Interleukin-33 (IL-33) is a central regulator of immune responses and inflammation, and genetic variation in IL33 and its receptor IL1RL1 (ST2) is strongly linked to disease susceptibility, notably asthma. Emerging evidence suggests that IL-33 also influences hematopoietic processes and platelet biology, indicating functions beyond canonical immunity. We investigated two missense variants identified in a patient with unexplained thrombocytopenia: a rare novel IL33 variant, c.385T>C (p.Tyr129His) and the common IL1RL1 variant c.1501_1502CA>AG (p.Gln501Arg), previously associated with reduced IL-33 signaling. Structural modeling revealed that IL-33 Y129H disrupts a conserved hydrogen bond within the IL-33/ST2/IL-1RAcP ternary complex, destabilizing receptor engagement. Functional assays confirmed markedly reduced binding affinity and biological activity, establishing Y129H as a loss-of-function variant. The IL1RL1 Q501R variant affects the Toll/IL-1 receptor (TIR) domain, critical for recruiting adapter proteins such as MyD88. Modeling revealed pronounced perturbation in a peripheral helix of the TIR domain, potentially impacting adapter recruitment and downstream signaling, providing a mechanistic basis for its protective association with asthma. Together, these findings provide structural and functional insights into clinically relevant IL33 and IL1RL1 variants. Their simultaneous occurrence in a patient with thrombocytopenia further supports a potential role for this pathway in platelet homeostasis and stress-responsive hematopoiesis.

Abstract Platelets, traditionally recognized for their involvement in hemostasis and wound healing, also play a central role in immune regulation and inflammation. Their function and production adapt in response to inflammatory cues such as cytokines and danger-associated molecular patterns. Interleukin-33 (IL-33), an alarmin released during tissue damage, particularly in lung inflammation, has been implicated in influencing platelet biology, though its exact effects remain poorly understood. To clarify IL-33’s role, we examined its impact on platelet production, proteome, adhesion, secretion, and aggregation using platelets from IL-33-deficient (IL-33 knockout [IL-33KO]) mice and IL-33 stimulation in vivo. Our results reveal that although platelets themselves do not express IL-33, platelets isolated from IL-33KO mice display altered proteomic signatures and reduced adhesion to fibrinogen, podoplanin, and laminin, alongside impaired thrombus formation under shear stress. IL-33 administration in vivo led to proteomic remodeling characterized by increased expression of inflammatory proteins, as well as changes in platelet morphology, including increased size, typically associated with de novo production. Using lung intravital microscopy, we visualized platelet fragmentation within the lung vasculature in real time, and observed enhanced fragmentation following IL-33 stimulation. Interestingly, ST2, the receptor for IL-33, is expressed in subsets of mouse and human megakaryocytes and hematopoietic progenitors, particularly those involved in a noncanonical pathway of thrombopoiesis that enables the rapid replenishment of platelets during inflammation, infection, and aging. Together, these findings identify IL-33 as a pivotal regulator of platelet function and production, linking inflammatory signaling to the dynamic regulation of thrombopoiesis.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB), results in more human mortality than any other single pathogen, in part because of the lack of an effective vaccine. Although T cells are essential for immunity to TB, the mechanisms that provide protective immunity are poorly understood. In this Review, we describe current gaps in our knowledge about T cell-mediated immune responses to M. tuberculosis and discuss how recent technologies, including multiphoton intravital microscopy, spatial multiomics and high-resolution in vivo analyses of cell–cell interactions, may be used to gain insights that can inform the design of T cell-targeted TB vaccines. T cells have an essential role in immune responses to Mycobacterium tuberculosis, but the mechanisms by which they may provide protective immunity remain poorly understood. This Review explores the knowns and unknowns of T cell immunity in tuberculosis and how recent technologies may inform the design of T cell-targeted TB vaccines.

CD8 + T cells are robustly activated during tuberculosis but how their responses differ in susceptible hosts remains unclear. Using mice lacking the transcriptional repressor Sp140, we assessed the magnitude and diversity of pulmonary CD8⁺ T cell responses to Mycobacterium tuberculosis infection. We show that control mice develop a robust CD8 + T cell response following infection, characterized by a diverse pool of effector and memory subsets and strong TNF and IFNγ production, whereas Sp140 -/- mice display a profound reduction in T cell numbers across all subsets. Single-cell RNA sequencing revealed redistribution and skewing of CD8 + T cell clusters in Sp140 -/- mice, with overrepresentation of gene expression programs associated with exhaustion and type I interferon (IFN-I) signaling. Blockade of the IFN-I receptor (IFNAR) restored CD8 + T cell numbers, diversity, cytokine production, spatial localization, and coincided with substantially reduced bacterial burden and lung pathology. Similarly, CD4 + T cell numbers were also rescued. Intravital microscopy of infected lungs further showed that T cell dynamics and motility within lesions were restricted under exuberant IFN-I signaling but fully restored by IFNAR blockade. Together, these findings reveal that SP140 sustains host resistance in mice by restraining IFN-I-driven pathology, coinciding with preserved T cell immunity and lesion surveillance.

Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9. A combination of large-scale proteomic analyses, lung intravital microscopy, and adoptive transfer of ILC9 cells revealed that high IL-9 expression distinguishes a multicytokine-producing state-of-activated ILC2s with an increased capacity to initiate IL-5-dependent allergic airway inflammation. Similar to IL-33 and TSLP, TL1A is expressed in airway basal cells in healthy and asthmatic human lungs. Together, these results indicate that TL1A is an epithelium-derived cytokine and an important cofactor of IL-33 in the airways.

Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces, and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis (Mtb) infection alters the phenotype and function of lung IL-18Rα+ ILC toward a protective interferon-γ-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program. Moreover, a BCG-driven type I milieu enhances the early generation of ILC1-like cells during secondary challenge with Mtb. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen-tailored immune response.

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

Megakaryocytes (MKs) are large cells giving rise to platelets. It is well established that in adults, MKs develop from hematopoietic stem cells and reside in the bone marrow. MKs are also rare but normal constituents of the venous blood returning to the lungs, and MKs are found in the lung vasculature (MKcirc), suggesting that these cells are migrants from the bone marrow and get trapped in lung capillaries where the final steps of platelet production can occur. An unprecedented increase in the number of lung and circulating MKs was described in coronavirus disease 2019 (COVID-19) patients, suggesting that lung thrombopoiesis may be increased during lung infection and/or thromboinflammation. In addition to the population of platelet-producing intravascular MKs in the lung, a population of lung-resident megakaryocytes (MKL) has been identified and presents a specific immune signature compared to its bone marrow counterparts. Recent single-cell analysis and intravital imaging have helped us gain a better understanding of these populations in mouse and human. This review aims at summarizing the recent data on increased occurrence of lung MKs and discusses their origin, specificities, and potential role in homeostasis and inflammatory and infectious lung diseases. Here, we address remaining questions, controversies, and methodologic challenges for further studies of both MKcirc and MKL.

Background Recent reports revealed that CD8+ T cell antitumor responses rely on a subset of stem-like CD8+ T cells during cancer immunity and immunotherapy. However, the origin of stem-like CD8+ T cells and the mechanisms controlling their trafficking are not well established. High endothelial venules (HEVs) are specialized blood vessels mediating lymphocyte trafficking in lymph nodes (LNs),1,2 and HEV-like blood vessels defined as tumor-associated HEVs (TA-HEVs) are important regulators of lymphocyte entry into tumors.3 Here, we investigated the role of LN-HEVs and TA-HEVs in the generation and migration of stem-like CD8+ T cells during spontaneous antitumor immunity and cancer immunotherapy. Methods In mouse syngeneic tumor models, we applied multidisciplinary approaches (immunofluorescence, flow cytometry, short-term homing assays and in vivo imaging) to identify the adhesion pathways used by LN-HEVs and TA-HEVs for CD8+ T cell trafficking. Then, using in vivo function-blocking antibodies targeting LN-HEVs and/or TA-HEVs, we defined their respective roles in the recruitment of naive CD44-PD-1- CD8+ T cells into tumor-draining lymph node and of CD44+PD-1+TCF-1high stem-like CD8+ T cells into tumor. Lastly, we blocked the function of LN-HEVs and/or TA-HEVs during combined anti-PD-1 plus anti-CTLA-4 cancer immunotherapy to analyze their contributions in treatment-induced CD8+ T cell responses. Results We found that naive and stem-like CD8+ T cells use distinct adhesion pathways to interact with LN-HEVs and TA-HEVs, respectively. Blocking naive CD8+ T cell adhesion to LN-HEVs and their subsequent entry into tumor-draining lymph node abrogated the generation of stem-like CD8+ T cells and prevented their accumulation in tumor, showing that peripheral and intratumoral responses are tightly related. In peripheral blood, circulating stem-like CD8+ T cells expressed adhesion molecules and chemokine receptors enabling interaction with TA-HEVs that mediated their entry into tumor. Finally, we found that checkpoint blockade therapy increased the number and activity of stem-like CD8+ T cells in tumor-draining lymph node and peripheral blood, suggesting that peripheral responses are involved during treatment. Accordingly, HEV-mediated trafficking in tumor, but also in tumor-draining lymph node, were required for expansion and differentiation of intratumoral stem-like CD8+ T cells during checkpoint blockade therapy. Conclusions In this study, we unveil the dual role of HEVs in tumor and periphery for CD8+ T cell responses during cancer immunity and immunotherapy, and provide mechanistic insights on the importance of peripheral responses during checkpoint blockade therapy. Our findings also strongly support the use of combined anti-PD-1 plus anti-CTLA-4 cancer immunotherapy in the neoadjuvant setting. Acknowledgements This work was supported by grants from Fondation ARC pour la Recherche sur le Cancer (PGA1 RF20180206911 to J-PG), Institut National du Cancer (INCa_2017-155 to J-PG). LB and EV were supported by fellowships from Fondation pour la Recherche Médicale (ECO201806006827, ECO202006011469, and FDT202106012889). References Moussion C, Girard JP. Dendritic cells control lymphocyte entry to lymph nodes through high endothelial venules. Nature. 2011;479:542–546. Blanchard L, Girard JP. High endothelial venules (HEVs) in immunity, inflammation and cancer. Angiogenesis. 2021;24:719–753. Asrir A*, Tardiveau C*, Coudert J*, Laffont R*, Blanchard L*, Bellard E, Veerman K, Bettini S, Lafouresse F, Vina E, Tarroux D, Roy S, Girault I, Molinaro I, Martins F, Scoazec JY, Ortega N, Robert C, Girard JP. Tumor-associated high endothelial venules mediate lymphocyte entry into tumors and predict response to PD-1 plus CTLA-4 combination immunotherapy. Cancer Cell. 2022;40:318–334. (* co-first authors) Ethics Approval This study obtained ethical approvals by the French Ministry of Research and the FRBT (C2EA-01) animal care committee (Projects APAFIS#12256-2017112021529157v3 and APAFIS#23416-2019122019025727v3).

