Inflammation
Abstract
Metabolic dysfunction-associated steatotic liver disease (MASLD)—characterized by excess accumulation of fat in the liver—now affects one third of the world’s population. As MASLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply characterized cohort of patients spanning the full histopathologic spectrum of MASLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of MASLD-associated liver fibrosis.
Authors
Sebastian Steinhauser, David Estoppey, Dennis P. Buehler, Yanhua Xiong, Nicolas Pizzato, Amandine Rietsch, Fabian Wu, Nelly Leroy, Tiffany Wunderlin, Isabelle Claerr, Philipp Tropberger, Miriam Müller, Alexandra Vissieres, Lindsay M. Davison, Eric H. Farber-Eger, Quinn S. Wells, Quanhu Sheng, Sebastian Bergling, Sophia A Wild, Pierre Moulin, Jiancong Liang, Wayne J. English, Brandon Williams, Judith Knehr, Marc Altorfer, Alejandro Reyes, Johannes Voshol, Craig Mickanin, Dominic Hoepfner, Florian Nigsch, Mathias Frederiksen, Charles R. Flynn, Barna D. Fodor, Jonathan D. Brown, Christian Kolter
Abstract
Temporomandibular joint (TMJ) osteoarthritis with pain is a highly prevalent disorder affecting patients’ quality of life. A comprehensive understanding of cell type diversity and its dynamics in painful TMJ osteoarthritis (TMJOA) is lacking. Here, we utilized an inflammatory TMJOA mouse model via intra-articular injection of CFA. TMJOA mice exhibited cartilage remodeling, bone loss, synovitis, increased osteoarthritis (OA) score, and orofacial pain, recapitulating hallmark symptoms in patients. Single-cell transcriptomic profiling of the TMJ was performed in conjunction with mouse genetic labeling, tissue clearing, light sheet and confocal 3D imaging, multiplex RNAscope, and immunodetection. We visualized, reconstructed, and analyzed the distribution and density of nociceptive innervation of TMJ at single-axon levels. We systematically mapped the heterogeneity and anatomical position of blood endothelial cells, synovial fibroblasts, and immune cells, including Cx3cr1-positive barrier macrophages. Importantly, TMJOA mice exhibited enhanced neurovascular coupling, sublining fibroblast hyperplasia, inflammatory immune cell expansion, disrupted signaling-dependent cell-cell interaction, and a breakdown of the sandwich-like organization consisting of synovial barrier macrophages and fibroblasts. By utilizing a mouse model with combined TMJ pain history and OA, we reveal the cellular diversity, anatomical structure, and cell dynamics of the TMJ at single-cell resolution, which facilitate our understanding and potential targeting of TMJOA.
Authors
Supawadee Jariyasakulroj, Yang Shu, Ziying Lin, Jingyi Chen, Qing Chang, Pao-Fen Ko, Jian-Fu Chen
Abstract
Induction of podoplanin (PDPN) expression is a critical response of macrophages to LPS stimulation or bacterial infection in sepsis, but how this key process of TLR4-stimulated PDPN upregulation is regulated and the impact of PDPN expression on macrophage function remain elusive. Here, we determined how this process is regulated in vitro and in vivo. PDPN failed to be upregulated in TLR4 stimulated macrophages deficient in adhesion and degranulation-promoting adapter protein (ADAP), which could be rescued by the reconstitution of ADAP. A distinct PDPNhi peritoneal macrophage (PM) subset, which exhibited an M2-like phenotype and enhanced phagocytic activity, was generated in WT but not in ADAP-deficient septic mice. The blockade of PDPNhi PMs mimicked the effect of ADAP deficiency, which exacerbated sepsis. Mechanistically, BTK-mediated ADAP Y571 phosphorylation worked together with mTOR to converge on STAT3 activation for the transactivation of the PDPN promoter. Moreover, agonist activation of STAT3 profoundly potentiated the PDPNhi PM subset generation and alleviated sepsis severity in mice. Together, our findings reveal a mechanism whereby ADAP resets macrophage function by controlling the TLR4-induced upregulation of PDPN as a host innate immune defense during sepsis.
