NLRP6 negatively regulates host defense against polymicrobial sepsis.

Introduction: Sepsis remains a major cause of death in Intensive Care Units. Sepsis is a life-threatening multi-organ dysfunction caused by a dysregulated systemic inflammatory response. Pattern recognition receptors, such as TLRs and NLRs contribute to innate immune responses. Upon activation, some NLRs form multimeric protein complexes in the cytoplasm termed "inflammasomes" which induce gasdermin d-mediated pyroptotic cell death and the release of mature forms of IL-1ß and IL-18. The NLRP6 inflammasome is documented to be both a positive and a negative regulator of host defense in distinct infectious diseases. However, the role of NLRP6 in polymicrobial sepsis remains elusive. Methods: We have used NLRP6 KO mice and human septic spleen samples to examine the role of NLRP6 in host defense in sepsis. Results: NLRP6 KO mice display enhanced survival, reduced bacterial burden in the organs, and reduced cytokine/chemokine production. Co-housed WT and KO mice following sepsis show decreased bacterial burden in the KO mice as observed in singly housed groups. NLRP6 is upregulated in CD3, CD4, and CD8 cells of septic patients and septic mice. The KO mice showed a higher number of CD3, CD4, and CD8 positive T cell subsets and reduced T cell death in the spleen following sepsis. Furthermore, administration of recombinant IL-18, but not IL-1ß, elicited excessive inflammation and reversed the survival advantages observed in NLRP6 KO mice. Conclusion: These results unveil NLRP6 as a negative regulator of host defense during sepsis and offer novel insights for the development of new treatment strategies for sepsis.

Role of toll-like receptors and nod-like receptors in acute lung infection.

The respiratory system exposed to microorganisms continuously, and the pathogenicity of these microbes not only contingent on their virulence factors, but also the host's immunity. A multifaceted innate immune mechanism exists in the respiratory tract to cope with microbial infections and to decrease tissue damage. The key cell types of the innate immune response are macrophages, neutrophils, dendritic cells, epithelial cells, and endothelial cells. Both the myeloid and structural cells of the respiratory system sense invading microorganisms through binding or activation of pathogen-associated molecular patterns (PAMPs) to pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and NOD-like receptors (NLRs). The recognition of microbes and subsequent activation of PRRs triggers a signaling cascade that leads to the activation of transcription factors, induction of cytokines/5chemokines, upregulation of cell adhesion molecules, recruitment of immune cells, and subsequent microbe clearance. Since numerous microbes resist antimicrobial agents and escape innate immune defenses, in the future, a comprehensive strategy consisting of newer vaccines and novel antimicrobials will be required to control microbial infections. This review summarizes key findings in the area of innate immune defense in response to acute microbial infections in the lung. Understanding the innate immune mechanisms is critical to design host-targeted immunotherapies to mitigate excessive inflammation while controlling microbial burden in tissues following lung infection.

Challenges in the combination of radiotherapy and immunotherapy for breast cancer.

INTRODUCTION: Immunotherapy (IT) is showing promise in the treatment of breast cancer, but IT alone only benefits a minority of patients. Radiotherapy (RT) is usually included in the standard of care for breast cancer patients and is traditionally considered as a local form of treatment. The emerging knowledge of RT-induced systemic immune response, and the observation that the rare abscopal effect of RT on distant cancer metastases can be augmented by IT, have increased the enthusiasm for combinatorial immunoradiotherapy (IRT) for breast cancer patients. However, IRT largely follows the traditional sole RT and IT protocols and does not consider patient specificity, although patients' responses to treatment remain heterogeneous. AREAS COVERED: This review discusses the rationale of IRT for breast cancer, the current knowledge, challenges, and future directions. EXPERT OPINION: The synergy between RT and the immune system has been observed but not well understood at the basic level. The optimal dosages, timing, target, and impact of biomarkers are largely unknown. There is an urgent need to design efficacious pre-clinical and clinical trials to optimize IRT for cancer patients, maximize the synergy of radiation and immune response, and explore the abscopal effect in depth, taking into account patients' personal features.

