To reach inflamed tissues, neutrophils must traverse capillaries as narrow as 2 micrometers. However, how they do so without compromising blood flow or capillary function has remained unclear. By combining intravital live-cell imaging with biomimetic microdevices, we show that neutrophils maintain migration speed in capillaries across increasing levels of confinement. This behavior was not shared by other leukocytes and was independent of nuclear properties. Instead, confinement rapidly engaged Rho-dependent actomyosin contractility at the cell rear, thereby offsetting the increased mechanical resistance imposed by confinement. Disrupting this adaptive response caused neutrophil jamming and eventual occlusion of confined capillary-like networks. These findings identify a neutrophil-intrinsic mechanism that couples capillary confinement to rapid migratory adaptation, helping preserve vascular patency and potentially limiting tissue dysfunction during inflammation.
Both memory and de novo antibody clones neutralize SARS-CoV-2 variants, but with distinct origins and targets.
During a primary immune response, B cells can undergo isotype switching from an immunoglobulin M (IgM) B cell receptor (BCR) to an IgG BCR. B cells that have switched to IgG give rise to more bone marrow long-lived plasma cells (PCs) compared with those expressing IgM, but how BCR isotype-driven bias occurs remains unclear. Here, we found that IgG1-expressing germinal center B cells presented higher levels of antigen to T follicular helper cells, resulting in greater proliferation of IgG1 PCs than IgM PCs. BCR signaling through IgM induced more Bim-dependent apoptosis in IgM PCs, together leading to the predominance of IgG1 PCs in secondary lymphoid tissues. In addition, IgG1 PCs were more prone to migrating to bone marrow. Hence, our findings suggest that isotype-specific differences in antigen presentation and BCR signaling contribute to the enrichment of IgG1 PCs in the bone marrow long-lived PC compartment.
Removal of cellular waste from the extracellular space is fundamental for tissue health. Because the rate of material ejected by parenchymal cells varies across tissues, we searched for mechanisms that couple waste production and removal. Here, we show that the uptake of parenchyma-released mitochondria by macrophages is prominent across organs that rely on oxidative respiration-including heart, skeletal muscle, and brown adipose tissue-and that macrophage numbers closely align with the mitochondrial activity of each of these organs. We found that the mitochondrial activity of myofibers dictates the abundance of macrophages by modulating colony-stimulating factor 1 (CSF1) availability and the number of CSF1-producing fibroblasts in the tissue. Consequently, inhibition of CSF1-CSF1 receptor (CSF1R) signaling depleted macrophages and collapsed the mitochondrial activity of skeletal muscles. We propose that, by coupling macrophage abundance to the mitochondrial activity of their parenchyma, tissues ensure efficient waste disposal and fitness.
Alterations to monocyte output and function occur during infections driving T helper 1 (TH1)-type inflammation. The degree to which monocytes respond to infections initiating alternative types of responses is poorly understood. Here, we describe a distinct state of the monocyte compartment associated with type 2-polarizing intestinal helminths. Unexpectedly, the adapted monocyte state in a type 2 setting was associated with acquisition of an interferon (IFN) signature. This IFN-induced state provided helminth-infected animals with systemic protection against secondary bacterial infection and allowed for the development of effective type 2 immunity. This pathway of monocyte education was distinct from that in TH1 settings and involved an endogenous bacteria-mediated induction of type I IFN that led to adaptive lymphocyte-dependent IFN-γ priming of monocytes. These findings reveal an IFN-driven mechanism of monocyte education that enables the host to be simultaneously protected against type 2 infections at barrier sites and type 1 infections in the circulation.
Elderly individuals affected by COVID-19 are vulnerable to severe respiratory failure for reasons that have remained poorly defined. Here, we show that platelet-released serotonin drives lung pathology in a mouse model of age-associated disease severity after SARS-CoV-2 infection. In middle-aged mice and aged human healthy donors, platelet serotonin release upon activation was enhanced, and increased morbidity with respiratory dysfunction was associated with activated platelets aggregating and promoting fibrin deposition in the lung microvasculature. Pharmacologic or genetic disruption of serotonin uptake, or blockade of serotonin-dependent signaling, attenuated platelet activation and protected against respiratory distress, independently of viral replication and immune responses. Inhibition of fibrin formation similarly reduced disease severity, implicating serotonin-driven platelet procoagulant activity as a key contributor to age-related lung dysfunction. Thus, serotonin-mediated platelet procoagulant activity is a major contributor to respiratory insufficiency during SARS-CoV-2 infection and a potential therapeutic target for preserving pulmonary function, particularly in the elderly.
