BackgroundNon-small cell lung cancer (NSCLC) has a high incidence rate, and most patients develop resistance to anti-PD-1 immunotherapy, resulting in shortened survival. Current evidence suggests that peripheral T cells, particularly CD8+ T cells, play a key role in the response to anti-PD-1 immunotherapy. However, the key molecules that impair peripheral CD8+ T cell function and thereby drive resistance to anti-PD-1 immunotherapy remain unclear. This study aims to demonstrate that loss of cathepsin L (CTSL) expression in peripheral CD8+ T cells is a critical factor driving resistance to anti-PD-1 therapy in NSCLC.MethodsUsing flow cytometry, we tracked the dynamic expression patterns of CTSL in peripheral CD8+ T cells from NSCLC patients receiving anti-PD-1 therapy, as well as its longitudinal distribution across different T cell subsets. We then investigated the association between CTSL expression levels in peripheral CD8+ T cells and the expression of anti-tumor effector molecules. Finally, through bioinformatics analysis, flow cytometry, ELISA, and pharmacological interventions, we explored the functional relationship between CTSL and NLRP3 inflammasome activation in mediating the anti-tumor function of CD8+ T cells.ResultsCTSL expression in peripheral CD8+ T cells was demonstrated as a predictor of improved clinical response to anti-PD-1 immunotherapy in NSCLC (p < 0.01). Subset analysis revealed that in anti-PD-1-resistant patients, CTSL expression was significantly reduced in effector memory CD8+ T cells and terminally differentiated effector memory CD8+ T cells. Mechanistically, CTSL upregulation enhanced the expression of functional molecules in CD8+ T cells, including perforin, granzyme, IFN-γ, and Ki67. Functional exploration experiments further showed not only a positive correlation between CTSL and NLRP3 expression in peripheral CD8+ T cells, but also that activation of NLRP3 reversed the anti-tumor dysfunction of CD8+ T cells induced by CTSL inhibition.ConclusionLoss of CTSL expression in peripheral CD8+ T cells is a key factor driving resistance to anti-PD-1 immunotherapy in NSCLC. CTSL loss may impair the anti-tumor function of CD8+ T cells by inhibiting NLRP3 inflammasome activation. This study not only provides a potential circulating biomarker for predicting response to anti-PD-1 therapy, but also offers new perspectives for understanding the mechanisms of resistance.

Background: Tuberculosis (TB) remains a major global health challenge, highlighting the urgent need for more effective vaccines. This study aimed to develop an artificial intelligence-guided epitope prediction and prioritization pipeline to identify immunodominant peptides from Mycobacterium tuberculosis (Mtb) and to evaluate the immunogenicity and protective efficacy of the resulting vaccine candidates. Methods: An AI-guided framework was used to predict and prioritize immunodominant Mtb epitopes, leading to the generation of 72 recombinant immunogens. Among these, RI-13, RI-20, and RI-31 were selected as the leading candidates. Their protective efficacy was assessed in a zebrafish TB infection model and in BALB/c mice following DNA vaccination. Humoral and cellular immune responses were further evaluated in C57BL/6 mice. Results: RI-13, RI-20, and RI-31 markedly reduced infection-associated pathology and lowered bacterial burden by up to 1.5 log10 in the zebrafish TB infection model, outperforming benchmark antigen combinations, including the Ag85A plus ESAT6/CFP10 cocktail used in the phase III vaccine candidate GamTBvac. In BALB/c mice, DNA vaccination with each construct reduced pulmonary mycobacterial burden by approximately 0.3 log10 and alleviated lung tissue damage. In addition, all three candidates elicited robust humoral and cellular immune responses, with RI-13 showing the strongest overall immunogenicity and inducing a balanced Th1, Th2, and Th17 response profile in C57BL/6 mice. Conclusions: These findings identify RI-13, derived from Rv1174c, as a promising next-generation TB vaccine candidate. More broadly, this study supports the utility of an AI-guided framework for the rational design and preclinical prioritization of novel TB immunogens.