Multiple neutrophil death programs contribute to host defense against infections. Although expressing all necessary components, neutrophils specifically fail to undergo pyroptosis, a lytic form of cell death triggered by the activation of the pro-inflammatory complex inflammasome. In the light of the arm race, we hypothesized that intrinsic neutrophil pyroptosis resistance might be bypassed in response to specific microbial species. We show that Pseudomonas aeruginosa ( P. aeruginosa ) stimulates Caspase-1-dependent pyroptosis in human and murine neutrophils. Mechanistically, activated NLRC4 inflammasome supports Caspase-1-driven Gasdermin-D (GSDMD) activation, IL-1β cytokine release and neutrophil pyroptosis. Furthermore, GSDMD activates Peptidyl Arginine Deaminase-4 which drives an “incomplete NETosis” where neutrophil DNA fills the cell cytosol but fails crossing plasma membrane. Finally, we show that neutrophil Caspase-1 account for IL-1β production and contributes to various P. aeruginosa strains spread in mice. Overall, we demonstrate that neutrophils are fully competent for Caspase-1-dependent pyroptosis, which drives an unsuspected “incomplete NETosis”. Summary Neutrophils play an essential roles against infections. Although multiple neutrophil death programs contribute to host defense against infections, they fail to undergo pyroptosis, a pro-inflammatory form of cell death. Upon Infections, pyroptosis can be induced in macrophages or epithelial cells upon activation of pro-inflammatory complexes, inflammasomes that trigger Caspase-1-driven Gasdermin dependent plasma membrane lysis. In the light of host-microbe interactions, we hypothesized that yet to find microbial species might hold the capacity to overcome neutrophil resistance to inflammasome-driven pyroptosis. Among several bacterial species, we describe that the bacterium Pseudomonas aeruginosa specifically engages the NLRC4 inflammasome, which promotes Caspase-1-dependent Gasdermin-D activation and subsequent neutrophil pyroptosis. Furthermore, inflammasome-driven pyroptosis leads to DNA decondensation and expansion into the host cell cytosol but not to the so called Neutrophil Extracellular Trap (NET) release as DNA fails breaching the plasma membrane. Finally, in vivo P. aeruginosa infections highlight that Caspase-1-driven neutrophil pyroptosis is functional and is detrimental upon P. aeruginosa infection. Altogether, our results unexpectedly underline neutrophil competence for Caspase-1-dependent pyroptosis, a process that contributes to host susceptibility to P. aeruginosa infection.