Host-derived lipids undergoing enzymatic or nonenzymatic oxidation play critical roles in regulating inflammation. Polyunsaturated fatty acids, cholesterol, and cholesterol intermediates can be enzymatically oxidized and serve as signaling mediators controlling tissue homeostasis and immunity. Spontaneously generated oxidized lipids, including nonenzymatically oxidized phospholipids (oxPLs), result from oxidative stress and accumulate during inflammation, affecting cellular metabolism, immune cell functions, and cell fate. These distinct classes of oxidized lipids not only share overlapping inflammatory roles but also exhibit divergent effects depending on their molecular structures and cellular targets. This Review highlights the double-edged nature of oxPLs: Although their transient production triggers protective responses, their accumulation sustains inflammation, contributing to tissue damage. We also discuss the emerging roles of oxPLs in cell death programs, immune cell activation, and stromal cell functions, which are critical processes favoring tumor growth. Overall, we highlight how oxidized lipids orchestrate immune responses and explore their contribution to infectious diseases and cancer.
The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway promotes tumor immunogenicity, but intratumoral STING agonists, despite strong preclinical efficacy, have shown limited clinical efficacy. The mechanisms restricting therapeutic STING activation remain unclear. In mice, intratumoral delivery of the endogenous STING ligand cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) (cGAMP) using viruslike particles (VLPs) preferentially activates STING in dendritic cells and primes circulating tumor-specific T cells. Using this system, we investigated mechanisms limiting effective STING-based immunotherapy. STING-induced type I interferon signaling was dispensable for cGAMP-VLP-mediated tumor control. In contrast, dendritic cell autophagy was required for generating circulating antitumor CD8 T cells and for regulating baseline neutrophil levels in lymph nodes. cGAMP-VLP overrode this regulation, inducing neutrophil accumulation in tumors and draining lymph nodes that limited efficacy. Neutrophil depletion enhanced tumor control through mechanisms involving neutrophil elastase and programmed cell death 1 ligand 1. These findings reveal that defective dendritic cell autophagy and neutrophil-mediated immunosuppression, rather than insufficient interferon signaling, hinder the effectiveness of intratumoral STING immunotherapy in preclinical mouse models.
Immunoglobulin G (IgG)-based monoclonal antibodies are effective therapies for cancer, autoimmune diseases, and migraine. However, they are actively transported across the placenta by the neonatal Fc receptor (FcRn), limiting their use during pregnancy. Using mouse models and an ex vivo human placental perfusion system, we show that although FcRn binds albumin independently of IgG, albumin is not transported to the fetus in mice or across human placental tissue. Fusion of IgG to albumin markedly reduced transplacental transport in both models while preserving the prolonged plasma half-life conferred by FcRn. Similarly, fragment antigen-binding fragments fused to engineered albumin with enhanced FcRn binding showed minimal fetal exposure. In a mouse model of fetal and neonatal alloimmune thrombocytopenia, albumin fusion of an anti-human platelet antigen IgG reduced fetal antibody transfer and attenuated thrombocytopenia in the offspring. These findings identify albumin as an attractive fusion partner for biologics intended to minimize fetal exposure during pregnancy.
Longitudinal nasopharyngeal swabs sample and reveal upper airway humoral memory generated after intranasal FluMist vaccination.
CDC42 is an evolutionarily conserved guanosine triphosphatase (GTPase) regulating critical cellular and immune processes. Variants in CDC42 therefore cause a spectrum of developmental phenotypes, including hematological and immunological symptoms. Here, we identify a previously unrecognized autoinflammatory condition caused by CDC42Met45→Leu(M45L). We confirm the pathogenicity of CDC42M45L given that it promotes pyrin inflammasome activation, consistent with highly elevated circulating interleukin-18 (IL-18) in all affected individuals. Coimmunoprecipitation demonstrates that CDC42 interacts with the PYD and B30.2 domains of pyrin. The B30.2 interaction is greatly enhanced by CDC42M45L. Models show that CDC42 methionine-45 sits in a hydrophobic cleft of the pyrin B30.2 domain, and substitution with a negatively charged residue prevents colocalization between CDC42 and pyrin. This study supports a model whereby CDC42 nucleates pyrin via the B30.2 domain, after which a threshold is reached to liberate the PYD for inflammasome activation. Blocking the inflammasome-associated cytokine IL-1 was therapeutically effective when tested against this autoinflammatory CDC42M45L endotype.
Three studies identify CDC42 as binding partner of the pyrin B30.2 domain, establishing a previously unrecognized mechanism regulating pyrin inflammasome activation (see related Research Articles by Aoki et al., Feng et al., and Iwata et al.).
Heterozygous carboxyl-terminal variants in the RHO guanosine triphosphatase (GTPase) CDC42 are known to cause severe autoinflammatory syndromes. Here, we identified a heterozygous amino-terminal p.T43I (Thr43→Ile) CDC42 variant in patients with autoinflammation and uncovered a molecular link between CDC42 and the inflammasome sensor pyrin, mutated in the hereditary autoinflammatory syndrome familial Mediterranean fever. We demonstrate that the region surrounding residue T43 of CDC42 interacts with the carboxyl-terminal B30.2 domain of pyrin and regulates its localization and activation. The p.T43I substitution strengthens the CDC42-pyrin interaction through additional van der Waals interactions, leading to increased pyrin inflammasome activation, as evidenced by increased ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain) speck formation, enhanced pyroptosis, and excessive interleukin-1β (IL-1β) and IL-18 production. These findings identify CDC42 as a pyrin ligand and provide critical insights into the role of the pyrin B30.2 domain in inflammasome activation, suggesting dual regulation of pyrin by two RHO family GTPases, RHOA and CDC42.