Extracellular acidification is a hallmark of the inflamed intestinal tract in individuals with inflammatory bowel disease (IBD). The proton channel Otopetrin-2 (OTOP2), which is enriched in intestinal tissues, becomes dysregulated during inflammation. However, the specific role of OTOP2 in IBD pathogenesis remains unclear. In this study, we demonstrate a significant reduction of OTOP2 mRNA and protein in the inflamed mucosa of pediatric patients with IBD, which inversely correlates with disease progression. Otop2 knockout (Otop2-/-) mice exhibit growth retardation and heightened susceptibility to intestinal inflammation, which is linked to intestinal pH dysregulation, altered gut microbiota composition, and compromised tight junction integrity. Accordingly, Otop2-/- mice exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis. Mechanistically, Otop2 deficiency reduces Paneth cell numbers and diminishes antimicrobial factor expression, likely due to impaired autophagy-lysosomal processes within these cells. Similarly, Otop2 deficiency impairs phagocytic function in bone marrow-derived macrophages (BMDMs). Together, these findings establish OTOP2 as a critical, pH-sensitive regulator of intestinal homeostasis and highlight its potential as a therapeutic target in IBD.

Objectives: Tuberculosis (TB) remains a major global health threat, necessitating accurate, rapid, and cost-effective diagnostic methods to improve early detection and control of the disease. The traditional methods for detecting Mycobacterium tuberculosis (MTB) in clinical testing are fluorescence smear microscopy (FSM) and Lowenstein–Jensen medium culture (LJMC) methods. With the development of molecular diagnostics, the Xpert MTB/rifampicin assay (Xpert) has become increasingly used. This study compared Xpert with traditional FSM and LJMC using sputum specimens to study the application value in the diagnosis of TB. Methods: A total of 342 examination reports were included. Sputum samples from all study subjects were tested using FSM, LJMC, and Xpert methods. Results: The Xpert method showed a significantly higher positive rate than FSM, and a slightly higher rate than LJMC. Conclusion: Among all sputum samples from suspected or confirmed TB patients, Xpert detected all cases that were positive by FSM, while both Xpert and LJMC missed positives found by each other. FSM also detected a small number of positives not detected by LJMC. Overall, Xpert had the highest positive detection rate, and FSM the lowest. Although FSM is the fastest and least expensive (costing only one-seventh of Xpert), it demonstrated the lowest sensitivity, with its positive rate being roughly half that of Xpert or LJMC.

Ketogenic diets (KDs) have been reported to influence tumor progression through metabolic and immunological modulation of the tumor microenvironment. β-hydroxybutyrate (βOHB), the predominant ketone body elevated by KD, functions not only as an energy substrate but also as a potent signaling metabolite. Despite its role in modulating the tumor microenvironment, the direct impact of βOHB on the function of CD8+ T cell, a key mediator of anti-tumor immunity, remains incompletely understood. Here, we demonstrate that βOHB suppresses tumor growth in multiple mouse tumor models by enhancing the accumulation, survival, and effector function of tumor-infiltrating CD8+ T cells. In contrast, acetoacetate does not exert comparable immunomodulatory effects. Mechanistically, βOHB upregulates the Tcf7–Lck signaling pathway by engaging with the cell surface receptor Hcar2, an effect potentially working in parallel with its role as an HDAC inhibitor. Knockdown of either Tcf7 or Hcar2 in CD8+ T cells abolishes the promoting effect of βOHB on CD8+ T function. Our findings elucidate a metabolite-immune axis that directly regulates the functional state of tumor-infiltrating CD8⁺ T cells and provide experimental evidence linking ketone metabolism to anti-tumor immune regulation.

Tuberculosis (TB) vaccine design relies on the selection of optimal antigens. However, evidence on candidate antigens is scattered across the literature and rarely linked to critical vaccine parameters, such as immune responses, antigen combinations, and adjuvants. Here, we introduce Mycobacterium tuberculosis (MTB)-ImmunogenKG, a provenance-linked, antigen-centric knowledge graph for MTB, that is constructed via an optimized information-extraction pipeline and integrated with a large language model (LLM) system to enable knowledge-augmented reasoning. From over 77,000 publications indexed in PubMed (as of July 2024), this graph consolidates 1.48 million sentence-level statements spanning 14 entity types. In practical applications, MTB-ImmunogenKG enables contradiction-aware antigen profiling for 3,154 proteins (representing about 77% of the total annotated proteins) and improves protective-efficacy prediction with a Matthews correlation coefficient (MCC) gain of 0.19 over sequence-based tools and 0.45 over an LLM-only baseline. By delivering a traceable synthesis of fragmented findings, MTB-ImmunogenKG facilitates the construction of antigen panels and the pairing of adjuvants, thereby streamlining the experimental design cycle for next-generation TB vaccines.

