Journal of immunology最新文献

TLR7 and myeloid-derived suppressor cells (MDSCs) play unique roles in determining host resistance to candidiasis. However, the precise mechanisms of TLR7 in MDSC differentiation and functionality during Candida albicans infection remain elusive. We found that compared with wild-type mice, kidney injuries and inflammation were significant in Tlr7 knockout mice. Tlr7 deficiency impeded the differentiation and maturation of mature myeloid cells and stimulated MDSC expansion. Furthermore, the absence of Tlr7 enhanced the immunosuppressive ability of infected MDSCs. Contrarily, the treatment of the TLR7 agonist R848 directly acted on MDSCs, leading to the differentiation and maturation of MDSCs and blocking their immunosuppressive activity. TLR7+ granulocytic MDSCs (G-MDSCs) significantly exhibited the enhanced expression of RUNX1 and KLF4. Subsequently, prevention of RUNX1 activity with Ro5-3335 or treatment with KLF4-activating agent APTO-253 affected the differentiation and maturation of G-MDSCs in vitro. Taken together, our results identified a function of TLR7 in modulating the MDSC response and suggested that RUNX1 and KLF4 were key transcription factors in regulating TLR7-mediated G-MDSC immune responses.

Tuberculosis (TB), a significant global health issue, needs novel therapeutic approaches to reduce its burden. Studying host-pathogen interactions provides new targets for host-directed therapeutics (HDTs). Nuclear receptors (NRs) are important master regulators of cellular function and bona fide drug targets. Herein, we identify high basal expression of the NR4A NR family in human alveolar macrophages and determine that all 3 members (NR4A1, NR4A2, and NR4A3) are upregulated in response to Mycobacterium tuberculosis (M.tb) infection. NR4A expression was also increased in our recently developed human alveolar macrophage-like (AML) cell model compared to monocyte-derived macrophages. We investigated the role of the NR4As in apoptosis given its importance in controlling M.tb growth. NR4A small interfering RNA knockdown in AML cells prior to their treatment with apoptosis-inducing compounds resulted in reduced caspase-3/7 activity, indicating reduced apoptosis. Additionally, knockdown prior to M.tb infection resulted in reduced apoptosis of AML cells and increased M.tb growth. Treatment of AML cells with NR4A ligands significantly reduced M.tb growth while treatment with an NR4A antagonist significantly increased it. In conclusion, we identify the expression, location, and apoptotic activity of NR4A NRs in human macrophages and their potential as new TB HDT therapeutic targets.

Group 2 innate lymphoid cells (ILC2s) produce large amounts of IL-5, IL-13, and amphiregulin, which are involved in the development of lung fibrosis. Activation of ILC2s is mediated by phosphorylation of STAT5. Although STAT5 has tyrosine and serine phosphorylation sites, the mechanisms responsible for phosphorylating serine residues and their significance in ILC2s remain unclear. The present study demonstrated that cyclin-dependent kinase (CDK) 8 and its paralog CDK19 (CDK8/19) were crucial for the activation of ILC2s in OVA-induced asthmatic BALB/c mice by phosphorylating serine residues of STAT5, leading to lung fibrosis. The following results were obtained: (1) The development of lung fibrosis and the number of ILC2s were significantly ameliorated by the CDK8/19 inhibitor, AS3334366, in OVA-induced asthmatic mice. Consistently, lung fibrosis did not develop in OVA-induced asthmatic ILC2-deficient (Il7rCre/+ Rorafl/fl) mice. (2) IL-33 markedly up-regulated the expression of IL-2 receptor chains, CD25 and CD132, in lung ILC2s. IL-2 significantly augmented the expression of ST2, the high-affinity receptor for IL-33. Furthermore, simultaneous stimulation with IL-33 and IL-2 resulted in the upregulation of CDK8/19 expression in ILC2s. (3) The combined treatment with IL-33 and IL-2 synergistically enhanced the proliferation and cytokine production of ILC2s. Conversely, both these effects, along with the phosphorylation of serine residues in STAT5, were suppressed by AS3334366 in a concentration-dependent manner. The present study is the first to report the involvement of CDK8/19 in the activation of ILC2s via the phosphorylation of STAT5, which led to the development of lung fibrosis.