Regulation of emergency granulopoiesis during infection.

During acute infectious and inflammatory conditions, a large number of neutrophils are in high demand as they are consumed in peripheral organs. The hematopoietic system rapidly responds to the demand by turning from steady state to emergency granulopoiesis to expedite neutrophil generation in the bone marrow (BM). How the hematopoietic system integrates pathogenic and inflammatory stress signals into the molecular cues of emergency granulopoiesis has been the subject of investigations. Recent studies in the field have highlighted emerging concepts, including the direct sensing of pathogens by BM resident or sentinel hematopoietic stem and progenitor cells (HSPCs), the crosstalk of HSPCs, endothelial cells, and stromal cells to convert signals to granulopoiesis, and the identification of novel inflammatory molecules, such as C/EBP-ß, ROS, IL-27, IFN-γ, CXCL1 with direct effects on HSPCs. In this review, we will provide a detailed account of emerging concepts while reassessing well-established cellular and molecular players of emergency granulopoiesis. While providing our views on the discrepant results and theories, we will postulate an updated model of granulopoiesis in the context of health and disease.

Host Defense against Klebsiella pneumoniae Pneumonia Is Augmented by Lung-Derived Mesenchymal Stem Cells.

Klebsiella pneumoniae is a common cause of Gram-negative pneumonia. The spread of antibiotic-resistant and hypervirulent strains has made treatment more challenging. This study sought to determine the immunomodulatory, antibacterial, and therapeutic potential of purified murine stem cell Ag-1+ (Sca-1+) lung mesenchymal stem cells (LMSCs) using in vitro cell culture and an in vivo mouse model of pneumonia caused by K pneumoniae. Sca-1+ LMSCs are plastic adherent, possess colony-forming capacity, express mesenchymal stem cell markers, differentiate into osteogenic and adipogenic lineages in vitro, and exhibit a high proliferative capacity. Further, these Sca-1+ LMSCs are morphologically similar to fibroblasts but differ ultrastructurally. Moreover, Sca-1+ LMSCs have the capacity to inhibit LPS-induced secretion of inflammatory cytokines by bone marrow-derived macrophages and neutrophils in vitro. Sca-1+ LMSCs inhibit the growth of K pneumoniae more potently than do neutrophils. Sca-1+ LMSCs also possess the intrinsic ability to phagocytize and kill K. pneumoniae intracellularly. Whereas the induction of autophagy promotes bacterial replication, inhibition of autophagy enhances the intracellular clearance of K. pneumoniae in Sca-1+ LMSCs during the early time of infection. Adoptive transfer of Sca-1+ LMSCs in K. pneumoniae-infected mice improved survival, reduced inflammatory cells in bronchoalveolar lavage fluid, reduced inflammatory cytokine levels and pathological lesions in the lung, and enhanced bacterial clearance in the lung and in extrapulmonary organs. To our knowledge, these results together illustrate for the first time the protective role of LMSCs in bacterial pneumonia.

NLRP6 modulates neutrophil homeostasis in bacterial pneumonia-derived sepsis.

Bacterial pneumonia is a significant cause of morbidity, mortality, and health care expenditures. Optimum neutrophil recruitment and their function are critical defense mechanisms against respiratory pathogens. The nucleotide-binding oligomerization domain-like receptor (NLRP) 6 controls gut microbiota and immune response to systemic and enteric infections. However, the importance of NLRP6 in neutrophil homeostasis following lung infection remains elusive. To investigate the role of NLRs in neutrophil homeostasis, we used Nlrp6 gene-deficient (Nlrp6-/-) mice in a model of Klebsiella pneumoniae-induced pneumonia-derived sepsis. We demonstrated that NLRP6 is critical for host survival, bacterial clearance, neutrophil influx, and CXC-chemokine production. Kp-infected Nlrp6-/- mice have reduced numbers of hematopoietic stem cells and granulocyte-monocyte progenitors but increased retention of matured neutrophils in bone marrow. Neutrophil extracellular trap (NET) formation and NET-mediated bacterial killing were also impaired in Nlrp6-/- neutrophils in vitro. Furthermore, recombinant CXCL1 rescued the impaired host defense, granulopoietic response, and NETosis in Kp-infected Nlrp6-/- mice. Using A/J background mice and co-housing experiments, our findings revealed that the susceptible phenotype of Nlrp6-/- mice is not strain-specific and gut microbiota-dependent. Taken together, these data unveil NLRP6 as a central regulator of neutrophil recruitment, generation, and function during bacterial pneumonia followed by sepsis.