INTRODUCTION:Transient receptor potential cation channel subfamily V member 1+ (TRPV1+) sensory nerves, usually involved in transmitting pain and itch signals, densely innervate the kidney. Because the kidney is not a classic pain-sensitive organ, the roles of these sensory nerves beyond pain perception remain unknown. Here, we reveal a neuroimmune axis wherein TRPV1+ nociceptive sensory neurons protect against kidney ischemia-reperfusion (I/R) injury by inducing an anti-inflammatory macrophage subset. METHODS:We used reporter mice to visualize TRPV1+ nociceptive sensory nerves and used retrograde neuronal tracing to identify the activation of kidney-innervating TRPV1+ neurons during kidney ischemia/reperfusion injury. To examine their function, we applied multiple nociceptor ablation strategies (genetic and chemical) and activation approaches. Macrophage responses were analyzed using flow cytometry and RNA sequencing, whereas molecular signaling pathways were investigated with pharmacological inhibitors and in vitro stimulation assays. Urinary calcitonin gene-related peptide (CGRP) levels and macrophage profiles were also assessed in postnephrectomy patients to validate clinical relevance. RESULTS:Nociceptive sensory nerves are activated by inflammation during kidney ischemia/reperfusion injury and initiate protective anti-inflammatory programs by releasing CGRP, which signals through receptor activity-modifying protein 1 (RAMP1) receptors on macrophages to induce a unique interleukin-4 receptor alpha (IL4Rαhi) population via the cAMP PKA-CREB-dependent pathway. CGRP synergizes with IL-4 to promote the anti-inflammatory and prohealing function of macrophages. Elevated urinary CGRP levels correlated with reduced kidney injury markers (KIM-1 and NGAL) and higher proportions of anti-inflammatory macrophages in postnephrectomy patients. CONCLUSIONS:Our findings redefine nociceptors as active regulators of kidney inflammation and homeostasis, leveraging CGRP to convert macrophages into anti-inflammatory phenotypes to protect against kidney injury, offering new therapeutic opportunities for AKI.

Bacillus Calmette-Guerin (BCG)-induced trained immunity in the macrophages is characterized by exaggerated inflammatory cytokine production with favourable effects on disease controls. However, how BCG-trained macrophages exert anti-tumor effects and underlying mechanisms remain to be clarified. Pan-anti-tumor activity induced by BCG training was evaluated in mouse models with grafted tumors. The proportion and function of tumor infiltrating macrophages (TAMs) and CD8+T cells, as well as the level of reactive oxygen species (ROS), were detected by flow cytometry. The secretion of IL-1β and TNF-α were detected by Enzyme-Linked Immunosorbent Assay (ELISA). The expression and activation of NOX2 complex and NF-κB were detected by qRT-PCR, immunoblotting and immunofluorescence. The methylation modification level and chromatin opening level were detected by CUT RUN and ATAC sequencing. RNA sequencing was used to detect the transcriptome of macrophages and bladder cancer tissues. BCG-trained mice exhibited pan-anti-tumor activity where TAMs originated from newly bone marrow hematopoiesis were the predominant effectors. The anti-tumor effects of BCG-trained TAMs were mediated by ROS production in the tumor microenvironment (TME), resulting from the overactivation of NADPH oxidase 2 (NOX2) complex. Epigenetic rewiring of NOX2 complex occurred both in the myeloid progenitor of BCG-trained mice as well as in BCG-trained macrophages marked by increased deposition of H3K4me3 at the promoter regions of NOX2 complex genes, which in turn facilitated the accessibility of transcription factor such as NF-kB and enhanced transcriptional activation. Clinically, NOX2 gene signatures correlated with a favourable prognosis in bladder cancer patients receiving BCG intravesical instillation. Our findings reveal that BCG training reprograms TAMs to overproduce ROS through epigenetic rewiring of NOX2-ROS axis. Systemic BCG training becomes an effective and promising strategy to remodel the TME with enhanced pan-anti-tumor activity of infiltrating macrophages.