Authors
Pengchao Zhang, Xinning Wang, Xiaodong Yang, Hebin Liu
CD34hi subset of synovial fibroblasts contributes to fibrotic phenotype of human knee osteoarthritis
Abstract
Osteoarthritis (OA) shows various clinical manifestations depending on the status of its joint components. We aimed to identify the synovial cell subsets responsible for OA pathophysiology by comprehensive analyses of human synovium samples in single-cell resolution. Two distinct OA synovial tissue groups were classified by gene expression profiles in RNA-Seq: inflammatory and fibrotic. The inflammatory group exhibited high expression of inflammatory cytokines, histologically inflammatory infiltrate, and a more severe pain score. The fibrotic group showed higher expression of fibroblast growth factor (FGFs) and bone morphogenetic proteins (BMPs), showed histologically perivascular fibrosis, and showed a lower pain score. In single-cell RNA-Seq (scRNA-Seq) of synovial cells, MERTKloCD206lo macrophages and CD34hi fibroblasts were associated with the inflammatory and fibrotic groups, respectively. Among the 3 fibroblast subsets, CD34loTHY1lo and CD34loTHY1hi fibroblasts were influenced by synovial immune cells, whereas CD34hi fibroblasts were influenced by mural and endothelial cells. Particularly, in CD34hi fibroblast subsets, CD34hiCD70hi fibroblasts promoted proliferation of Tregs, potentially suppressing synovitis and protecting articular cartilage. Elucidation of the mechanisms underlying the regulation of these synovial cell subsets may lead to novel strategies for OA therapeutics.
Authors
Junya Miyahara, Yasunori Omata, Ryota Chijimatsu, Hiroyuki Okada, Hisatoshi Ishikura, Junya Higuchi, Naohiro Tachibana, Kosei Nagata, Shoichiro Tani, Kenichi Kono, Kohei Kawaguchi, Ryota Yamagami, Hiroshi Inui, Shuji Taketomi, Yasuhide Iwanaga, Asuka Terashima, Fumiko Yano, Masahide Seki, Yutaka Suzuki, Roland Baron, Sakae Tanaka, Taku Saito
Abstract
Endometriosis is a chronic gynecological disease that affects 1 in 10 reproductive-aged women. Most studies investigate established disease; however, the initiation and early events in endometriotic lesion development remain poorly understood. Our study used neutrophils from human menstrual effluent from subjects with and without endometriosis for immunophenotyping, and a mouse model of endometriosis and a mouse endometriosis cell line to determine the role of neutrophils in the initiating events of endometriosis, including attachment and survival of minced endometrial pieces. In menstrual effluent from women with endometriosis, the ratio of aged and pro-angiogenic neutrophils increased compared to controls, indicating a potentially permissive pro-inflammatory microenvironment. In our endometriosis mouse model, knocking-down neutrophil recruitment with α-CXCR2 into the peritoneum decreased endometrial tissue adhesion—supported by decreased levels of myeloperoxidase and neutrophil elastase in both developing lesions and peritoneal fluid. Fibrinogen was identified as the preferred substrate for endometrial cell adhesion in an in vitro adhesion assay and in developing lesions in vivo. Together, aged and pro-angiogenic neutrophils and their secretions likely promote attachment and formation of endometriotic lesions by releasing neutrophil extracellular traps and upregulating fibrinogen expression as a provisional matrix to establish attachment and survival in the development of endometriosis lesions.