Memory CD8+ T cells are essential for long-term protective immunity. Here, we show that activation of p38 MAPK during the primary response of CD8+ T cells orchestrates a delicate balance between the formation of short-lived effector cells and memory precursor effector cells. p38αfl/flp38βfl/flGzmBcre/- mice, in which p38α and p38β were efficiently deleted in CD8+ T cells and also in early stages of T-cell development, were used in studying the role of the p38 pathway in T cells. The deletion of p38α and p38β (simplified as p38α/β) has very minor effects on thymic and peripheral T-cell development. In contrast, p38α/β-deficient CD8+ T cells were skewed toward a central memory phenotype and mounted stronger recall responses upon secondary challenge. Transcriptomic analyses of antigen-specific CD8+ T cells revealed that p38α/β deficiency is associated with reduced effector gene expression and enhanced memory-associated programs. Furthermore, in vitro differentiated p38α/β-deficient CD8+ T cells showed superior persistence and functional responses after adoptive transfer. These results establish a role for p38 in controlling effector CD8+ T-cell differentiation and memory formation, and reinforce the therapeutic potential of targeting this pathway, aligning with recent studies demonstrating the beneficial effects of p38 inhibitors in adoptive cell therapy.

Transforming growth factor beta (TGFβ) is an immunosuppressive cytokine that is overexpressed in tumor microenvironments. We have shown that CD8+ T cells with genetic ablation of the TGFβ type I receptor, Alk5 (CD8ΔALK5), were more sensitive to αCD3 stimulation resulting in enhanced proliferation and cytokine production. Based on these data, we hypothesized that TGFβ impaired T-cell receptor (TCR) signaling. We tested in vitro cytotoxicity of wild-type (WT) and CD8ΔALK5 OT-I T cells against murine oral carcinoma models transduced with ovalbumin altered peptide ligands (APLs) of differing affinities and found that loss of TGFβ renders CD8+ T cells more cytotoxic, but with diminishing effect at lower TCR agonism. TGFβ limits proximal TCR signaling intensity and duration, mediated by an interaction between the TGFβ type II receptor, PTPN22, and Zap70 that requires the Alk5 receptor. Downstream TCR signal integration is impaired by TGFβ following high and moderate, but not low TCR agonism. In vitro and in vivo models of chronic antigen stimulation demonstrate that TGFβ promotes both stem-like differentiation and terminal exhaustion, with loss of the more cytotoxic transitory exhausted population. Tumors of mixed APL clonality were implanted into Rag-/- animals followed by adoptive cell transfer of WT or CD8ΔALK5 OT-I T cells and monitored for clonal outgrowth. CD8ΔALK5 OT-I T cells were better able to control tumor clones with moderate TCR agonism compared to WT OT-I T cells. Targeting TGFβ signaling is one approach to enhance TCR signaling following strong or moderate agonism, alter differentiation toward more cytotoxic transitory exhaustion, and reduce terminal exhaustion, to improve antitumor immunity.

Chronic inflammatory diseases are becoming more prevalent in Western countries, yet there is limited research on clinical tools for their cure. Understanding the bacteria in the gastrointestinal tract is crucial for managing these diseases, as pathogenic bacteria can lead to inflammation and cancer, while commensal bacteria help mitigate these harmful effects. For science to continue progressing, there is a need to develop new approach methods that simulate human organ models with high throughput, are cost-effective, and are still precise and accurately representative. Various cells, such as human B lymphocytes (represented by Raji B cells) and human colorectal cells (represented by smooth Caco-2 cells, microfold Caco-2 cells, and HT29-MTX cells), play distinct roles in maintaining intestinal health. In vitro models using these cells help simulate gastrointestinal functions more accurately. We have developed such a model termed "CHaRM" (Caco-2, HT29-MTX, and Raji cell Model). Recent studies have shown that introducing different bacterial strains into the gastrointestinal tract increases cytokine activity, with nontoxigenic bacteria (nontoxigenic Bacteroides fragilis) triggering a stronger response than toxigenic bacteria (enterotoxigenic Bacteroides fragilis [ETBF]). While both treatments increased cytokine levels, ETBF did not significantly alter proinflammatory cytokine levels compared to the control. However, PD-L1/B7-H1, a transmembrane protein, decreased with ETBF treatment, as did some growth factor proteins.

Systemic lupus erythematosus is characterized by activation of many self-reactive B cell clones that produce autoantibodies. This can be modeled using mixed bone marrow chimeras, where autoreactive 564Igi B cells initiate autoimmunity that spreads to wild-type (WT) B cells. The mechanisms controlling the inclusion of new B cell clones into spontaneous germinal centers (GCs) remain unclear. Using CRISPR-Cas9, we generated 2 autoreactive B cell receptor knock-in strains, M05 and G55, based on B cell receptors from WT GC B cells in WT:564Igi chimeras. M05 and G55 mice lacked spontaneous GCs and overt autoantibody production, with receptor editing (λ light chain expression) contributing to tolerance. However, autoreactivity was not purged from the B cell compartment since presence of 564Igi clone allowed M05 and G55 B cells to join GCs and produce autoantibodies. These findings reveal that GCs can override peripheral tolerance, recruiting previously silent autoreactive clones and facilitating diversification of autoantibodies.