Neutrophils mediate essential immune and microbicidal processes. Consequently, to counteract neutrophil attack, pathogens have developed various virulence strategies. Here, we showed that Pseudomonas aeruginosa (P. aeruginosa) phospholipase ExoU drives pathological NETosis in neutrophils. Surprisingly, inhibition of ExoU activity uncovered a fully functional Caspase-1-driven pyroptosis pathway in neutrophils. Mechanistically, activated NLRC4 inflammasome promoted Caspase-1-dependent Gasdermin-D activation, IL-1β cytokine release and neutrophil pyroptosis. Whereas both pyroptotic and netotic neutrophils released alarmins, only NETosis liberated the destructive DAMPs Histones, which exacerbated Pseudomonas-induced mouse lethality. To the contrary, subcortical actin allowed pyroptotic neutrophils to physically limit poisonous inflammation by keeping Histones intracellularly. Finally, mouse models of infection highlighted that both NETosis and neutrophil Caspase-1 contributed to P. aeruginosa spreading. Overall, we established the host deleterious consequences of Pseudomonas-induced-NETosis but also uncovered an unsuspected ability of neutrophils to undergo Caspase-1-dependent pyroptosis, a process where neutrophils exhibit a self-regulatory function that limit Histone release.P.aeruginosaExoU (right) triggers phospholipid degradation and subsequent neutrophil lysis that associates to NETosis through F-Actin collapse/degradation and PAD4-dependent DNA decondensation. P.aeruginosaExoU- (left) triggers NLRC4-dependent pyroptosis in neutrophils, which leads to PAD4- depndent DNA decondensation but not expulsion due to a still function subcortical F- Actin network. Created with Biorender.com.

Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis and protect against pathogens at mucosal surfaces and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues in their tissue of residence remains to be fully understood. Here we show that Mycobacterium tuberculosis infection alters the biology of lung ILCs and, in particular, induces the emergence of a non-classical, protective, interferon-γ-producing ILC1-like population. Adoptive transfer, fate-mapping and in vitro differentiation experiments revealed that ILC1-like cells originate from immature ILC2 rather than from mature ILC2. This plasticity is controlled by type 1 cytokines and a glycolytic program involving the transcription factor HIF1α. Collectively, our data reveal how tissue-resident ILCs adapt to their inflammatory and metabolic environment to undergo phenotypic and functional changes toward a pathogen-adapted immune response.

Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis infection alters the phenotype and function of immature lung ILC2 toward a protective interferon-γ-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program involving the transcription factor HIF1α. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen tailored immune response.

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

Platelets are recruited to inflammatory foci and contribute to host defense and inflammatory responses. Compared with platelet recruitment in hemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defense are poorly understood. Neutrophil recruitment to lung airspaces after inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice after LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils. BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and, 48 hours later, lungs were collected and platelets and neutrophils quantified in tissue sections by immunohistochemistry. The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules, P-selectin or PSGL 1, or treatment with a neutrophil-depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre X mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs. Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation. In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL‐1&bgr; secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL‐1&bgr; in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program‐3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA‐low and ‐high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program‐3 cohort is “eDNA‐high,” as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA‐low patients with asthma, eDNA‐high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values <0.05). Sputum eDNA in asthma was associated with airway neutrophilic inflammation, increases in soluble NET components, and increases in caspase 1 activity and IL‐1&bgr; (all P values <0.001). In in vitro studies, NETs caused cytotoxicity in airway epithelial cells that was prevented by disruption of NETs with DNase. Conclusions: High extracellular DNA concentrations in sputum mark a subset of patients with more severe asthma who have NETs and markers of inflammasome activation in their airways.

Neutrophils dominate the early immune response in pathogen-induced acute lung injury, but efforts to harness their responses have not led to therapeutic advancements. Neutrophil extracellular traps (NETs) have been proposed as an innate defense mechanism responsible for pathogen clearance, but there are concerns that NETs may induce collateral damage to host tissues. Here, we detected NETs in abundance in mouse models of severe bacterial pneumonia/acute lung injury and in human subjects with acute respiratory distress syndrome (ARDS) from pneumonia or sepsis. Decreasing NETs reduced lung injury and improved survival after DNase I treatment or with partial protein arginine deiminase 4 deficiency (PAD4(+/-)). Complete PAD4 deficiency (PAD4(-/-)) reduced NETs and lung injury but was counterbalanced by increased bacterial load and inflammation. Importantly, we discovered that the lipoxin pathway could be a potent modulator of NET formation, and that mice deficient in the lipoxin receptor (Fpr2(-/-)) produced excess NETs leading to increased lung injury and mortality. Lastly, we observed in humans that increased plasma NETs were associated with ARDS severity and mortality, and lower plasma DNase I levels were associated with the development of sepsis-induced ARDS. We conclude that a critical balance of NETs is necessary to prevent lung injury and to maintain microbial control, which has important therapeutic implications.