Authors
Taylor R. Wilson, Kurt R. Peterson, Stephanie A. Morris, Damaris Kuhnell, Susan Kasper, Katherine A. Burns
Abstract
Fibrosis results from excessive extracellular matrix (ECM) deposition, causing tissue stiffening and organ dysfunction. Activated fibroblasts, central to fibrosis, exhibit increased migration, proliferation, contraction, and ECM production. However, it remains unclear if the same fibroblast performs all of the processes that fall under the umbrella term of "activation". Due to fibroblast heterogeneity in connective tissues, subpopulations with specific functions may operate under distinct regulatory controls. Using a transgenic mouse model of skin fibrosis, we found that Mindin (spondin-2), secreted by Snail transgenic keratinocytes, differentially regulates fibroblast subpopulations. Mindin promotes migration and inflammatory gene expression in SCA1+ dermal fibroblasts via Fyn kinase. In contrast, it enhances contractility and collagen production in papillary CD26+ fibroblasts through c-Src signalling. Moreover, in the context of the fibrotic microenvironment of the tumour stroma, we found that differential responses of resident fibroblasts subpopulations to Mindin extend to the generation of functionally heterogeneous cancer-associated fibroblasts (CAFs). This study unveils Mindin as a key orchestrator of dermal fibroblast heterogeneity, reshaping cellular dynamics and signalling diversity in the complex landscapes of skin fibrosis and cancer.
Authors
Sunny Kataria, Isha Rana, Krithika Badarinath, Rania F. Zaarour, Gaurav Kansagara, Sultan Ahmed, Abrar Rizvi, Dyuti Saha, Binita Dam, Abhik Dutta, Ravindra K. Zirmire, Edries Yousaf Hajam, Pankaj Kumar, Akash Gulyani, Colin Jamora
Abstract
Patients with immune-mediated inflammatory diseases (IMIDs) like rheumatoid arthritis (RA) are at higher risk for severe COVID-19 and long-term complications in bone health. Emerging clinical evidence demonstrated that SARS-CoV-2 infection reduces bone turnover and promotes bone loss, but the mechanism underlying worsened bone health remains elusive. This study sought to identify specific immune mediators that exacerbated preexisting IMIDs after SARS-CoV-2 exposure. Plasma samples from 4 groups were analyzed: healthy, IMID only, COVID-19 only, and COVID-19 + IMID. Using high-throughput multiplexed proteomics, we profiled 1,500 protein biomarkers and identified 148 unique biomarkers in COVID-19 patients with IMIDs, including elevated inflammatory cytokines (e.g., IL-17F) and bone resorption markers. Long-term circulating SARS-CoV-2 ORF8, a virulence factor for COVID-19, was detected in the COVID + IMID group. RA was one of the most common IMIDs in our study. ORF8 treatment of RA-derived human osteoblasts (RA-hOBs) increased levels of inflammatory (TNF, IL6, CCL2) and bone resorption (RANKL/osteoprotegerin ratio) markers compared with healthy controls. Supernatants from ORF8-treated RA-hOBs drove the differentiation of macrophages into osteoclast-like cells. These findings suggest that SARS-CoV-2 exposure can exacerbate IMIDs through ORF8-driven inflammation and osteoclastogenesis, highlighting potential therapeutic targets for managing COVID-19–induced bone pathologies.
Authors
Ivonne Melano, Tamiris Azamor, Camila C.S. Caetano, Nikki M. Meyer, Chineme Onwubueke, Anabelle Visperas, Débora Familiar-Macedo, Gielenny M. Salem, Brandy-Lee Soos, Cassandra M. Calabrese, Youn Jung Choi, Shuyang Chen, Younho Choi, Xianfang Wu, Zilton Vasconcelos, Suzy A.A. Comhair, Karin Nielsen-Saines, Leonard H. Calabrese, M. Elaine Husni, Jae U. Jung, Nicolas S. Piuzzi, Suan-Sin Foo, Weiqiang Chen
Abstract
Despite recent advances in the treatment of thyroid eye disease thyroid-related eye disease (TED), marked gaps remain in our understanding of the underlying molecular mechanisms, particularly concerning the insulin-like growth factor-1 receptor (IGF-1R) pathway. To dissect the pathophysiology of TED, we used single-nucleus RNA-Seq to analyze orbital fat specimens from both patients with TED and matched individuals acting as controls. The analysis demonstrated a marked increase in the proportion of fibroblasts transitioning to adipogenesis in the orbital fat of patients with TED compared with that in control patients. This was associated with diverse alterations in immune cell composition. Significant alterations in the IGF-1R signaling pathway were noted between TED specimens and those from control patients, indicating a potential pathological mechanism driven by IGF-1R signaling abnormalities. Additionally, our data showed that linsitinib, a small-molecule inhibitor of IGF-1R, effectively reduced adipogenesis in TED orbital fibroblasts in vitro, suggesting its potential utility as a therapeutic agent. Our findings reveal that, beyond immune dysfunction, abnormal IGF-1R signaling leading to enhanced adipogenesis is a crucial pathogenic mechanism in TED.
