Germinal center (GC) B cells are critical for the production of long-lived plasma cells and high-affinity antibodies. Upon exiting the GC reaction, B cells differentiate into either memory B (MB) cells or plasma cells depending on affinity signals and transcription factor profiles. However, the underlying transcriptional and metabolic mechanisms regulating this fate decision remain poorly understood. Here, we generated mice with B and GC B-cell–specific knockout of heterogeneous nuclear ribonucleoprotein K (hnRNPK). We found that hnRNPK deletion partially impaired B-cell development and, more strikingly, markedly attenuated the GC B-cell response. The reduction in GC B-cell numbers was attributed to excessive differentiation of precursor MB cells and plasma cells during the GC reaction. Mechanistically, hnRNPK directly suppressed the expression of the key MB cell–associated transcription factors Hhex and Zfp318. Moreover, we observed that compared with CD25-negative GC B cells, CD25-positive GC B cells displayed elevated glycolytic gene expression and significantly increased glucose uptake. HnRNPK deficiency further upregulated glycolytic activity, promoted CD25 expression, and consequently enhanced plasma cell differentiation. Blocking IL‑2/CD25 signaling mitigated the excessive plasmablast differentiation induced by hnRNPK loss and rescued the GC defects in hnRNPK‑deficient mice. Together, these findings demonstrate that hnRNPK controls GC B-cell output by transcriptionally repressing the expression of the MB cell–differentiation factors Hhex and Zfp318 and by modulating the glycolysis‑CD25 regulatory axis.
AAA+ ATPase p97 is a central regulator of protein homeostasis, yet its role in late-stage thymocyte development remains undefined. Here, we demonstrate that T-cell-specific ablation of p97 in mice severely blocks the double-positive (DP) to single-positive (SP) transition, with a pronounced defect in CD8+ lineage commitment. Using both genetic deletion and acute pharmacological inhibition, we revealed a stage- and lineage-specific requirement for p97, with DP thymocytes being most sensitive to p97 loss. This failure in late-stage positive selection leads to intrathymic developmental arrest of immature DP cells and profound peripheral T-cell lymphopenia. Mechanistically, p97 deficiency results in the accumulation of ubiquitinated proteins, triggering the unfolded protein response and apoptosis in thymocytes. Furthermore, we identified a critical requirement for p97 in sustaining IL-7 receptor (IL-7R) expression and JAK signaling. Strikingly, pharmacological activation of JAK partially rescued SP thymocyte development in p97-deficient mice. Our findings establish p97-mediated protein homeostasis as a previously uncharacterized, cell-intrinsic checkpoint that is indispensable for late-stage positive selection by preventing proteostatic collapse and ensuring the fidelity of IL-7R signaling.
Regulatory T-cell (Treg) stability is maintained by dynamic remodeling of the FOXP3 transcriptional complex, and disruption of this complex leads to Treg dysfunction and immune dysregulation. However, how specific FOXP3 mutations alter the dynamic remodeling of the FOXP3 complex and thereby contribute to pathogenic Treg reprogramming in IPEX syndrome remains unclear. Here, we demonstrate that predominant Th1 inflammation manifests in both FOXP3V408M IPEX patients and FOXP3V408M knock-in mice and reveal that the mutation intrinsically impairs Treg-mediated control of Th1 inflammation, revealing a distinct pathogenesis of this mutation in IPEX syndrome. Mechanistically, the V408M mutation disrupts the FOXP3-T-bet interaction, impairing the FOXP3-mediated restraint of T-bet-driven IFN-γ production and thereby contributing to increased Th1 inflammation. Using an AI-driven virtual screening approach, we identified a first-in-class small molecule, FM029, that directly binds to FOXP3 and reinforces its interaction with T-bet. FM029 strongly suppressed Treg-derived IFN-γ production and alleviated IFN-γ-driven tissue inflammation in both FOXP3V408M mice and an acute colitis model. Collectively, these findings establish the FOXP3-T-bet interaction as a central checkpoint that governs Treg stability and IFN-γ-driven Th1 pathology, providing a proof-of-concept that pharmacologic stabilization of the FOXP3-T-bet interaction can mitigate IFN-γ-driven immune disorders.
