Renal tubular epithelial cells are among the earliest renal parenchymal cells to be injured in the context of acute kidney injury (AKI). Numerous studies have confirmed that fibrinogen-like protein 2 (FGL2) can regulate the occurrence and development of inflammation during disease progression. We found that FGL2 expression is elevated under AKI conditions. However, the role of FGL2 in AKI remains unclear. To elucidate the role of FGL2 in AKI, we employed lentiviral and adeno-associated virus for transfection in cellular and murine models. Furthermore, by leveraging datasets from the gene expression omnibus and gene set enrichment analysis databases, we identified the inflammation-related genes in AKI and predicted their interaction with FGL2. Both in vivo and in vitro studies showed that overexpression of FGL2 markedly increased complement C3a (C3a) levels and exacerbated inflammation and injury. In contrast, knockdown of FGL2 resulted in a marked decline in C3a levels, which not only conferred a substantial protective effect against hypoxia/reoxygenation-induced injury in tubular cells but also effectively alleviated kidney injury in mice. At the molecular level, FGL2 interacts with complement C3, leading to elevated C3a production. This stimulates the inflammatory response of renal tubular epithelial cells, thereby inducing tubular damage. Targeting FGL2 may hold potential prevention and treatment strategy for tubular injury in AKI.
AutoimmunityMing Zhu, Suqing Zhou, Changxing Gao, Yanshan Zhu, Xieling He, Qianjin Lu, Ming Zhao, Zijun Wang
Increasing evidence shows that CD8+ T cells are the pathogenic mediators of tissue injury in systemic lupus erythematosus (SLE), sustaining the chronic inflammation through the accumulation of long-lived cytotoxic memory populations. However, the transcriptional mechanisms that prevent the aberrant differentiation of pathogenic CD8+ T cells remain poorly understood. Here, the transcription factor E4BP4 (NFIL3) was identified as a critical restraint of cytotoxic effector-memory CD8+ T cells in lupus. E4BP4 expression was reduced in CD8+ T cells from SLE patients and inversely correlated with disease activity. Using a lupus-like disease model, we found that E4BP4 deficiency accelerated disease progression, resulting in heightened autoantibody production, immune complex deposition, and renal pathology. This phenotype was associated with the systemic accumulation of cytotoxic effector-memory CD8+ T cells. Depletion of CD8+ T cells significantly ameliorated the disease phenotype, confirming the functional contribution of CD8+ T cells to lupus-like immunopathology. Competitive adoptive transfer experiments revealed that E4BP4 functions cell-intrinsically to limit the cytotoxicity and proliferation of CD8+ T cells in autoimmunity. Beyond autoimmunity, E4BP4 deficiency also resulted in an exuberant CD8+ effector-memory T cell response to Listeria monocytogenes infection, indicating a broader role for E4BP4 in limiting CD8+ T cell effector-memory responses. Collectively, these findings establish E4BP4 as a transcriptional checkpoint that restricts pathogenic CD8+ effector-memory T cell responses to maintain immune homeostasis in autoimmunity and infection.
Gut microbesIzabella Sall, Randi Foxall, Lindsey Felth, Anirudh Gaur, Jennifer L Whistler, Cheryl A Whistler
Although opioids are effective analgesics, they can lead to problematic drug use behaviors that underlie opioid use disorder (OUD). Opioids also cause gut microbiota dysbiosis, which is linked to altered opioid responses. We used a longitudinal paradigm of voluntary oral morphine self-administration to capture multiple facets of drug seeking and preserve both individual behavioral responses and individual gut microbiota variation to investigate the role of the gut microbiota in a mouse model of OUD. Although all the mice consumed morphine, only a subset of the mice that transitioned to a state we defined statistically as compulsive. In compulsive mice, morphine constricted natural variability and fragmented the microbiota community networks, which convergently reorganized to form robust novel connections post-morphine. In contrast, the more variable communities of non-compulsive mice were highly interconnected during morphine disturbance and displayed more continuity post-morphine, suggesting greater flexibility and adaptability. Compulsive mice displayed a greater loss of functional diversity and a shift in favor of potential pathobionts, whereas non-compulsive mice better preserved genera associated with gut health and broader functional diversity. These findings highlight the potential role of persistent and stable opioid-induced microbiota dysbiosis in long-term behavioral changes underlying OUD and contributing to vulnerability to relapse.