Authors
Dong Won Kim, Soohyun Kim, Jeong Han, Karan Belday, Emily Li, Nicholas Mahoney, Seth Blackshaw, Fatemeh Rajaii
Abstract
Specialized memory CD4 T cells that reside long-term within tissues are critical components of immunity at portals of pathogen entry. In the lung, such tissue-resident memory (TRM) cells are activated rapidly after infection and promote local inflammation to control pathogen levels before circulating T cells can respond. However, optimal clearance of Influenza A virus can require TRM and responses by other virus-specific T cells that reach the lung only several days after their activation in secondary lymphoid organs. Whether local CD4 TRM sentinel activity can impact the efficiency of T cell activation in secondary lymphoid organs is not clear. Here, we found that recognition of antigen by influenza -primed TRM in the airways promotes more rapid migration of highly activated antigen-bearing dendritic cells to the draining lymph nodes. This in turn accelerated the priming of naive T cells recognizing the same antigen, resulting in newly activated effector T cells reaching the lungs earlier than in mice not harboring TRM. Our findings thus reveal a circuit linking local and regional immunity whereby antigen recognition by TRM improves effector T cell recruitment to the site of infection though enhancing the efficiency of antigen presentation in the draining lymph node.
Authors
Caroline M. Finn, Kunal Dhume, Eugene Baffoe, Lauren A. Kimball, Tara M. Strutt, K. Kai McKinstry
Abstract
Macrophage plays a crucial role in promoting perfusion recovery and revascularization after ischemia through anti-inflammatory polarization, a process essential for the treatment of peripheral arterial disease (PAD). Mitochondrial dynamics, particularly regulated by the fission protein DRP1, are closely linked to macrophage metabolism and inflammation. However, the role of DRP1 in reparative neovascularization remains unexplored. Here we show that DRP1 expression was increased in F4/80+ macrophages within ischemic muscle at day 3 after hindlimb ischemia (HLI), an animal model of PAD. Mice lacking Drp1 in myeloid cells exhibited impaired limb perfusion recovery, angiogenesis and muscle regeneration post-HLI. These effects were associated with increased pro-inflammatory M1-like macrophages, p-NFkB and TNFα, and reduced anti-inflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1–/– mice. In vitro, Drp1-deficient macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction, and excessive mitochondrial ROS production, resulting in increased pro-inflammatory M1-gene and reduced anti-inflammatory M2-gene expression. Conditioned media from HSS-treated Drp1–/– macrophages exhibited increased pro-inflammatory cytokine secretion, leading to suppressed angiogenesis in endothelial cells. Thus, macrophage DRP1 deficiency under ischemia drives pro-inflammatory metabolic reprogramming and macrophage polarization, limiting revascularization in experimental PAD.
Authors
Shikha Yadav, Vijay C. Ganta, Sudhahar Varadarajan, Vy Ong, Yang Shi, Archita Das, Dipankar Ash, Sheela Nagarkoti, Malgorzata McMenamin, Stephanie Kelley, Tohru Fukai, Masuko Ushio-Fukai
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