Targeting the gut microbiota is a promising strategy to enhance the efficiency of cancer immunotherapy; however, success has been limited. Here we combined metagenomic analysis and in silico prediction to identify bacterial species associated with immunotherapy response in patients with non-small-cell lung cancer. We constructed a defined consortium (RCom) of 15 bacterial species, most of which were isolated from responder patient faeces, associated with improved clinical response to anti-programmed cell death protein 1 (PD-1) treatment. Metabolic models and in vitro experiments revealed that RCom is a stable and cooperative community, and in vivo experiments showed that RCom engrafts and produces immunomodulatory metabolites. Oral administration of RCom improved the anti-tumour activity of anti-PD-1 by increasing the intratumoural infiltration and cytotoxic function of CD8+ T cells in syngeneic tumour models and across mice with heterogeneity in baseline gut microbiota composition. RCom supplementation also limited anti-PD-1 resistance in mice conferred by faecal microbiota transplantation from individual non-responsive patients. These findings suggest that RCom is a potential adjuvant to improve responsiveness to anti-PD-1 therapy in cancer.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, yet only a limited proportion of patients benefit from ICI monotherapy. Identifying new factors that enhance antitumor responses for combination therapy is key to improving clinical efficacy. Herein, we report that exogenous 5-HT (5-hydroxytryptamine, also named serotonin) synergizes with anti-PD-1 therapy to significantly suppress tumor growth and enhance CD8 + T cell antitumor responses. Serotonylation, as a newly noted 5-HT-involved post-translational modification, is essential for T cell functionality through modulating mitochondrial function. Mechanistically, exogenous 5-HT treatment leads to STAT1 serotonylation at glutamine 285 (Q285), which is catalyzed by tissue transglutaminase 2 (TGM2) in CD8 + T cells. This in turn promotes STAT1 phosphorylation at serine 727 and then represses Mtfr2 (mitochondrial fission regulator 2) transcription to inhibit mitochondrial fission via the MTFR2-DRP1 axis, while activating Mfn2 transcription to promote mitochondrial fusion. Clinically, higher tryptophan hydroxylase 1 (TPH1) and peripheral 5-HT levels are associated with favorable responses in patients with non-small cell lung cancer (NSCLC) receiving anti-PD-1 treatment. Together, these findings reveal a previously underappreciated serotonylation on STAT1 that remodels mitochondrial function and enhances CD8 + T cell antitumor immunity. Our study raises the possibility that 5-HT supplementation may represent a potential combination strategy with ICIs in cancer immunotherapy.

OBJECTIVES:T cell immunity is impaired due to T cell exhaustion during chronic infection, including infections caused by Mycobacterium tuberculosis (M.tb). However, the immunological characteristics of multidrug resistant-tuberculosis (MDR-TB) patients remain unclear. METHODS:Multiplex flow cytometry was employed to measure the expression of immune checkpoint (IC) molecules (cytotoxic T-lymphocyte-associated protein-4 [CTLA-4], programmed cell death protein-1 [PD-1], T cell immunoglobulin and mucin-domain containing-3 [TIM-3]) and the proliferation marker Ki67 in MDR-TB (n = 27) and drug-sensitive TB (nondrug resistant [NR]-TB) (n = 51) samples. RESULT:We showed that MDR-TB patients exhibited higher percentages of CTLA-4, PD-1, and TIM-3 expressing T cells than NR-TB subjects before anti-TB treatment. Additionally, significantly higher percentages of CTLA-4+ PD-1+ and CTLA-4+ TIM-3+ co-expressing T cells were observed in MDR-TB patients when compared to NR-TB patients. Impaired cell proliferation of T cells was detected in MDR-TB patients with more exhaustion status of T cells. Interestingly, the expression levels of these IC molecules on T cells decreased along with the anti-TB treatment in MDR-TB patients, and gradually converged to the similar levels of NR-TB subjects. CONCLUSIONS:Our results thus indicate that T cells exhibit more exhausted status in MDR-TB patients which could be reversed after the treatment. These results thus provide an alternative way to ameliorate MDR-TB treatment through improving anti-TB T cell immunity.