Gut microbesDemengjie Le, Mohammad Hassan Heidari, Shunmin Zhu, Yingying Luo, Changzhou Li, Yue Li, Bin Yang, Xuan Wang
The gut-skin axis plays a pivotal role in psoriasis pathogenesis, yet the precise metabolic crosstalk by which intestinal commensals regulate cutaneous immunity remains elusive. Here, we identify a functional "microbe-enzyme-metabolite-immune" axis that orchestrates skin homeostasis. Through multi-omics analyses of psoriatic patients and imiquimod-induced murine models, we reveal a significant depletion of the gut commensal Parabacteroides distasonis (P. distasonis) and its metabolite, genistein. We demonstrate that P. distasonis utilizes its inherent β-glucosidase activity to convert dietary genistin into bioactive genistein, a process we validated using an engineered β-glucosidase-expressing E. coli strain. Therapeutically, supplementation with P. distasonis or genistein significantly ameliorates psoriatic phenotypes, restores skin barrier integrity (filaggrin/loricrin), and suppresses IL-23/IL-17-mediated inflammation. Mechanistically, we uncovered that genistein enhanced CD200-CD200R signaling and suppressed macrophage activation. It effectively reactivates CD200 expression, thereby inhibiting the canonical NF-κB signaling pathway and blunting macrophage-driven inflammation. Notably, the therapeutic efficacy of this axis was abrogated by CD200 blockade, confirming its indispensability. Collectively, our findings elucidate a causal mechanism linking gut microbial enzyme activity to host skin immunity, highlighting P. distasonis-derived genistein as a promising precision intervention for psoriasis management.
While interferon regulatory factor 1 (IRF1) has been implicated in reactive oxygen species (ROS)-dependent neutrophil extracellular trap (NET) release during acute lung injury, its regulatory role in mitochondrial dysfunction-driven NETosis specific to pediatric pneumonia remains unexplored. Neutrophils were isolated from the bronchoalveolar lavage fluid (BALF) of pediatric pneumonia patients and the bone marrow of mice. IRF1 expression was quantified via qRT-PCR and Western blotting. Mitochondrial ROS (mtROS) and total ROS were measured by flow cytometry. Mitochondrial dysfunction was assessed by ATP quantification. NETosis was evaluated through immunofluorescence staining and ELISA quantification of neutrophil elastase (NE), myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3) levels. For in vivo studies, pneumonia-related lung injury was induced by intratracheal LPS instillation in mice, with pathological severity graded by H&E staining, pulmonary edema quantified via the wet/dry weight ratio, and inflammation assessed by the BALF protein concentration. Mechanistically, the IRF1/MMP9 interaction was predicted by bioinformatics (STRING database) and validated by co-immunoprecipitation (Co-IP) and immunofluorescence staining, while MMP9 overexpression was achieved via lentivirus transduction to delineate pathway regulation. IRF1 was significantly upregulated in BALF neutrophils from patients and correlated with elevated ROS production and mitochondrial dysfunction, as well as NETs release. IRF1 knockdown attenuated ROS-driven NETosis in vitro. Matrix metalloproteinase 9 (MMP9) was predicted to interact with IRF1, and MMP9 overexpression effectively reversed the beneficial effects of IRF1 deficiency on ROS release, mitochondrial dysfunction, neutrophil apoptosis, and NETosis. Consistently, in mouse models, MMP9 overexpression abolished the protective effects of IRF1 deficiency, exacerbating acute lung injury and restoring NETs levels in BALF.