The NLRP3 inflammasome is a central signaling pathway of innate immunity that orchestrates host defense and inflammatory responses through the activation of proinflammatory cytokines and the induction of pyroptotic cell death. Accumulating evidence indicates that NLRP3 functions within an extensive network of innate immune and cell death pathways. Reciprocal interactions between NLRP3 and other pattern-recognition receptor pathways, including Toll-like receptors, DNA-sensing cGAS-STING signaling, and RNA-sensing RIG-I/MDA5-MAVS signaling, shape the magnitude and duration of inflammatory responses during pathogen infection. Moreover, substantial crosstalk exists between NLRP3 and other inflammasomes and programmed cell death pathways, reflecting the integrated nature of cellular stress and inflammatory signaling. In this review, we summarize recent advances in our understanding of the molecular mechanisms that mediate these interactions, focusing on shared signaling components, organelle dynamics, posttranslational modifications, and feedback regulatory circuits. We further discuss how these signaling networks contribute to infectious and inflammatory diseases and highlight key unanswered questions and emerging areas of investigation that may guide the development of therapies targeting inflammasome-associated pathologies.
Macrophage-enteric neuron dialog is crucial for intestinal homeostasis, but its specific role and underlying mechanism in inflammatory bowel disease (IBD) pathogenesis remain elusive. Here, we demonstrate that colonic macrophage-mediated extracellular matrix (ECM) remodeling orchestrates enteric neuronal maturation and dictates colitis progression. The depletion of Pcif1, recognized as a unique methyltransferase for m6Am mRNA methylation, in macrophages attenuates colitis and protects enteric neurons from inflammation-driven degeneration. Specifically, Pcif1-deficient macrophages increase ECM accumulation, promoting their own extravasation into enteric neuronal plexuses and the maturation of enteric neurons, thereby curbing inflammation-driven neuronal injury and colitis progression. Mechanistically, Pcif1-mediated m6Am modification inhibits the translation of Znf219 mRNA and represses the Znf219-Egr1 axis, a master transcriptional module that governs ECM remodeling. Finally, pharmacological blockade of PCIF1 promotes macrophage-mediated ECM deposition and suppresses colitis and neuronal loss. Our findings reveal a novel mechanism whereby ECM remodeling in macrophages drives enteric neuronal maturation and illuminate the m6Am machinery as a promising therapeutic target for preventing neuronal loss in IBD patients.
The immunosuppressive microenvironment is a hallmark of glioblastoma (GBM), limiting the efficacy of contemporary immunotherapies. While the central nervous system (CNS) relies on specialized cytokine networks to maintain immune homeostasis under physiological conditions, how these homeostatic signals are subverted during gliomagenesis remains poorly understood. Here, we report that interleukin-17D (IL-17D) is a CNS-intrinsic tumor suppressor whose expression is downregulated during gliomagenesis. Restoring IL-17D substantially extends survival in orthotopic GBM models by selectively reprogramming the myeloid compartment. Mechanistically, IL-17D engages its receptor CD93 to assemble a novel costimulatory receptor complex with SCARB1 in a lipid raft-dependent manner. This structural assembly engages Fyn/BCAP to trigger PI3K/AKT/NF-κB signaling, effectively inverting the function of SCARB1 from tolerogenic efferocytosis to active antigen presentation. Our study demonstrated that the restoration of IL-17D expression reshaped the GBM immune landscape, highlighting its potential as a therapeutic target to enhance the efficacy of immunotherapy.
