Sepsis followed by multiple organ failure is a leading cause of death in noncoronary intensive care units. While the NLRC4 inflammasome has been shown to play a crucial role in the innate immune response, the role of NLRC4 in sepsis remains unclear. Here, we used NLRC4 gene-deficient mice to explore its role in cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Survival, bacterial clearance in the lung and extrapulmonary organs, and leukocyte influx to the peritoneum were determined. Chemokines and cytokines in the peritoneal fluid (PF) were quantified using ELISA. Mice were co-housed to compare the gut microbiota's effect on bacterial burden following sepsis. Here, we report that NLRC4 deficiency improves host survival and bacterial clearance in mice with CLP-induced sepsis. Nlrc4-/- mice displayed reduced numbers of total leukocytes in the PF, including neutrophils compared to wild-type (WT) mice at 12 and 24 h post-CLP, although the recruitment of macrophages in NLRC4 knockout mice was higher at 12 h. Nlrc4-/- mice displayed lower levels of cytokines and chemokines in PF following sepsis. Co-housing of WT and Nlrc4-/- mice suggests that NLRC4 regulates host defense in CLP-induced sepsis independently of gut microbiota. Depletion of macrophages demonstrated that NLRC4 deficiency protects macrophages from sepsis-induced immune dysfunction. Moreover, we observed higher CD4+, CD8+, IFN-γ+CD8+, and NK cell subsets in the spleen and lower level of apoptosis of spleen cells of NLRC4-deficient mice after sepsis. Overall, these findings identify that NLRC4 activation has a detrimental role in sepsis through modulating macrophages and T-cell responses.

MRSA is the leading cause of community and hospital-acquired pneumonia. However, the role of NLRP12 during MRSA-induced pneumonia remains elusive. Female wild-type (WT) and NLRP12 knockout (KO) mice were infected with MRSA (USA300, 5 x 107 CFU). We quantified cytokines and chemokines in the bronchoalveolar lavage fluid (BALF) and lung tissues and enumerated bacterial burden in the organs. We performed extracellular bacterial killing assays using bone marrow-derived neutrophils (BMDNs) from WT and KO mice. We also performed infection in mice after bone marrow transplantation. Finally, we depleted neutrophils from WT and KO mice and monitored survival after MRSA infection. NLRP12 is upregulated in neutrophils, macrophages, and epithelial cells of both human and murine pneumonic lungs. The KO mice had a higher survival rate than the WT mice, which was associated with lower bacterial burden in the lungs and spleens of the KO mice. The KO mice had higher levels of neutrophils and macrophages as well as cytokines/chemokines (IFN-γ and IL-17A) in BALF. The KO BMDNs are more potent in the extracellular killing of MRSA. Using chimeric mice, the deletion of NLRP12 in the hematopoietic cells increased bacterial clearance in the lungs. Furthermore, neutrophil depletion in the KO mice ameliorated the protection against MRSA pneumonia. Deletion of NLRP12 augments neutrophil and macrophage recruitment and increases survival during MRSA pneumonia. Supported by NIH F31HL168986-02, R01AI157353, R01A1180123, and 1RO1AI140500 Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

The role of Nrf2 in host defense against carbapenem-resistant Klebsiella pneumoniae (CRKP) (aka superbug) remains elusive. We conducted intracellular and extracellular bacterial killing assays using bone marrow-derived macrophages (BMDMs) and neutrophils (BMDNs) from wild type (WT) and Nrf2 knock out (Nrf2 KO) mice. We infected the WT and Nrf2 KO mice with CRKP and sacrificed the mice 48 h post-infection. We assessed bacterial burden in pulmonary and distal organs, performed bronchoalveolar lavage (BAL) phenotyping, analyzed lung pathology, examined emergency granulopoiesis, measured cytokines in BAL fluids (BALF) and monitored survival after infection with CRKP. Nrf2 was found to be upregulated in human and WT mouse pneumonic lungs. WT BMDMs are more potent in the phagocytosis and intracellular killing of opsonized CRKP than KO BMDMs although both WT and KO BMDNs are inefficient in extracellular killing. Also, KO BMDNs exhibit reduced NETosis after infection. Furthermore, KO mice infected with CRKP show augmented neutrophil and macrophage recruitment, production of inflammatory cytokines (TNFα and IL-1β) in BALF, lung pathology, pulmonary fibrosis, bacterial burden in the lungs and distal organs, and mortality. Moreover, KO mice display impaired emergency granulopoiesis after infection. Nrf2 is critical to host defense against CRKP pneumonia by modulating leukocyte homeostasis and function. Supported by NIH grants (R01AI157353, R01A1180123, 1RO1AI140500-01, 1P20GM130555-01, 1P20GM130555-06, 1R21AI133681-01A1) Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Although subsets with immunosuppressive properties exist, neutrophils are typically known for their pro-inflammatory role and pathogen clearance capabilities. Here, we reveal that neutrophils can paradoxically aid in resolving inflammation by actively producing anti-inflammatory extracellular vesicles. These large agingneutrophil-derived vesicles (LAND-Vs) do not fit into classical vesicle categorizations due to their specific size, structure, or biogenesis pathway. They are protected from efferocytotic clearance by phagocytes due to surface "do not eat me"signals and accumulate in the resolution phase of inflammation. CD55 on LAND-Vs exerts a robust, sustained anti-inflammatory effect by inhibiting complement 3 convertase, thereby reducing neutrophil recruitment and tissue damage. CD55+ LAND-Vs originate in ordered lipid raft domains, where CD55 accumulates asymmetrically during neutrophil aging, and are subsequently formed through RhoA-dependent budding. Collectively, LAND-V emerges as a pivotal physiological immunomodulator and showcases functions that transcend the limited lifespan of neutrophils, offering a therapeutic target for inflammatory and infectious diseases.