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease, and currently, there are no effective means to reverse its progression. Cuproptosis is a newly discovered copper-dependent programmed cell death mechanism, but its role in IPF remains unknown. This study aimed to investigate whether Cuproptosis is involved in the pathogenesis of IPF and to evaluate its potential as a therapeutic target. METHODS: In vitro, a fibrosis model was induced in human lung epithelial cells by bleomycin treatment. In vivo, a C57BL/6J mouse IPF model was established by intratracheal instillation of bleomycin. The effects on fibrosis progression were observed using the Cuproptosis inhibitor ammonium tetrathiomolybdate and siRNA knockdown of the copper ion transporter Slc31a1. RESULTS: Significant Cuproptosis was observed in both BLM-induced lung epithelial cells and mouse lung tissue. Gene expression profiling identified key Cuproptosis-related genes, including Slc31a1. Pharmacological inhibition of Cuproptosis effectively reversed the Cuproptosis process in both in vitro and in vivo models and significantly reduced fibrotic pathological changes. At the cellular level, knockdown of Slc31a1 mimics the protective effect of TTM; however, its efficacy in whole animal models is limited. CONCLUSION: This study identifies cuproptosis as a significant contributor to the pathogenesis of pulmonary fibrosis. Pharmacological inhibition of this pathway alleviates disease phenotypes, supporting the feasibility of targeting copper metabolism.
VirulenceAgustin Resendiz-Sharpe, Carmen Kandelbauer, Maria Isabel Navarro Mendoza, Giel Lemmens, Jakob Scheler, Ingo Bauer, Eliane Vanhoffelen, Laura Seldeslachts, Lan…
Mucormycosis, a disease encompassing life-threatening fungal infections by members of the Mucorales group, is characterized by significant heterogeneity, poor clinical outcomes, and globally rising incidences. Although showing clear outbreak potential, the disease remains poorly understood. Mucorales infections are characterized by rapid progression and extensive tissue destruction, often requiring disfiguring surgical interventions. Moreover, intrinsic resistance and high drug tolerance limit antifungal effectiveness, contributing to the disease's high mortality. To overcome the challenges associated with these infections, a deeper understanding of mucormycosis is urgently needed. Valuable translational insights traditionally derive from in vivo host models. However, current systems are limited by single-endpoint invasive analysis, offering only a narrow view of infection progression and treatment effects. To address these critical shortcomings and enhance experimental models, bioluminescent Mucor lusitanicus reporter strains were generated to establish models that allow for real-time, longitudinal monitoring of infection in individual animals. Codon optimization, targeted integration of the firefly luciferase gene, and control of expression using highly active promoters resulted in the successful establishment of two mucormycosis models: Galleria mellonella larvae as an intermediate infection model and a translational mouse model. In the murine model, the integration of micro-CT imaging further enhanced the characterization of host-pathogen interactions by enabling noninvasive assessment of tissue responses over time. Overall, our approach enables enhanced temporal resolution for quantitative assessment of fungal burden and host responses. Using M. lusitanicus as a model organism, this methodology establishes a foundation and technical expertise for application toward clinically relevant Mucorales infection and antifungal research.
VirulenceYvette Kazungu, Shrilakshmi Hegde, Parul Sharma, Amy Marriott, Andrew Steven, Jesus Reine, Jessica Dagley, Matthias Mack, Anja Kipar, James P Stewart, Joseph D…
SARS-CoV2 can induce an acute respiratory distress syndrome (ARDS), provoked by a dysregulated hyper-inflammatory pulmonary immune response. Here, we used the keratinocyte-18 humanized angiotensin converting enzyme-2 (K18-hACE2) mouse model of SARS-CoV2, where expression of the CoV2 spike protein receptor, hACE-2, is restricted to epithelia, to characterize inflammatory pulmonary immune responses post-intranasal infections with the delta isolate SARS-CoV2(∆) B.1.617.2. Immune-profiling by focused transcript analysis, inflammatory protein array, and multi-color flow cytometry, confirmed that clinically relevant markers of COVID-19 (IL-6, GM-CSF, neutrophils, inflammatory monocytes) were significantly elevated in lungs of mice at day 5 post-infection and that remdesivir antiviral active metabolite (GS441524) treatment significantly modified SARS-CoV2∆ viral loads and pulmonary inflammation. Chemokine ligands of CCR2 (CCL2/7/8) were among the top 5% upregulated pulmonary transcripts in a focused human infection response array to SARS-CoV2∆. CCL2 was confirmed as elevated in protein assays in SARS-CoV2∆ infected lungs. To address the functional relevance of the CCR2 pathway of inflammatory cell recruitment to the lungs mediating disease, mice were administered with anti-CCR2 antibody daily at the point of infection for up to 6 d. Anti-CCR2 treated mice showed significant improved welfare scores, were protected from weight loss, modified myeloid pneumonitis, and displayed significantly blunted cytokine and chemokine response in the lungs, despite not affecting pulmonary viral loads. Our data supports therapeutic benefit of modifying CCR2-dependent cell recruitment in the treatment of viral-induced ARDS.