Mutations in the MEFV gene, which encodes pyrin, are associated with a spectrum of inflammatory conditions called pyrin-associated autoinflammatory diseases (PAADs). Of the 400 MEFV variants listed in the Infevers database, most are classified as variants of uncertain significance. Thus, genetic diagnosis of PAADs remains challenging, and the molecular mechanisms underlying pyrin activation remain poorly understood. Here, we used a cell-based pyroptosis assay to stratify 265 missense MEFV variants and identified previously uncharacterized pathogenic variants. We then characterized the interaction between the pyrin B30.2 domain and CDC42, a key regulator of pyrin intracellular trafficking and activation. We found that classical familial Mediterranean fever (FMF)-related variants bind tightly to CDC42 to induce pyrin hyperactivation, whereas certain non-FMF variants induce pyrin hyperactivation independently of CDC42, indicating involvement of multiple pathways in pyrin activation. Our approach provides a proof of concept for a genotype-first approach, which may advance our understanding of complex human diseases.
The noncanonical functions of PKM2 drive pathogenic T cell function in patients with MS.
Peritoneal cavity fluid and mesothelial surfaces host distinct resident macrophage populations, among which include the well-described Gata6+ large cavity macrophages (LCMs) in peritoneal fluid. Here, we reveal that LCMs arise from two separable differentiation pathways. In the quantitatively minor pathway, monocytes gave rise to LYVE1+ LCMs but few Gata6+ LCMs. This pathway did not require the transcription factor Gata6 but was severely impaired in mice bearing three mutations in the -165 kb Zeb2 enhancer (Zeb2TM) with impaired monocyte development. The second, dominant pathway supported Gata6-dependent LCMs and was intact in Zeb2TM mice, even when turnover was enforced by irradiation, and was supported by adoptive transfer of a specialized LCM intermediate expressing Gata6 before the residency marker TIMD4. Functionally, the quantitatively minor LCM pathway distinctly surveilled the mesothelium, replenishing mesothelial border macrophages upon encountering an open niche. Thus, beyond embryonic versus adult hematopoietic paradigms, LCMs with overlapping and distinct phenotypes arise from two pathways linked to divergent fates.
CD8 T cells are critical players in immune responses against pathogens. Interleukin-21 (IL-21) is predominantly produced by CD4 T cells and exerts multifaceted effects on CD8 T cell regulation and function. Using Il21-reporter and Il21-fate mapping mice, we report that a subpopulation of activated CD8 T cells also produces IL-21 in the context of lymphocytic choriomeningitis virus (LCMV) infection. During the early effector phase of both acute and chronic infections, IL-21-expressing CD8 T cells exhibit substantial proliferative and cytotoxic capacities, with higher levels of interferon-γ (IFN-γ) and granzyme B (GZMB) compared with IL-21- counterparts. Moreover, CD8 T cell-derived IL-21 played a critical protective role in chronic, but not acute, infection. IL-21 expression in CD8 T cells appeared to be largely restricted to exhausted progenitor T cells during the exhaustion phase of chronic infection. These findings identify IL-21-expressing CD8 T cells as pivotal players in the control of chronic infection.
Effective pulmonary immunity requires the precise spatial organization of immune cells, yet the mechanisms guiding their intratissue positioning during inflammation remain unclear. Here, we identify a cholesterol-derived chemotactic axis that spatially organizes T helper 2 (TH2) cells during fungal-induced pulmonary type 2 inflammation. Inflammation-expanded macrophages expressing cholesterol-25-hydroxylase (CH25H) produced 25-hydroxycholesterol, which was converted into the oxysterol 7α,25-dihydroxycholesterol to attract GPR183-expressing TH2 cells into infectious lesions. This TH2 cell positioning suppressed interferon-γ responsiveness in inflammatory Ly6C+ macrophages, promoting fungal persistence. Disruption of this axis via TH2-specific GPR183 deletion restored type 1 macrophage activation and enhanced fungal clearance. Our findings reveal a macrophage-driven, metabolite-based mechanism of immunosuppressive cell positioning in inflamed lung tissue.
B cells generate plasma cells (PCs) and memory B cells (MBCs) to combat recurrent pathogens. B cell receptor (BCR) affinity dictates fate decisions in response to model antigens, but the factors regulating B cell fate during infection remain unknown. Here, we used respiratory and gastrointestinal infection models to study B cell selection across barrier tissues in mice. Memory selection was governed by tissue-specific cues: Selection in the lung was skewed toward MBCs, whereas the gut favored PC entry, even in response to the same pathogen. Divergence was linked to differential BCR isotype usage across barrier tissues rather than differential affinity maturation. In the gut, the commensal-induced TGF-β-rich milieu promoted class-switching to IgA, which skewed selection toward PCs, a process that was counteracted by the IgA cytosolic tail domain. Thus, mucosal B cell selection integrates tissue-specific cues by relying on BCR isotype usage, with implications for nasal and oral vaccine development.