Small cell lung cancer (SCLC) is characterized by high malignancy and early propensity for metastasis, and modest response to immunotherapy due to the immunosuppressive microenvironment. Surgical intervention has shown benefits in treating early-stage SCLC. However, most patients experience recurrence after surgery. The factors associated with relapse free survival in these patients remain unclear. We collected operation specimens from ten early-stage SCLC patients (N0M0), conducted long-term follow-up, and grouped them based on disease status. Subsequently, we performed a retrospective analysis using single-cell spatial imaging mass cytometry to explore the characteristics of tumor cells and differences in the tumor microenvironment, especially the single-cell constitute of immune cells, between the two groups. We found that, in early-stage SCLC, tumor cells display pronounced heterogeneity, both intra-group and inter-group. Patients with early recurrence are characterized by a distinct subpopulation of tumor cells with high Ki-67 expression. Non-relapse patients demonstrate better infiltration of M1 macrophages and stromal cells. Neighborhood analysis suggested that positive interactions between macrophages, stromal cells, and T cells with tumor cells may benefit patient prognosis. Additionally, recurrent tumor cells might enhance their metastatic capacity and remodel the microenvironment through upregulation of GranzymeB or reduction of c-Myc expression. In conclusion, SCLC tumor cells demonstrate tumor heterogeneity and microenvironmental changes in the early clinical stages. A higher proportion of M1 macrophages is associated with prolonged postoperative survival in early-stage SCLC patients. This research provides novel insights and evidence for treating and preventing postoperative recurrence in SCLC.

Emerging evidence suggests that alterations in intestinal epithelial glycosylation are implicated in the pathogenesis of inflammatory bowel disease (IBD). However, the intricate roles of gut glycosylation in maintaining intestinal homeostasis remain inadequately elucidated. Beta 1, 3-N-acetylglucosaminyltransferases (B3GNTs) are Golgi glycosyltransferases involved in the biosynthesis of poly-N-acetyl-lactosamine chains. In this study, we here create B3gnt8 knockout (B3gnt8-/-) mice to investigate its precise effects on intestinal homeostasis. Our findings reveal that both messenger RNA (mRNA) and protein levels of B3GNT8 are significantly diminished in the inflamed mucosa of pediatric IBD patients. Furthermore, the levels of B3GNT8 were negatively correlated with ulcerative colitis (UC) progression. B3gnt8-/- mice exhibited heightened vulnerability to Dextran sodium sulfate (DSS)-induced intestinal inflammation, characterized by compromised tight junction integrity and impaired secretion of Mucin from goblet cells. The loss of B3gnt8 resulted in a significant reduction in Paneth cell populations as well as diminished lysozyme content, leading to an altered composition and adhesion properties of intestinal bacteria. Additionally, B3gnt8 deficiency impaired lysosomal stability, potentially reducing glycosylation of lysosomal-associated membrane proteins half (LAMP1/2). From a mechanistic perspective, deficiency in B3gnt8 disrupted autophagy-lysosomal processes within Paneth cells may via the ATG16L1-ATG12-ATG5 pathway. Notably, the absence of B3gnt8 rendered these mice more susceptible to DSS-induced colitis. In conclusion, our findings identify B3GNT8 as a key player in intestinal epithelial glycosylation, thereby revealing a potential target for new IBD therapeutics.