Gut microbesMengXuan Du, Wenzhao Wang, Min-Zhi Jiang, Xin-Wei Sun, Lei Sun, Chang Liu, Shuang-Jiang Liu
Next-generation probiotics derived from gut commensals show promise for metabolic disease intervention, yet effective anti-obesity strains remain limited. Here, we demonstrate that oral administration of Christensenella massiliensis markedly alleviates obesity and metabolic dysfunction in high-fat diet-induced obese mice. Treatment reduced food intake, improved glucose tolerance and insulin sensitivity, lowered blood glucose and lipid levels, and attenuated hepatic steatosis and adipose accumulation. C. massiliensis increased the levels of plasma GLP-1 and ileal GLP-1 receptor expression while decreasing ghrelin level, suggesting modulation of gut hormone regulation. C. massiliensis also suppressed systemic and colonic inflammation, accompanied by upregulation of metabolic homeostasis-related genes (ppara, pparg, ucp2). Targeted and quantitative metabolomics identified altered gut metabolic profiles, particularly reduced kynurenine levels. In vitro assays further showed that C. massiliensis converted kynurenine into kynurenic acid, and its lysate reversed kynurenine-induced lipid accumulation, inflammation, and PPARγ suppression in hepatocytes, providing mechanistic support for the observed in vivo metabolic benefits. These findings support C. massiliensis as a promising next-generation probiotic for obesity management.
Gut microbesFernanda Raya Tonetti, Hui Han, Marcos F Fondevila, Wenchao Wei, Burcin Özdirik, Jasmohan S Bajaj, Mitchell L Schubert, Masoumeh Sikaroodi, Patrick M Gillevet,…
Gastric acid-suppressive medications, particularly proton pump inhibitors (PPIs), are commonly used in patients with alcohol-associated liver disease (ALD) to prevent and manage upper gastrointestinal bleeding, gastroesophageal reflux disease, and non-steroidal anti-inflammatory/aspirin-induced gastroesophageal damage. By inhibiting the gastric H⁺/K⁺-ATPase, PPIs suppress acid secretion and impair bacterial killing, thereby promoting gut dysbiosis that disrupts barrier integrity and enhances bacterial translocation, ultimately exacerbating liver injury. PPIs are frequently co-administered with antibiotics for indications such as gastrointestinal bleeding, Spontaneous Bacterial Peritonitis (SBP), other infections, or hepatic encephalopathy prophylaxis, but the consequences of this combined therapy on gut microbial ecology and disease outcomes remain unclear. Our study addresses this gap by showing how PPI use, alone or with antibiotics, reshapes the gut microbiome and aggravates liver disease progression. In previous studies, we showed that PPIs promote dysbiosis and ALD progression in mice and humans by facilitating intestinal expansion and hepatic translocation of Gram-positive Enterococcus. Fecal cytolysin, an Enterococcus faecalis exotoxin that induces hepatocyte death, predicts mortality in patients with alcohol-associated hepatitis (AH). In this study, we have examined the mechanism by which PPIs alone and in combination with non-absorbable antibiotics targeting Gram-positive bacteria influence ALD, as well as the disease mechanisms associated with cytolytic Enterococcus faecalis and the development of therapeutic strategies. In mice, alcohol administration during gastric acid suppression promoted expansion of Gram-positive taxa, including cytolysin-producing Enterococcus. Similarly, PPI use in patients with AH was associated with increased fecal Enterococcus and higher 30-d mortality, underscoring the translational relevance of our findings. Unexpectedly, treatment of acid-suppressed mice with non-absorbable antibiotics designed to suppress Gram-positive bacteria worsened ethanol-induced steatohepatitis: while Enterococcus abundance decreased, Streptococcus and other potentially pathogenic taxa expanded, leading to increased bacterial translocation and aggravated liver injury. In patients with cirrhosis or metabolic dysfunction-associated steatotic liver disease (MASLD), PPIs did not promote Enterococcus expansion, indicating etiology-dependent microbiome responses. Finally, we identified dipalmitoylphosphatidylcholine and Caspase-1 inhibitor as in vitro and in vivo modulators of cytolysin activity, highlighting potential therapeutic avenues. Collectively, our study demonstrates how PPIs and non-absorbable antibiotics targeting Gram-positive bacteria interact with the gut microbiome to drive ALD, underscoring the need for careful therapeutic management.
