Role of NOD2/RIP2 signaling in acute bacterial pneumonia and sepsis

Bacterial pneumonia and sepsis are two important causes of mortality in the world. Emergence of multidrug resistant bacteria has necessitated the development of new treatment and/or prevention strategies to augment host immune defense. In this context, the innate host defense is critical in clearing pathogenic bacteria from the host. Early neutrophil recruitment is a critical step in a multistep requence leading to bacterial clearance. Pattern recognition receptors (PRRs) play a critical role in the innate immune system. Receptor interacting protein 2 (RIP-2) is an adaptor for the nod-like receptors (NLR) NOD1 and NOD2. Nucleotide oligomerisation domain 2 (NOD2) is an intracellular PRR that is shown to be important for host defense against intracellular bacterial pathogens. However, the role of NOD2 and RIP-2 during Gram-negative bacterial pneumonia and polymicrobial sepsis has not been explored. Thus, we hypothesize that the NOD2/RIP-2 axis is critical for host defense during bacterial pneumonia and sepsis/septic peritonitis. To test this hypothesis, we infected NOD2(NOD2-/-), RIP-2(RIP-2-/-) deficient mice intratracheally (i.t) with E. coli (106 CFUs/mouse) and Klebsiella pneumoniae (103 CFUs/mouse). We observed that NOD2/RIP2 signaling is critical for the host defense during gram-negative pneumonia and poly microbial sepsis. The NOD2/RIP2 axis regulates neutrophil recruitment via IL-17A production. We also found that NOD2/RIP-2 signaling is essential for the production of IL-6 and activation of STAT3. We demonstrated that RIP-2 regulates inflammasome activity that is independent of NOD2 signaling. Taken together, these data demonstrate that the NOD2/RIP-2 axis plays a critical role in neutrophil-mediated host defense through IL-17A production and by inflammasome activation. In cecal ligation puncture (CLP) induced sepsis, RIP2-/- mice show increased mortality with higher bacterial burden in the peritoneum and systemic organs compared to WT controls. We found reduced neutrophil influx IL-17A and IL-1beta levels in the peritoneum of RIP2-/- mice after CLP. Furthermore, we also observed increased systemic inflammation accompanied by vital organ damage in the knockout mice. As a whole our data suggest a critical role of RIP2 in neutrophil recruitment, along with IL-17A and IL-1beta during sepsis

Review: Toll-like receptors and NOD-like receptors in pulmonary antibacterial immunity

Lung diseases caused by bacteria are a leading cause of death in both immunocompromised and immunocompetent individuals as well as in children. Although neutrophil recruitment is critical to augment the host defence, excessive neutrophil accumulation results in life-threatening diseases, such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Therefore, it is important to modulate excessive neutrophil influx in ALI/ARDS to mitigate lung damage and mortality. A better understanding of the basic mechanisms underlying neutrophil influx is crucial to designing novel and innovative treatment strategies for ALI/ARDS. Recognition of bacteria in the lung is the critical first step leading to neutrophil influx. Pattern recognition receptors, such as Toll-like receptors and NOD-like receptors, play an important role in the recognition of bacterial pathogens. Understanding the molecular and cellular mechanisms associated with the recognition of bacterial pathogens by the host is critical for the development of effective therapeutic strategies to control parenchymal damage via modulating neutrophil accumulation in the lung

Intrapulmonary G-CSF rescues neutrophil recruitment to the lung and neutrophil release to blood in Gram-negative bacterial infection in MCP-1-/- mice

We previously demonstrated that MCP-1 is important for E. coli-induced neutrophil migration to the lungs. However, E. coli neither disseminates nor induces death in mice. Furthermore, the cell types and the host defense mechanisms that contribute to MCP-1-dependent neutrophil trafficking have not been defined. In this study, we sought to explore the cell types and the mechanisms associated with Klebsiella pneumoniae-mediated MCP-1-dependent neutrophil influx. MCP-1(-/-) mice are more susceptible to pulmonary K. pneumoniae infection and show higher bacterial burden in the lungs and dissemination. MCP-1(-/-) mice also display attenuated neutrophil influx, cytokine/chemokine production, and activation of NF-κB and MAPKs following intratracheal K. pneumoniae infection. rMCP-1 treatment in MCP-1(-/-) mice following K. pneumoniae infection rescued impairment in survival, bacterial clearance, and neutrophil accumulation in the lung. Neutrophil numbers in the blood of MCP-1(-/-) mice were associated with G-CSF concentrations in bronchoalveolar lavage fluid and blood. Bone marrow or resident cell-derived MCP-1 contributed to bacterial clearance, neutrophil accumulation, and cytokine/chemokine production in the lungs following infection. Furthermore, exogenous MCP-1 dose dependently increased neutrophil counts and G-CSF concentrations in the blood. Intriguingly, administration of intratracheal rG-CSF to MCP-1(-/-) mice after K. pneumoniae infection rescued survival, bacterial clearance and dissemination, and neutrophil influx in MCP-1(-/-) mice. Collectively, these novel findings unveil an unrecognized role of MCP-1 in neutrophil-mediated host immunity during K. pneumoniae pneumonia and illustrate that G-CSF could be used to rescue impairment in host immunity in individuals with absent or malfunctional MCP-1