H-NS is a prokaryotic histone-like protein that binds to bacterial chromosomal DNA with important regulatory roles in gene expression. Unlike histone proteins, hitherto post-translational modifications of H-NS are still largely uncharacterized, especially in bacterial pathogens. Salmonella Typhimurium is a primary enteric pathogen and its virulence is mainly dependent on specialized type III secretion systems (T3SSs), which were evolutionarily acquired via horizontal gene transfer. Previous studies have shown that H-NS plays a critical role in silencing foreign T3SS genes. Here, we found that H-NS is phosphorylated at multiple residues in S. Typhimurium, including S45, Y61, S78, S84, T86, and T106. Notably, we demonstrated that phosphorylation of H-NS S78 promotes its dissociation from DNA via a mechanism dependent on dimer formation, thereby leading to transcriptional activation of target genes. Functionally, phosphoryl-H-NS contributes to the expression of T3SS-associated proteins and hence increases bacterial virulence during infection. Therefore, our study reveals a novel mechanism by which covalent modifications of prokaryotic histone-like proteins regulate bacterial virulence of an important human pathogen.

The bile acid isoallolithocholic acid (isoalloLCA) has been observed to be reduced in patients with inflammatory bowel diseases (IBD). However, its role in the pathogenesis of pediatric IBD remains poorly understood. Here we show evidence that isoalloLCA treatment decreases lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production in blood cells from children diagnosed with IBD. In experimental models of IBD, isoalloLCA alleviates acute intestinal inflammation caused by LPS or dextran sulfate sodium (DSS) and shows therapeutic efficacy in a chronic colitis model using Il10 knockout (Il10-/-) mice. Within the mucosa of these murine models, isoalloLCA enhances the expression of the regulatory T cell transcription factor Forkhead box P3 (Foxp3), while simultaneously inhibiting ETS2, a critical regulator of inflammatory macrophages in IBD. In bone marrow-derived macrophages (BMDMs), isoalloLCA mitigates LPS-induced inflammation, potentially through the enhancement of mitochondrial reactive oxygen species (mitoROS) production and inhibition of the ETS2-HIF1A/PFKFB3 signaling pathway. Simultaneously, isoalloLCA metabolically reprograms macrophages by enhancing oxidative phosphorylation (OXPHOS) that is linked to anti-inflammatory effects. Our research indicates that metabolic modulation of macrophages amplifies the anti-inflammatory properties of isoalloLCA, thereby revealing a promising therapeutic avenue for addressing pediatric IBD.

Long COVID has emerged as a major global health concern, yet the long-term trajectory of immune recovery and its contribution to persistent symptoms remain to be elucidated. Here, we conducted a three-year longitudinal follow-up of the 47 COVID-19 patients and applied single-cell RNA sequencing (scRNA-seq) and multiplex cytokine profiling to comprehensively characterize the peripheral immune landscape during convalescence. We observed persistent immune dysregulation up to three years post-infection, characterized by chronic inflammation and impaired restoration of naïve CD4⁺ T cells, naïve CD8⁺ T cells, and SLC4A10⁺ MAIT cells—features reminiscent of immunosenescence. Notably, Th17 cells, rather than monocytes, emerged as key drivers of chronic inflammation beyond one year. We identified two distinct Th17 subsets: RORC⁺ Th17 cells and LTB⁺ Th17 cells. While RORC⁺ Th17 cells were negatively correlated with inflammatory cytokine levels, LTB⁺ Th17 cells showed proinflammatory features and were positively associated with long COVID symptoms. Sustained elevation of S100A8 and IL-16 in follow-up patients may contribute to the persistent presence of LTB⁺ Th17 cells. Together, our study provides an in-depth longitudinal map of immune remodeling in COVID-19 convalescents, revealing key cellular and molecular drivers of sustained inflammation up to three years post-infection.

Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase that contains two catalytic domains and a zinc finger ubiquitin binding domain (ZnF-UBP). The deacetylation function of HDAC6 has been extensively studied with well-characterized substrates such as α-tubulin and Hsp90. Apart from its deacetylase activity, HDAC6 ZnF-UBP binds to unanchored ubiquitin of specific sequences and serves as a carrier for transport of aggregated proteins. subsequently, aggresomes is degraded by the autophagy-lysosome pathway. Additionally, Cells can utilize this HDAC6-dependent microtubule transport to assemble and activate inflammasomes, which play a critical role in immune regulation. HDAC6 displays a unique structure and cellular localization as well as diverse substrates, and exhibits a wider range of biological functions than other HDAC isoforms. HDAC6 has been intimately linked to a spectrum of diseases, including rheumatoid arthritis, systemic lupus erythematosus, psoriasis, neuritis, and the cancer immune microenvironment. This review systematically synthesizes the current research advancements of HDAC6, focusing on three key dimensions: the mechanism of action of HDAC6, therapeutic advancements, and translational prospects in clinical applications.