Objective: The transition from acute kidney injury (AKI) to chronic kidney disease (CKD) is a major cause of poor prognosis in renal injury, and preventing the progression from AKI to CKD has important clinical significance. Finerenone exerts renoprotective effects, however, its role in the AKI-CKD transition remains unclear.Methods: An ischemia-reperfusion (IR)-induced AKI mouse model was established in male C57BL/6 mice by unilateral renal artery clamping combined with contralateral nephrectomy. Finerenone was administered at doses of 10 mg/kg or 15 mg/kg, and renal function, aldosterone levels, podocyte injury, inflammatory responses, and renal fibrosis were evaluated at days 8 and 28 to investigate pathological mechanisms underlying the AKI-CKD transition.Result: Finerenone significantly attenuated IR-induced renal injury and improved renal function during the acute phase and throughout the subsequent AKI-CKD transition. Compared with 10 mg/kg, finerenone at 15 mg/kg more effectively reduced podocyte injury, dampened the inflammatory response, and attenuated renal fibrosis. Mechanistically, finerenone suppressed the p38 phosphorylation and reduced nuclear factor kappa-B (NF-κB) activation during the AKI-CKD transition.Conclusion: Finerenone exerts dose-dependent renoprotective effects during AKI progression and the subsequent AKI-CKD transition, potentially by suppressing podocyte mineralocorticoid receptor overactivation and concomitant reductions in renal inflammation and fibrosis, accompanied by attenuation of p38 phosphorylation and NF-κB activation during the AKI-CKD transition.
Clostridioides difficile infection (CDI) is a major cause of antibiotic-associated diarrhea, with frequent recurrences closely linked to antibiotic-induced dysbiosis of the gut microbiota and bile acid metabolism. Parabacteroides distasonis, a potential probiotic capable of converting primary to secondary bile acids, has shown therapeutic promise in several metabolic and inflammatory diseases. This study evaluated the preventive and therapeutic effects of P. distasonis against CDI and explored the underlying mechanisms. We characterized the probiotic properties of four P. distasonis strains and investigated the inhibitory activity of strain 1190003 against C. difficile, as well as its protective and therapeutic efficacy in mouse models. Gut microbiota structure and bile acid metabolic profiles were analyzed by integrating 16S rRNA gene sequencing and metabolomics. The four P. distasonis strains exhibited strong acid and bile salt tolerance as well as auto-aggregation ability. Supernatants from P. distasonis co-cultured with cholic acid (4 mM and 8 mM) significantly inhibited C. difficile growth, toxin expression and spore formation, with deoxycholic acid identified as the key inhibitory metabolite. Both live P. distasonis and its culture supernatant alleviated disease severity in CDI mouse models and ameliorated gut microbiota dysbiosis. Notably, the relative abundance of Parabacteroides goldsteinii was increased following supernatant treatment. Furthermore, intervention with either live P. distasonis or its supernatant elevated the level of hyodeoxycholic acid. In summary, P. distasonis acts as a potential probiotic that alleviates CDI by ameliorating gut microbiota dysbiosis and remodeling bile acid metabolism. These findings provide experimental evidence for its use as a microbiota-based therapeutic strategy.
