2 is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments, and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-κB activation induced by B. pseudomallei stimulation of HEK293 cells. After low dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared to wild type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1,562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole blood stimulation with the NOD2 ligand, MDP, or B. pseudomallei. These findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.
doi:10.4049/jimmunol.1301436
PMCID: PMC3872087  PMID: 24298015
Burkholderia pseudomallei; melioidosis; NOD2; innate immunity; genetic variation; animal model; pneumonia; sepsis
20.  Contribution of Phagocytosis and Intracellular Sensing for Cytokine Production by Staphylococcus aureus-Activated Macrophages▿  
Infection and Immunity  2006;75(2):830-837.
Toll-like receptors (TLRs) are involved in the sensing of microbially derived compounds. We analyzed the contribution of these receptors to cytokine production by macrophages following stimulation with whole bacteria. Using knockout mice, we confirmed that the TLR4 and TLR2 contribution was predominant in the induction of tumor necrosis factor alpha and interleukin-10 by gram-negative bacteria. In contrast, the absence of TLR2 and/or TLR4 or TLR9 did not affect the response to gram-positive bacteria. In the absence of TLR2, phagocytosis was essential for cytokine production in response to heat-killed Staphylococcus aureus (HKSA). Because intracellular sensing was important in the absence of TLR2, we evaluated the contribution of Nod1 and Nod2, intracytoplasmic sensors of peptidoglycan-derived muropeptides, to the response to HKSA. By transfecting RAW 264.7 macrophages with dominant negative (DN) forms of Nod1 and Nod2, we showed that both molecules inhibited NF-κB activation in response to HKSA. The unexpected interference of DN Nod1 in the response of macrophages to gram-positive bacteria was confirmed with a Nod2 agonist (muramyl dipeptide) in transfection experiments with HEK293T cell. Taken together, these results show the contribution of phagocytosis and Nod molecules to the response to HKSA in macrophages and also identify possible cross talk between Nod1 and Nod2.
doi:10.1128/IAI.01199-06
PMCID: PMC1828506  PMID: 17118979
21.  Enhancement of Neutrophil Function by Interleukin-18 Therapy Protects Burn-Injured Mice from Methicillin-Resistant Staphylococcus aureus ▿ 
Infection and Immunity  2011;79(7):2670-2680.
Methicillin-resistant Staphylococcus aureus (MRSA) infection is a grave concern in burn-injured patients. We investigated the efficacy of interleukin-18 (IL-18) treatment in postburn MRSA infection. Alternate-day injections of IL-18 into burn-injured C57BL/6 mice significantly increased their survival after MRSA infection and after methicillin-sensitive S. aureus infection. Although IL-18 treatment of burn-injured mice augmented natural IgM production before MRSA infection and gamma interferon (IFN-γ) production after MRSA infection, neither IgM nor IFN-γ significantly contributed to the improvement in mouse survival. IL-18 treatment increased/restored the serum tumor necrosis factor (TNF), IL-17, IL-23, granulocyte colony-stimulating factor (G-CSF), and macrophage inflammatory protein (MIP-2) levels, as well as the neutrophil count, after MRSA infection of burn-injured mice; it also improved impaired neutrophil functions, phagocytic activity, production of reactive oxygen species, and MRSA-killing activity. However, IL-18 treatment was ineffective against MRSA infection in both burn- and sham-injured neutropenic mice. Enhancement of neutrophil functions by IL-18 was also observed in vitro. Furthermore, when neutrophils from IL-18-treated burn-injured mice were adoptively transferred into nontreated burn-injured mice 2 days after MRSA challenge, survival of the recipient mice increased. NOD-SCID mice that have functionally intact neutrophils and macrophages (but not T, B, or NK cells) were substantially resistant to MRSA infection. IL-18 treatment increased the survival of NOD-SCID mice after burn injury and MRSA infection. An adoptive transfer of neutrophils using NOD-SCID mice also showed a beneficial effect of IL-18-activated neutrophils, similar to that seen in C57BL/6 mice. Thus, although neutrophil functions were impaired in burn-injured mice, IL-18 therapy markedly activated neutrophil functions, thereby increasing survival from postburn MRSA infection.
doi:10.1128/IAI.01298-10
PMCID: PMC3191962  PMID: 21536791
22.  Induction of Nod1 and Nod2 Intracellular Pattern Recognition Receptors in Murine Osteoblasts following Bacterial Challenge  
Infection and Immunity  2005;73(5):2967-2973.