As the key component of the complement system, C3 plays important roles in complement activation to regulate phagocytosis, lyse cells, mediate inflammation, and clear immune complexes. In the present study, CgC3 in the cell-free hemolymph of the Pacific oyster Crassostrea gigas was found to be able to bind various polysaccharides and microbes and then interacted with membrane receptor CgCD18 to mediate the entry of the CgC3-coated Vibrio into hemocytes. CgATPV1D in hemocytes sensed the CgC3-coated Vibrio vacuole and recruited CgATG16L1. The free CgC3 in hemocytes could also recognize and bind the intracellular invading Vibrio and then directly recruited CgATG16L1. CgATG16L1 recruited CgLC3 to promote the extension of autophagosome membrane. The autophagosome then fused with lysosome to form autolysosome to degrade the CgC3-coated Vibrio. When the C3-CD18-ATG16L1 axis was destroyed by their antibodies, dsRNAs or siRNAs, the co-localizations of Vibrio, CgC3, CgATPV1D, CgATG16L1, CgLC3, and lysosome were all inhibited in hemocytes. The cleavage of CgLC3 and the amount of autophagosomes and autolysosomes were also reduced after Vibrio stimulation. The results collectively demonstrated that CgC3 was able to bind intra/extracellular microbes to form intracellular microbe-associated complexes of C3-CD18-ATPV1D-ATG16L1-LC3 and C3-ATG16L1-LC3, and then trigger the intracellular antibacterial autophagy-lysosome pathway to eliminate the invading microbes.

Janus kinase 2 (JAK2) has been linked to various neutrophil functions, but the intracellular mechanisms underlying its modulation are unknown. Neutrophils are essential cells for host defense. Neutrophil effector functions include migration, neutrophil extracellular trap production (NETosis), reactive oxygen species (ROS) production, and degranulation. The goal of this study was to elucidate the signaling mechanism through which JAK2 modulates neutrophil function and the effect of aging on this pathway. We hypothesized that JAK2-mediated modulation changes the molecular mechanisms associated with neutrophil function in an age-dependent manner. Neutrophils from young (3 mo) and aged (≥22 mo) male and female C57BL/6J mice were isolated, treated with a JAK2 inhibitor (AZD1480) or a pan-JAK inhibitor (baricitinib), and stimulated with PMA. Functional assays were conducted to assess migration, degranulation, NETosis, and metabolism. Mass spectrometry and Luminex assays provided proteomic and cytokine profiles. Our data showed that JAK2 promotes migration via membrane composition and actin remodeling, with age-dependent shifts in chemokine secretion. JAK2 primes ROS production by altering NADPH oxidase components, which contributes to NET production. JAK2 influences degranulation through actin remodeling. While aged neutrophils display impaired ROS-granule release, both young and aged neutrophils have distinct JAK-dependent release of granule contents. Metabolically, JAK2 enhances pentose phosphate pathway activity in young neutrophils and decreases glycogen breakdown in aged cells. These findings reveal mechanisms by which JAK2 modulates neutrophil function and suggest that organismal age plays a role in this modulation.

Organ transplantation is a life-saving treatment for patients with end-stage organ failure but requires lifelong immunosuppression that can result in significant complications. Achieving stable and durable donor-specific tolerance, whereby immunosuppression can be stopped without precipitating graft rejection, holds the promise to circumvent these problems. While transient inhibition of the CD40/CD154 costimulatory pathway delays transplant rejection in animal models, and antibodies blocking this interaction are currently in clinical trials, the efficacy of anti-CD154 (αCD154) in mouse models is significantly enhanced by the addition of donor splenocyte transfusion (DST). Indeed, αCD154 + DST, but not αCD154 alone, can successfully induce donor-specific transplantation tolerance to fully mismatched cardiac allografts in mice. Why DST needs to be added to αCD154 is not fully understood. By integrating tracking of graft-specific T cells and of donor cells, we show that systemic, but not subcutaneous, injection of DST enables alloantigen dissemination to secondary lymphoid organs beyond those directly draining the transplant. This wider biodistribution results in a greater number of alloreactive T cells interacting with donor alloantigens in all lymphoid organs, such that more alloreactive T cells can be the target of CD154 blockade. Furthermore, the duration of DST persistence, facilitated by the sharing of MHC alleles between the donor and the recipient, emerges as a critical factor in promoting αCD154-mediated graft acceptance. These results provide insights into rational approaches to improve translation of αCD154 in the clinic.