Gut microbesDoyeon Kim, Minshu Li, Thi Ha Nguyen, Yu-Jeong Choi, Sojeong Jang, Minhyeong Kim, Young-Kwang Kim, Min-Kyoo Shin, Arvie Camille V de Guzman, Sunghyouk Park
The gut microbiome modulates host neuropathology, but the mechanisms linking specific microbial genes and metabolites to host phenotypes remain poorly defined. Here, we identify microbiome-derived vitamin B6 (VB6) and its biosynthesis gene as key regulators of host dopaminergic homeostasis. Metagenomic analysis of fecal samples from Parkinson's disease (PD) patients revealed enrichment of biosynthetic pathways for pyridoxal-5'-phosphate (PLP), the active form of VB6, and tyrosine decarboxylase genes. Using E. coli-C. elegans symbiotic models, we demonstrate that the bacterial pdxJ gene, encoding a key enzyme in de novo VB6 synthesis, is essential in regulating host dopaminergic homeostasis. Colonization with pdxJ-deficient bacteria led to reduced host VB6 and dopamine levels, reduced dopaminergic enzyme activity, and altered motor behavior, which were all rescued by VB6 supplementation. In PD-relevant C. elegans models, bacterial PLP biosynthesis modulated α-synuclein aggregation and behavioral deficits associated with human LRRK2 mutations. In mice, colonization with pdxJ-deficient bacteria reduced serum VB6 levels, decreased tyrosine hydroxylase staining in the substantia nigra, and impaired motor coordination, which were rescued by VB6 supplementation. Overall, our results define a bacterial pdxJ-PLP-dopamine axis that links gut microbial metabolism to host dopaminergic phenotypes and suggest bacterial VB6 biosynthesis as a potential modifier of PD risk and a context-dependent therapeutic target.
Renal tubulointerstitial fibrosis, as the common final pathological outcome of almost all progressive chronic kidney diseases (CKD), is challenging to reverse. Although CKD remains incurable, reversal or amelioration of renal fibrosis is beneficial to retard CKD progression. In this study, based on our in vitro and in vivo studies, we found that relaxin, a human hormone with promising renal protection effects, was closely related to ameliorating tubular injury and reversing tubulointerstitial fibrosis in the obstructed kidney in the mouse model of unilateral ureteral obstruction (UUO). Downregulation of relaxin was accompanied by the development of tubulointerstitial fibrosis and tubular epithelial fatty acid oxidation (FAO) disorder. Furthermore, overexpression of relaxin significantly alleviated, whereas knockdown of relaxin extremely aggravated the process of renal interstitial fibrosis following UUO and FAO disorder in mouse tubular epithelial cells (mTECs). Mechanistically, AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α) pathway was validated as a downstream pathway of relaxin, which participated in the regulation of renal interstitial fibrosis. Our findings suggest that upregulation of relaxin positively alleviated renal tubulointerstitial fibrosis in UUO-induced CKD via AMPK-PGC1α axis, which gives insight into novel therapeutic and diagnostic targets of CKD.
Clinical and experimental hypertension (New York, N.Y. : 1993)Keng Zhong, Libo Zhao, Chengliang Cai, Jianhui Peng, Shangyi Yu, Jiajun Pan, Xuechao Zhang, Yanren Lu, Qing Xue, Guanxin Zhang
OBJECTIVE: Aortic dissection (AD) is a fatal cardiovascular emergency that is predominantly induced by long-term uncontrolled hypertension. MATERIALS AND METHODS: The mouse AD model was established by combining β-aminopropionitrile with angiotensin II. The incidence rate, rupture rate, and survival status of the mice were evaluated. The structural damage of the aorta was observed using tissue staining technology, the extracellular matrix status, and macrophage pyroptosis were evaluated by immunofluorescence staining, the infiltration of inflammatory cells was detected by immunohistochemistry, the expression of pyroptosis-related molecules, and inflammatory factors was analyzed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Cell proliferation was detected by EdU staining, and cell apoptosis was detected by flow cytometry. RESULTS: In the aortic tissues of AD model mice, the expression of APN, and the content of APN in the serum were significantly decreased. APN intervention can alleviate the thickening of the aortic media, rupture of elastic fibers, and degradation of the extracellular matrix. APN inhibits the proliferation, apoptosis, and phenotypic transformation of vascular smooth muscle cells (VSMCs). At the same time, it can inhibit the infiltration of neutrophils and macrophages and the inflammatory response. Underlying mechanism, it was found that APN inhibited NLRP3-mediated pyroptosis by reducing the release of inflammatory factors; this effect was dependent on the phosphorylation activation of AMPKα and APN receptor. CONCLUSION: APN plays a protective role in AD. Its underlying mechanism is related to the activation of the AMPK pathway, which in turn inhibits macrophage pyroptosis and vascular inflammation. This study offers a new perspective for the pathological mechanism of AD.