Plasma membrane perforation elicited by caspase cleavage of the gasdermin D (GSDMD) N-terminal domain (GSDMD-NT) triggers pyroptosis. The mechanisms underlying GSDMD membrane translocation and pore formation are not fully understood. Here, using a proteomic approach, we identified fatty acid synthase (FASN) as a GSDMD-binding partner. S-palmitoylation of GSDMD at Cys191/Cys192 (human/mouse), catalyzed by palmitoyl acyltransferases ZDHHC5 and ZDHHC9 and facilitated by reactive oxygen species (ROS), directly mediated membrane translocation of GSDMD-NT but not full-length GSDMD (GSDMD-FL). Palmitoylation of GSDMD-FL could be induced before inflammasome activation by stimuli such as lipopolysaccharide (LPS), consequently serving as an essential molecular event in macrophage priming. Inhibition of GSDMD palmitoylation suppressed macrophage pyroptosis and IL-1β release, mitigated organ damage, and enhanced the survival of septic mice. Thus, GSDMD-NT palmitoylation is a key regulatory mechanism controlling GSDMD membrane localization and activation, which may offer an additional target for modulating immune activity in infectious and inflammatory diseases.

Both lytic and apoptotic cell death remove senescent and damaged cells in living organisms. However, they elicit contrasting pro- and anti-inflammatory responses, respectively. The precise cellular mechanism that governs the choice between these two modes of death remains incompletely understood. Here we identify Gasdermin E (GSDME) as a master switch for neutrophil lytic pyroptotic death. The tightly regulated GSDME cleavage and activation in aging neutrophils are mediated by proteinase-3 and caspase-3, leading to pyroptosis. GSDME deficiency does not alter neutrophil overall survival rate; instead, it specifically precludes pyroptosis and skews neutrophil death towards apoptosis, thereby attenuating inflammatory responses due to augmented efferocytosis of apoptotic neutrophils by macrophages. In a clinically relevant acid-aspiration-induced lung injury model, neutrophil-specific deletion of GSDME reduces pulmonary inflammation, facilitates inflammation resolution, and alleviates lung injury. Thus, by controlling the mode of neutrophil death, GSDME dictates host inflammatory outcomes, providing a potential therapeutic target for infectious and inflammatory diseases.

IntroductionSepsis 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-1β 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.MethodsWe have used NLRP6 KO mice and human septic spleen samples to examine the role of NLRP6 in host defense in sepsis.ResultsNLRP6 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-1β, elicited excessive inflammation and reversed the survival advantages observed in NLRP6 KO mice.ConclusionThese 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.

Following respiratory infection or injury, neutrophil hyperactivation can damage surrounding lung tissue by releasing harmful compounds. In this issue of the JCI, Moussavi-Harami and colleagues identified tyrosine phosphatase SHP1 as a key negative regulator of neutrophil activation in acute respiratory distress syndrome (ARDS). Neutrophil-specific Shp1 disruption leads to hyperinflammation, pulmonary hemorrhage, and increased mortality in both sterile and pathogen-induced acute lung injury (ALI). Large intravascular neutrophil clusters and excessive PAD4-independent neutrophil extracellular traps (NETs) were identified as key features of lung injury. Mechanistically, Shp1 deficiency resulted in uncontrolled SYK kinase activation, driving chaotic neutrophil hyperactivation and inflammation.

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-β, 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.

This book explores the development of multilingual policy in education in Nepal in sociopolitical and historical contexts and examines the frameworks of language use in schools. It investigates the dynamics and factors that influence the process of construction and appropriation of the policy of multilingualism in education. The book surveys the language situation in schools and discusses how it is impacted by local language positions, societal power relations, ideological and identity contestations, and the attitude, language behaviour and resistance of key actors. It highlights the role of pedagogy, linguistics and politics that govern the policy of multilingual education. The author assesses the prospects of a multilingual approach to learning via teacher preparation, curriculum and learning material development, coordination of actors and institutions, and resources available in schools. The book presents Nepal’s linguistic background while discussing how multilingualism in education recognises local languages to improve the quality of learning in classrooms in ethnolinguistic communities. Evaluating the use of local languages in classrooms, it explores monolingual, multilingual and language maintenance frameworks of multilingualism in education. This book will be of interest to teachers, students, and researchers of education and educational studies, linguistics, sociology of education, school education, language studies, sociolinguistics, language policy and planning, public administration, ethnolinguistics, and sociology of language. It will also be useful to educationists, policymakers, linguists, sociolinguists and those working in related areas.