Tristetraprolin Overexpression in Non-hematopoietic Cells Protects Against Acute Lung Injury in Mice.

Tristetraprolin (TTP) is a mRNA binding protein that binds to adenylate-uridylate-rich elements within the 3' untranslated regions of certain transcripts, such as tumor necrosis factor (Tnf) mRNA, and increases their rate of decay. Modulation of TTP expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. Accordingly, we tested the role of TTP in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. LPS-challenged TTP-knockout (TTPKO) mice, as well as myeloid cell-specific TTP-deficient (TTPmyeKO) mice, exhibited significant increases in lung injury, although these responses were more robust in the TTPKO. Mice with systemic overexpression of TTP (TTPΔARE) were protected from ALI, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ALI. Interestingly, while irradiated wild-type (WT) mice reconstituted with TTPKO hematopoietic progenitor cells (HPCs) showed exaggerated ALI, their reconstitution with the TTPΔARE HPCs mitigated ALI. The reconstitution of irradiated TTPΔARE mice with HPCs from either WT or TTPΔARE donors conferred significant protection against ALI. In contrast, irradiated TTPΔARE mice reconstituted with TTPKO HPCs had exaggerated ALI, but the response was milder as compared to WT recipients that received TTPKO HPCs. Finally, the reconstitution of irradiated TTPKO recipient mice with TTPΔARE HPCs did not confer any protection to the TTPKO mice. These data together suggest that non-HPCs-specific overexpression of TTP within the lungs protects against ALI via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration.

The NLRP6 inflammasome in health and disease.

NACHT, LRR (leucine-rich repeat), and PYD (pyrin domain) domain-containing 6 (Nlrp6) is a member of the NLR (nucleotide-oligomerization domain-like receptor) family that patrols the cytosolic compartment of cells to detect pathogen- and damage-associated molecular patterns. Because Nlrp6 is a recently discovered inflammasome, details of its signaling mechanism, structural assembly, and roles in host defense have yet to be determined. To date, Nlrp6 has been proposed to perform a multitude of functions ranging from control of microbiota, maintenance of epithelial integrity, and regulation of metabolic diseases to modulation of host defense during microbial infections, cancer protection, and regulation of neuroinflammation. While recent studies have questioned some of the proposed functions of Nlrp6, Nlrp6 has been shown to form an inflammasome complex and cleaves interleukin-1ß (IL-1ß) and IL-18 during microbial infection, indicating that it is a bonafide inflammasome. In this review, we summarize the recent advancements in knowledge of the signaling mechanisms and structure of the Nlrp6 inflammasome and discuss the relevance of NLRP6 to human disease.

CXCL1 regulates neutrophil homeostasis in pneumonia-derived sepsis caused by Streptococcus pneumoniae serotype 3.