The clinical threat posed by Klebsiella pneumoniae is dual-faceted, encompassing both hypervirulence and carbapenem resistance. The emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) merges these threats, with the ST11-K64 clone being a dominant and concerning lineage. However, its clinical presentation diverges from the classic hypervirulent Klebsiella pneumoniae (hvKp); hv-CRKP is isolated from respiratory sites but is notably absent from pyogenic liver abscesses. This distinct clinical niche prompted us to investigate the underlying pathogenicity differences. Among 847 clinical Klebsiella pneumoniae isolates, 157 (18.5%) were identified as hv-CRKP. From this hv-CRKP collection, we selected five representative ST11-K64 isolates for downstream phenotypic and mechanistic analyses. Despite its prevalence and multidrug resistance, the ST11-K64 clone exhibited significantly attenuated lethality in mouse models compared to ST23-K1. Crucially, in a murine intestinal colonization model that mimics natural infection, only ST23-K1 successfully colonized the gut, caused bacteremia, and formed liver abscesses. In contrast, ST11-K64 strains showed impaired intestinal colonization and failed to translocate to the liver. Phenotypic profiling showed reduced capsule viscosity and siderophore production in ST11-K64 relative to ST23-K1, accompanied by diminished macrophage and Kupffer cell-associated fitness. RT-qPCR identified higher expression of rmpA, rmpA2, iroB, and iucA in ST23-K1, and isogenic deletion and complementation of rmpA2 or iucA in the ST23-K1 background supported their contribution to capsule/siderophore-associated phenotypes and intracellular survival in RAW264.7 macrophages. Together, these results indicate that while ST11-K64 hv-CRKP represents a serious antimicrobial-resistance threat, its invasive pathogenicity is route- and context-dependent and does not fully recapitulate the classic entero-hepatic hypervirulence of ST23-K1.
OncoimmunologyAmarilis Pérez-Baños, María Alejandra Gleisner, Andrés Tittarelli, Daniela Tapia, María Elisa Cuevas, Antonia Mardonez, Fabián Tempio, Rosario Astaburuaga-Garc…
Enhancing innate-adaptive immune crosstalk is key for improving cancer vaccine efficacy. TRIMELVax is a heat shock-conditioned whole-tumor-cell vaccine combining xenogeneic melanoma cell lysate, syngeneic B16F10 melanoma cell lysate, and Concholepas concholepas hemocyanin. Although TRIMELVax elicits robust antitumor responses in preclinical models, the mechanisms underlying its efficacy remain poorly defined. We characterized the early immune events triggered by TRIMELVax in mice using RT-qPCR, high-dimensional flow cytometry, immunohistochemistry, CFSE-based dendritic cell (DC) migration assays, and therapeutic melanoma models with transient neutrophil depletion. TRIMELVax elicited a rapid inflammatory response at the vaccination site, characterized by local upregulation of CXCL3, CXCL5, CXCL9, CCL3, CCL4, CCL12, IL-1β, IL-6/OSM, IL-12a, and G-CSF. This response drove an early influx of neutrophils and monocytes, followed by increased accumulation of cDC1, cDC2, and monocyte-derived DCs. Notably, we identified a transient population of neutrophils expressing markers associated with antigen-presenting cells (CD45⁺, CD11b⁺, Ly6G⁺, CD11c⁺, MHC-II⁺) that emerged within 12-24 hours postvaccination. These APC-like neutrophils colocalized with cDC1 at the injection site and subsequently migrated to the popliteal draining lymph nodes (pLN). Neutrophil depletion impaired cDC1 migration, reduced APC accumulation in pLN, and abolished the therapeutic efficacy of TRIMELVax. Together, these findings identify neutrophils as key early regulators of the innate inflammatory environment induced by TRIMELVax and suggest that neutrophils with APC-like features may impact DC trafficking and downstream antitumor immunity. Neutrophils, particularly those with APC-like phenotypes, emerge as promising cellular adjuvant targets for enhancing cancer vaccination strategies, offering a new avenue for rational vaccine design and combination with checkpoint blockade therapies.