Intrapulmonary G-CSF Rescues Neutrophil Recruitment to the Lung and Neutrophil Release to Blood in Gram-Negative Bacterial Infection in MCP-1 −/−

We previously demonstrated that MCP-1 is important for E. coli-induced neutrophil migration to the lungs. However, E. coli neither disseminates nor induces death in mice. Furthermore, the cell types and the host defense mechanisms which contribute to MCP-1-dependent neutrophil trafficking have not been defined. In this study, we sought to explore the cell types and the mechanisms associated with K. pneumoniae-mediated MCP-1-dependent neutrophil influx. MCP-1(−/−) mice are more susceptible to pulmonary K. pneumoniae infection and show higher bacterial burden in the lungs and dissemination. MCP-1(−/−) mice also display attenuated neutrophil influx, cytokine/chemokine production and activation of NF-κB and MAPKs following intratracheal (i.t) K. pneumoniae infection. Recombinant MCP-1 (rMCP-1) treatment in MCP-1(−/−) mice following K. pneumoniae infection rescued impairment in survival, bacterial clearance and neutrophil accumulation in the lung. Neutrophil numbers in the blood of MCP-1(−/−) mice were associated with G-CSF concentrations in BALF and blood. Bone marrow or resident cell-derived MCP-1 contributed to bacterial clearance, neutrophil accumulation, and cytokine/chemokine production in the lungs following infection. Furthermore, exogenous MCP-1 dose dependently increased neutrophil counts and G-CSF concentrations in the blood. Intriguingly, administration of i.t. rG-CSF to MCP-1(−/−) mice after K. pneumoniae infection rescued survival, bacterial clearance and dissemination, and neutrophil influx in MCP-1(−/−) mice. Collectively, these novel findings a) hitherto unveil an unrecognized role of MCP-1 in neutrophil mediated host immunity during K. pneumoniae pneumonia; and b) illustrate that G-CSF could be used to rescue impairment in host immunity in individuals with lacking or malfunctional MCP-1

The E3 ubiquitin ligase Itch inhibits p38α signaling and skin inflammation through the ubiquitylation of Tab1.

Deficiency in the E3 ubiquitin ligase Itch causes a skin-scratching phenotype in mice. We found that there was increased phosphorylation and activation of the mitogen-activated protein kinase p38α in spontaneous and experimentally induced skin lesions of Itch-deficient (Itch(-/-)) mice. Itch bound directly to the TGF-β-activated kinase 1-binding protein 1 (Tab1) through a conserved PPXY motif and inhibited the activation of p38α. Knockdown of Tab1 by short hairpin RNA attenuated the prolonged p38α phosphorylation exhibited by Itch(-/-) cells. Similarly, reconstitution of Itch(-/-) cells with wild-type Itch, but not the ligase-deficient Itch-C830A mutant, inhibited the phosphorylation and activation of p38α. Compared to the skin of wild-type mice, the skin of Itch(-/-) mice contained increased amounts of the mRNAs of proinflammatory cytokines, including tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-1β, IL-11, and IL-19. Inhibition of p38 or blocking the interaction between p38α and Tab1 with a cell-permeable peptide substantially attenuated skin inflammation in Itch(-/-) mice. These findings provide insight into how Itch-mediated regulatory mechanisms prevent chronic skin inflammation, which could be exploited therapeutically

Stromal Fibroblasts Mediate Anti–PD-1 Resistance via MMP-9 and Dictate TGFβ Inhibitor Sequencing in Melanoma

Although anti-PD-1 therapy has improved clinical outcomes for select patients with advanced cancer, many patients exhibit either primary or adaptive resistance to checkpoint inhibitor immunotherapy. The role of the tumor stroma in the development of these mechanisms of resistance to checkpoint inhibitors remains unclear. We demonstrated that pharmacologic inhibition of the TGF beta signaling pathway synergistically enhanced the efficacy of anti-CTLA-4 immunotherapy but failed to augment anti-PD-1/PD-L1 responses in an autochthonous model of BRAF(V600E) melanoma. Additional mechanistic studies revealed that TGF beta pathway inhibition promoted the proliferative expansion of stromal fibroblasts, thereby facilitating MMP-9-dependent cleavage of PD-L1 surface expression, leading to anti-PD-1 resistance in this model. Further work demonstrated that melanomas escaping anti-PD-1 therapy exhibited a mesenchymal phenotype associated with enhanced TGF beta signaling activity. Delayed TGF beta inhibitor therapy, following anti-PD-1 escape, better served to control further disease progression and was superior to a continuous combination of anti-PD-1 and TGF beta inhibition. This work illustrates that formulating immunotherapy combination regimens to enhance the efficacy of checkpoint blockade requires an in-depth understanding of the impact of these agents on the tumor microenvironment. These data indicated that stromal fibroblast MMP-9 may desensitize tumors to anti-PD-1 and suggests that TGF beta inhibition may generate greater immunologic efficacy when administered following the development of acquired anti-PD-1 resistance. (C) 2018 AACR