Trained immunity refers to memory-like responses of innate immune cells when they re-encounter pathogenic stimuli. Bacillus Calmette-Guérin (BCG) vaccination implies enhanced antiviral immunity, whereas the underlying mechanisms remain unclear. Herein, we have uncovered elevated expression of low-density lipoprotein receptor (LDLR) on BCG-trained macrophages with robust type I interferon (IFNI) production and antiviral effects both in vivo and in vitro. Consequently, cholesterol is accumulated in BCG-trained macrophages, leading to the augmentation of NFE2L1 expression and the formation of NFE2L1/IRAK1/TRIM25 complex where TRIM25 mediates IRAK1 K63 polyubiquitination to exaggerate IFNI responses in an RIG-I-dependent manner. We have also observed LDLR+ macrophages displaying heightened IFNI responses in BCG-treated human macrophages. To antagonize LDLR degradation by PCSK9 inhibitors increases IFNI responses in the macrophages and accelerated viral clearance. Our study thus couples LDLR upregulation to antiviral activity in BCG-trained macrophages, making commercial PCSK9 inhibitors potential antiviral indications in clinic.

Objective·To identify relevant biomarkers for patients with coronavirus disease 2019-associated kidney injury(COVID-19-associated KI)and explore the mechanisms underlying the involvement of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)proteins in infection-related KI by affecting the interactions between renal cells and macrophages.Methods·A retrospective analysis was conducted on the clinical characteristics of COVID-19 patients with KI treated in Shanghai Ninth,People's,hospital from December 2022 to February 2023.Serum levels of inflammatory factors and chemokines were measured by using enzyme-linked immunosorbent assay(ELISA).In vitro,human macrophage cell line THP-1 cells were stimulated with recombinant S1 subunit protein derived from SARS-CoV-2 spike protein.The cells and culture supernatants were collected to detect the levels of inflammatory factors and chemokines by using quantitative real-time PCR(qRT-PCR)and ELISA.Conditioned medium was prepared from the cell culture supernatants of S1-stimulated THP-1 cells and used to stimulate human renal epithelial cells(HK-2)in vitro to assess cytokine secretion.Antibody blocking experiments were performed to analyze the effects of the conditioned medium on the production of cytokines in HK-2 cells.Results·Among 39 patients with COVID-19,8(20.50%)had creatinine levels above the reference interval,which indicated the occurrence of KI.The levels of peripheral tumor necrosis factor-α(TNF-α)in the COVID-19 patient with KI group[(18.33±8.20)pg/mL]were significantly higher than those in the non-KI group[(11.88±6.50)pg/mL](P=0.015).In vitro assay has shown that S1-spike protein stimulation promoted the level of gene transcription and production of TNF-α,interleukin-1β(IL-1β)and chemokine C-X-C motif ligand 10(CXCL10)in THP-1 macrophage cells(P＜0.001).Furthermore,the conditioned medium from S1-stimulated THP-1 cells promoted the secretion of TNF-α,IL-1β and CXCL10 by HK-2 cells(P=0.005).When anti-TNF-α antibody(Infliximab)was used to block TNF-α in the culture supernatants from S1-stimulated THP-1 cells,the secretion level of TNF-α by HK-2 cells decreased dramatically(P＜0.001).Conclusion·TNF-α levels increase significantly in COVID-19 patients with KI,implying the significance of TNF-α in the occurrence of COVID-19-associated KI.In vitro experiments confirm that the S1 protein induces TNF-α secretion from THP-1 cells,leading to increased inflammatory responses in renal cells,which may contribute to the development of COVID-19-associated KI.Therefore,targeting TNF-α may become an alternative strategy to reduce the occurrence of COVID-19-associated KI.