Neutrophil migration to the site of bacterial infection is a critical step in host defense. Exclusively produced in the bone marrow, neutrophil release into the blood is tightly controlled. Although the chemokine CXCL1 induces neutrophil influx during bacterial infections, its role in regulating neutrophil recruitment, granulopoiesis, and neutrophil mobilization in response to lung infection-induced sepsis is unclear. Here, we used a murine model of intrapulmonary Streptococcus pneumoniae infection to investigate the role of CXCL1 in host defense, granulopoiesis, and neutrophil mobilization. Our results demonstrate that CXCL1 augments neutrophil influx to control bacterial growth in the lungs, as well as bacterial dissemination, resulting in improved host survival. This was shown in Cxcl1 -/- mice, which exhibited defective amplification of early neutrophil precursors in granulocytic compartments, and CD62L- and CD49d-dependent neutrophil release from the marrow. Administration of recombinant CXCL2 and CXCL5 after infection rescues the impairments in neutrophil-dependent host defense in Cxcl1 -/- mice. Taken together, these findings identify CXCL1 as a central player in host defense, granulopoiesis, and mobilization of neutrophils during Gram-positive bacterial pneumonia-induced sepsis.

NLRC4 suppresses IL-17A-mediated neutrophil-dependent host defense through upregulation of IL-18 and induction of necroptosis during Gram-positive pneumonia.

Gram-positive pathogens, including Staphylococcus aureus, cause necrotizing pneumonia. The central feature of S. aureus pneumonia is toxin-induced necroptosis of immune and resident cells, which impedes host defense. However, the role of the NLRC4 in the lung following S. aureus infection remains elusive. Here, we demonstrate that S. aureus activates the NLRC4 to drive necroptosis and IL-18 production, which impaired IL-17A-dependent neutrophil-mediated host susceptibility. In particular, Nlrc4-/- mice exhibit reduced necroptosis, enhanced neutrophil influx into the lungs, decreased bacterial burden, and improved host survival. Loss of NLRC4 signaling in both hematopoietic and non-hematopoietic cells contributes to the host protection against S. aureus pneumonia. Secretion of IL-17A by γδ T cells is essential for neutrophil recruitment into the lungs of Nlrc4-/- mice following infection. Moreover, treatment of wild-type mice with necroptosis inhibitors or genetic ablation of MLKL and IL-18 improves host defense against S. aureus infection, which is associated with increased IL-17A+γδ T cells and neutrophils. Taken together, these novel findings reveal that S. aureus activates the NLRC4 to dampen IL-17A-dependent neutrophil accumulation through induction of necroptosis and IL-18. Thus, modulating the function of the NLRC4 may be an attractive therapeutic approach for treating S. aureus infections.

NLRP6 negatively regulates pulmonary host defense in Gram-positive bacterial infection through modulating neutrophil recruitment and function.

Gram-positive bacteria, including Staphylococcus aureus are endemic in the U.S., which cause life-threatening necrotizing pneumonia. Neutrophils are known to be critical for clearance of S. aureus infection from the lungs and extrapulmonary organs. Therefore, we investigated whether the NLRP6 inflammasome regulates neutrophil-dependent host immunity during pulmonary S. aureus infection. Unlike their wild-type (WT) counterparts, NLRP6 knockout (KO) mice were protected against pulmonary S. aureus infection as evidenced by their higher survival rate and lower bacterial burden in the lungs and extrapulmonary organs. In addition, NLRP6 KO mice displayed increased neutrophil recruitment following infection, and when neutrophils were depleted the protective effect was lost. Furthermore, neutrophils from the KO mice demonstrated enhanced intracellular bacterial killing and increased NADPH oxidase-dependent ROS production. Intriguingly, we found higher NK cell-mediated IFN-γ production in KO mouse lungs, and treatment with IFN-γ was found to enhance the bactericidal ability of WT and KO neutrophils. The NLRP6 KO mice also displayed decreased pyroptosis and necroptosis in the lungs following infection. Blocking of pyroptosis and necroptosis in WT mice resulted in increased survival, reduced bacterial burden in the lungs, and attenuated cytokine production. Taken together, these novel findings show that NLRP6 serves as a negative regulator of neutrophil-mediated host defense during Gram-positive bacterial infection in the lungs through regulating both neutrophil influx and function. These results also suggest that blocking NLRP6 to augment neutrophil-associated bacterial clearance should be considered as a potential therapeutic intervention strategy for treatment of S. aureus pneumonia.