Osteoblasts produce an array of immune molecules following bacterial challenge that could recruit leukocytes to sites of infection and promote inflammation during bone diseases, such as osteomyelitis. Recent studies from our laboratory have shed light on the mechanisms by which this cell type can perceive and respond to bacteria by demonstrating the functional expression of members of the Toll-like family of cell surface pattern recognition receptors by osteoblasts. However, we have shown that bacterial components fail to elicit immune responses comparable with those seen following challenge with the intracellular pathogens salmonellae and Staphylococcus aureus. In the present study, we show that UV-killed bacteria and invasion-defective bacterial strains elicit significantly less inflammatory cytokine production than their viable wild-type counterparts. Importantly, we demonstrate that murine osteoblasts express the novel intracellular pattern recognition receptors Nod1 and Nod2. Levels of mRNA encoding Nod molecules and protein expression are significantly and differentially increased from low basal levels following exposure to these disparate bacterial pathogens. In addition, we have shown that osteoblasts express Rip2 kinase, a critical downstream effector molecule for Nod signaling. Furthermore, to begin to establish the functional nature of Nod expression, we show that a specific ligand for Nod proteins can significantly augment immune molecule production by osteoblasts exposed to either UV-inactivated bacteria or bacterial lipopolysaccharide. As such, the presence of Nod proteins in osteoblasts could represent an important mechanism by which this cell type responds to intracellular bacterial pathogens of bone.
doi:10.1128/IAI.73.5.2967-2973.2005
PMCID: PMC1087386  PMID: 15845503
23.  Host Genetics and Environmental Factors Regulate Ecological Succession of the Mouse Colon Tissue-Associated Microbiota 
PLoS ONE  2012;7(1):e30273.
Background
The integration of host genetics, environmental triggers and the microbiota is a recognised factor in the pathogenesis of barrier function diseases such as IBD. In order to determine how these factors interact to regulate the host immune response and ecological succession of the colon tissue-associated microbiota, we investigated the temporal interaction between the microbiota and the host following disruption of the colonic epithelial barrier.
Methodology/Principal Findings
Oral administration of DSS was applied as a mechanistic model of environmental damage of the colon and the resulting inflammation characterized for various parameters over time in WT and Nod2 KO mice.
Results
In WT mice, DSS damage exposed the host to the commensal flora and led to a migration of the tissue-associated bacteria from the epithelium to mucosal and submucosal layers correlating with changes in proinflammatory cytokine profiles and a progressive transition from acute to chronic inflammation of the colon. Tissue-associated bacteria levels peaked at day 21 post-DSS and declined thereafter, correlating with recruitment of innate immune cells and development of the adaptive immune response. Histological parameters, immune cell infiltration and cytokine biomarkers of inflammation were indistinguishable between Nod2 and WT littermates following DSS, however, Nod2 KO mice demonstrated significantly higher tissue-associated bacterial levels in the colon. DSS damage and Nod2 genotype independently regulated the community structure of the colon microbiota.
Conclusions/Significance
The results of these experiments demonstrate the integration of environmental and genetic factors in the ecological succession of the commensal flora in mammalian tissue. The association of Nod2 genotype (and other host polymorphisms) and environmental factors likely combine to influence the ecological succession of the tissue-associated microflora accounting in part for their association with the pathogenesis of inflammatory bowel diseases.
doi:10.1371/journal.pone.0030273
PMCID: PMC3260280  PMID: 22272321
24.  MDP-NOD2 stimulation induces HNP-1 secretion which contributes to NOD2 anti-bacterial function 
Inflammatory bowel diseases  2010;16(5):736-742.