Radiotherapy elicits dual immunomodulatory effects in cancer, activating antitumor immunity while paradoxically inducing immunosuppression, which limits therapeutic efficacy. The molecular pathways mediating postradiation immune escape in hepatocellular carcinoma (HCC) remain poorly defined. Here, we elucidate a previously uncharacterized mechanism whereby radiotherapy drives the accumulation of CD200+ tumor-associated macrophages (TAMs) that suppress eosinophil-mediated antitumor immunity in patients with HCC. Through single-cell RNA sequencing of postradiotherapy HCC specimens, we demonstrated that radiation-induced DNA damage activated the cytosolic DNA-sensing STING pathway in TAMs, triggering NF-κB-dependent CD200 upregulation independent of canonical type I interferon signaling. These radiation-induced CD200+ TAMs exhibited an immunosuppressive phenotype and correlated with adverse clinical outcomes in HCC patients. Mechanistically, CD200+ TAMs established an immunosuppressive axis by recruiting CCR1+ eosinophils through CCL3-mediated chemotaxis, subsequently inhibiting their antitumor functions via CD200-CD200R engagement. This interaction comprehensively suppressed NF-κB activation in eosinophils, impaired their antigen-presenting capacity and Th2 cytokine secretion and abrogated their ability to support CD8+ T-cell-mediated cytotoxicity. Therapeutic blockade of CD200R following radiotherapy restored eosinophil effector functions, promoted central memory T-cell formation, and significantly enhanced tumor control across multiple preclinical HCC models. Remarkably, CD200R antagonism sensitized PD-1-refractory "cold" tumors to radioimmunotherapy combinations, overcoming primary resistance. Our findings establish STING-driven CD200+ TAM accumulation and subsequent eosinophil dysfunction as critical determinants of radioresistance, positioning CD200R blockade as a promising therapeutic strategy to potentiate radioimmunotherapy responses in patients with HCC. Graphical abstract of the study findings. Radiotherapy-induced STING signaling activation promotes the accumulation of CD200+ TAMs. These CD200+ TAMs facilitate the recruitment of eosinophils via the CCL3-CCR1 chemotaxis axis while simultaneously suppressing eosinophil-mediated antitumor activity through CD200-CD200R engagement (left panel). Targeted blockade of CD200R following radiotherapy unleashed the antitumor potential of eosinophils, leading to enhanced infiltration and effector function of CTLs (right panel).
IgG Fc gamma receptor I (FcγRI) belongs to the immunoglobulin superfamily and plays a pivotal role in immune regulation. The post-translational regulation of FcγRI and its effects on immune regulation are unclear. In this study, we identified the membrane-associated RING-CH-type finger (MARCH) E3 ubiquitin ligases MARCH2 and MARCH3 as physiological regulators of FcγRI. MARCH2 and MARCH3 associate with FcγRI and mediate its K27-linked polyubiquitination at K336 and K368, respectively, leading to subsequent lysosomal degradation. While deficiency of either MARCH2 or MARCH3 modestly increases FcγRI levels as well as LPS- and IgG-induced transcription of downstream genes, double knockout of MARCH2/3 has a more dramatic effect. Double knockout of MARCH2/3 increases LPS-induced transcription of downstream genes in wild-type but not FcγRI knockout cells, and reconstitution of FcγRIK336R/K368R into FcγRI-deficient cells increases LPS-induced transcription of the downstream genes to a higher degree than reconstitution with wild-type FcγRI. Individual knockout of MARCH2 or MARCH3 sensitizes mice to LPS-induced lung injury and Salmonella typhimurium-induced inflammation, and these effects are more severe in MARCH2/3 double-knockout mice. These findings suggest that MARCH2 and MARCH3 redundantly target FcγRI for K27-linked polyubiquitination and lysosomal degradation, thereby acting as host factors to limit the FcγRI-mediated inflammatory response and pathogenesis.
Allelic variants in the leucine-rich repeat kinase-2 (LRRK2) gene are linked to Parkinson’s disease, Crohn’s disease and leprosy; however, a consensus role of LRRK2 in infection has not yet emerged. Parkinson’s disease-linked p.G2019S mutation of LRRK2 results in increased kinase activity. We evaluated the impact of the p.G2019S mutation during the infection of mice with Salmonella typhimurium (ST) or Listeria monocytogenes. We demonstrate that the p.G2019S mutation promotes the phosphorylation of the p40phox and p47phox subunits of the NADPH oxidase-2 complex in the cytosol of neutrophils, leading to their relocation to lysosomes for enhanced bacterial control. Activation of Cathepsin B, Rab10 or PKC was not modulated by p.G2019S. Activation of the cytosolic subunits of NADPH oxidase-2 subunits was inhibited by the virulence factor SifA, which promotes the phagosomal localization of ST. Deletion of SifA resulted in an even greater effect of the p.G2019S mutation on the control of ST by neutrophils. These results suggest a mechanism by which the p.G2019S mutation promotes better control of infections at the cost of excessive tissue damage via NADPH oxidase-2 activity under chronic inflammatory conditions.