Escalating resistance in Pseudomonas aeruginosa (PA) underscores the need for resistance-agnostic immunotherapies. In this study, we generated a panel of 20 monoclonal antibodies against the flagellin protein FliC and identified 16G10 as a high-affinity lead. Epitope mapping revealed a conserved linear site within the D1 domain (Gly71-Ile88), a region essential for filament assembly. In vitro, 16G10 induced bacterial aggregation, suppressed swimming motility, and significantly reduced adhesion to and invasion of A549 cells, while limiting biofilm biomass by SEM and confocal 3D imaging. In vivo, in murine pneumonia, where 16G10 administered either after pre-incubation with imipenem-resistant PA strain or as post-infection therapy, it improved survival, lowered lung bacterial loads, reduced IL-6 and TNF-α concentrations, and attenuated histologic inflammation. Protection extended across serotype A and B flagellated strains, indicating broad coverage. These data nominate 16G10 as a promising candidate for immunotherapy of drug-resistant PA lung infections and establish a druggable flagellin epitope for future vaccine and antibody designs.
Gut microbesJessica K Josephson, Jacqueline A Barnett, Emad Yuzbashian, Andrea Verdugo-Meza, Susan J Murch, Tyler J Wenzel, Andis Klegeris, Keith A Sharkey, Deanna L Gibson
Globally, the incidence of inflammatory bowel disease (IBD) is projected to reach 0.5% of the population by 2030, with increasing recognition of neurobehavioral comorbidities, including anxiety, depression, and cognitive dysfunction. The mechanisms underlying these comorbidities remain unclear but may involve interacting pathways, including microbial dysbiosis, inflammation and imbalanced neurometabolite production. Here, we investigated whether microbiome-associated alterations in neurometabolites are correlated with behavioral changes in a chronic colitis model. Specific pathogen-free (SPF) and germ-free (GF) mucin 2 knockout mice (Muc2-/-) alongside mucin 2 expressing mice (Muc2+/+) were evaluated for behavioral patterns of anxiety, depressive-like patterns and memory dysfunction. Tryptophan and metabolite concentrations were measured in the colon, serum and brain. Blood-brain barrier integrity and neuroimmune activation were assessed through tight-junction protein claudin-5 expression, glial fibrillary acid protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA-1) protein expression. Microbiome composition was characterized in relation to the tryptophan utilization pathways. To assess causality, early-life nutrient supplementation was used to address potential metabolite depletion. Female Muc2-/- displayed reduced anxiety-like behavior, while males displayed memory dysfunction. These changes coincided with decreased intestinal tryptophan, kynurenine, and serotonin within the gastrointestinal tract. GF Muc2-/- mice displayed normalized intestinal metabolite levels without concurrent brain metabolite changes. Notably, behavioral phenotypes were lost in GF Muc2-/- mice, revealing a key role for the microbiome played in these comorbidities. Muc2-/- exhibited reduced claudin-5, suggesting impaired blood‒brain barrier integrity. Microbiome analysis revealed a shift towards indole production and NAD+ salvage pathways with reduced abundance of Anaerotruncus, Enterocloster and Intestinimonas. Although early-life nutrient supplementation partially restored colonic tryptophan, it failed to fully rescue behavioral outcomes. Collectively, these findings demonstrate that chronic colitis is associated with microbiome-mediated disruption of host tryptophan metabolism, which correlates with neurobehavioral dysfunction. Targeting microbiome-driven metabolic alterations may represent a therapeutic strategy for both intestinal and neurobehavioral manifestations of IBD.
BACKGROUND: A subset of patients with chronic myelomonocytic leukemia (CMML) carries NRAS mutations, which are associated with shorter overall survival and an increased risk of transformation to acute myeloid leukemia. However, the effects of NRAS mutations on the bone marrow microenvironment (BME) remain unclear. METHODS: We used a CMML mouse model driven by a single Nras G12D allele mutation to investigate alterations in the BME and the potential role of CD69 in immune suppression. Nras G12D-mutated CMML mice were treated with an anti-CD69 monoclonal antibody. Flow cytometry, hematoxylin-eosin staining, and RNA sequencing were performed to evaluate treatment-related changes. RESULTS: Nras G12D-mutated CMML mice showed increased infiltration of regulatory T (Treg) cells and CD69+ T cells in the BME, whereas CD69 expression on peripheral blood T cells remained lower than that on bone marrow T cells. Anti-CD69 monoclonal antibody treatment was associated with reduced generation of granulocyte-macrophage progenitor cells, prolonged survival, and decreased Treg accumulation in the BME. CONCLUSION: Our findings suggest that CD69 may serve as a biomarker of BME immunological dysfunction in CMML.