Background
Human Neutrophil Peptide 1 (HNP-1) is a defensin with antibacterial activity secreted by various cells as a component of the innate immune host defense. NOD2 is a cytoplasmic protein that recognizes bacterial derived muramyl dipeptide, and is involved in bacterial clearance. The aim of the present study was to investigate the relationship between antibacterial activity of NOD2 and HNP-1 expression in epithelial cell lines.
Methods
Gentamicin protection assay using Salmonella Typhimurium was performed in Caco-2 cells. mRNA level was determined by quantitative RT-PCR and defensin expression was assessed by Western Blot and ELISA. Nuclear Factor-κB activation was assessed using pIV luciferase and renilla plasmids. NOD2 mutant was generated by site-directed mutagenesis.
Results
Among the defensins tested, only HNP-1 expression is induced in colonic epithelial model HCT116 cells after MDP-LD stimulation. HNP-1 secretion is significantly increased after MDP-LD stimulation in the cell supernatant of intestinal epithelial cells expressing endogenous NOD2, but not in cells which lack endogenous NOD2 expression. HNP-1 is required for NOD2 dependent NF-κB activation after MDP-LD stimulation since hnp-1 siRNA transfection abrogated response to MDP-LD stimulation. The antibacterial function of NOD2 against S. Typhimurium was impaired when expression of HNP-1 was blocked by siRNA.
Conclusions
HNP-1 secretion depends on NOD2 stimulation by MDP-LD and contributes to antibacterial activity in intestinal epithelial cells expressing endogenous NOD2, but not NOD2 3020insC mutant associated with increased susceptibility to Crohn’s disease.
doi:10.1002/ibd.21144
PMCID: PMC2895931  PMID: 19856414
Crohn’s disease; NOD2; HNP-1; invasive bacteria
25.  Role of Mouse Peptidoglycan Recognition Protein PGLYRP2 in the Innate Immune Response to Salmonella enterica Serovar Typhimurium Infection In Vivo 
Infection and Immunity  2012;80(8):2645-2654.
Peptidoglycan recognition proteins (PGRPs) are a family of innate pattern recognition molecules that bind bacterial peptidoglycan. While the role of PGRPs in Drosophila innate immunity has been extensively studied, how the four mammalian PGRP proteins (PGLYRP1 to PGLYRP4) contribute to host defense against bacterial pathogens in vivo remains poorly understood. PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal in vitro, whereas PGLYRP2 is an N-acetylmuramyl-l-alanine amidase that cleaves peptidoglycan between the sugar backbone and the peptide stem. Because PGLYRP2 cleaves muramyl peptides detected by host peptidoglycan sensors Nod1 and Nod2, we speculated that PGLYRP2 may act as a modifier of Nod1/Nod2-dependent innate immune responses. We investigated the role of PGLYRP2 in Salmonella enterica serovar Typhimurium-induced colitis, which is regulated by Nod1/Nod2 through the induction of an early Th17 response. PGLYRP2 did not contribute to expression of Th17-associated cytokines, interleukin-22 (IL-22)-dependent antimicrobial proteins, or inflammatory cytokines. However, we found that Pglyrp2-deficient mice displayed significantly enhanced inflammation in the cecum at 72 h postinfection, reflected by increased polymorphonuclear leukocyte (PMN) infiltration and goblet cell depletion. Pglyrp2 expression was also induced in the cecum of Salmonella-infected mice, and expression of green fluorescent protein under control of the Pglyrp2 promoter was increased in discrete populations of intraepithelial lymphocytes. Lastly, Nod2−/− Pglyrp2−/− mice displayed increased susceptibility to infection at 24 h postinfection compared to Pglyrp2−/− mice, which correlated with increased PMN infiltration and submucosal edema. Thus, PGLYRP2 plays a protective role in vivo in the control of S. Typhimurium infection through a Nod1/Nod2-independent mechanism.
doi:10.1128/IAI.00168-12
PMCID: PMC3434585  PMID: 22615249

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