Atherosclerotic lesions contain heterogeneous macrophage populations with divergent functional phenotypes that play opposing roles in disease progression, creating a major obstacle for the development of macrophage-targeted therapy. The identification of novel molecules that exhibit both preferential expression and functional activity in inflammatory plaque macrophages is crucial for the development of more specific anti-inflammatory therapies against atherosclerosis. Here, we report that the expression of C-C motif chemokine receptor-like 2 (CCRL2), a nonsignaling atypical chemokine receptor initially cloned from LPS-activated macrophages, progressively increased in atherosclerotic lesions during disease progression in both human and mouse models, with predominant expression in inflammatory macrophages characterized by high expression of NLRP3 inflammasome components. Moreover, specific deletion of hematopoietic CCRL2 attenuated atherosclerotic progression in Apoe-/- mice fed a high-cholesterol diet, as evidenced by significant decreases in plaque burden, macrophage accumulation, and NLRP3 inflammasome activation within the plaques, without affecting blood lipid levels. Mechanistically, ox-LDL or other danger signals induce macrophages to express CCRL2, which can be endocytosed and relocalized to early endosomes, where it interacts with NLRP3 to promote inflammasome assembly and activation. Importantly, macrophage-targeted nanoparticle delivery of Ccrl2-siRNA effectively attenuated atherosclerotic progression in vivo, concomitant with reduced levels of IL-1β, the key effector cytokine of the NLRP3 inflammasome, and diminished macrophage accumulation within plaques. Taken together, the results of our study establish CCRL2 as both a new marker for identifying inflammatory macrophages and a promising anti-inflammatory therapeutic target for atherosclerosis through the modulation of aberrant NLRP3 inflammasome activation in pathogenic inflammatory macrophages.
Appropriate T-cell functional polarization is critical for maintaining immune stability and immune tolerance. The role of Fam234a in the functional polarization of T cells is unknown. In a DSS-induced inflammatory bowel disease model in Rag2-/- mice with either naive WT or Fam234a-deficient CD4+ T cells, mice with Fam234a-deficient CD4+ T cells presented milder symptoms of colitis, accompanied by a decreased ratio of Th17/Treg cells. Consistent with the in vivo observations, Th17 differentiation was significantly decreased and Treg induction was increased in the in vitro naive Fam234a-deficient CD4+ T-cell polarizing induction system. Similarly, knocking down FAM234A in human T cells using siRNA also revealed that FAM234A deficiency significantly decreased the Th17/Treg cell ratio in human T cells. Coimmunoprecipitation-mass spectrometry (Co-IP-MS), protein interaction, and biochemical studies revealed that FAM234A may directly interact with the deubiquitinase USP4 to affect its deubiquitination function. The reduction in Th17 cells and increase in Treg cells among Fam234a-deficient T cells were significantly reversed by restoring USP4 overexpression. RNA sequencing and molecular studies indicated that Fam234a knockout reduced USP4-mediated Rheb and RORγt deubiquitination, mTOR activation, and Hif1α expression and ultimately affected Th17 and Treg differentiation. Therefore, Fam234a intrinsically balances the Th17 and Treg differentiation of naive CD4+ T cells by directly preventing USP4-mediated deubiquitination of Rheb to regulate mTOR-HIF1α-related oxidative phosphorylation and glycolytic gluconeogenesis metabolism pathways as well as USP4-mediated deubiquitination of RORγt pathways. This research revealed the critical role of FAM234A in the orchestration of Th17/Treg cell fate decisions and may offer potential therapies for their related diseases.
T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells play critical roles in regulating the activity of the germinal center (GC), which is essential for the generation of high-affinity antibodies. In the GC, Tfh cells help B cells to proliferate and to differentiate into memory B cells and long-lived plasma cells. In contrast, Tfr cells, a specialized subset of regulatory T cells (Tregs), modulate the humoral immune response by suppressing excessive or autoreactive B-cell activity. Here, we established an in vitro differentiation protocol for mouse CD4⁺ T cells that yielded CXCR5⁺FoxP3⁺ Tfr cells that exhibited a Bcl6hiPD-1hiCD25loGITRint phenotype and were distinct from Treg and Tfh cells. Functionally, in vitro-generated Tfr cells potently suppressed Tfh cell-driven B-cell class switching to IgG1 and downregulated the expression of B-cell costimulatory ligands. While in vitro-generated Bcl6-deficient Tfh cells were impaired in providing help to B cells for efficient class switching to IgG1, in vitro-generated Bcl6-deficient Tfr cells failed to inhibit Tfh cell-driven B-cell class switching to IgG1. Mechanistically, we showed that Tfr cells emerged from FoxP3+ precursors in low-IL-2 environments through a TGF-β- and c-Maf-dependent pathway, allowing for reprogramming and reinforcement of the follicular regulatory cell program in CD4+ T cells in vitro.
