CD8+ T cell dysfunction impedes anti-tumor immunity in solid cancers but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional co-activator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend upon oncogene-mediated ECM composition and remodeling.
Ashley M. Fuller, Hawley C. Pruitt, Ying Liu, Valerie M. Irizarry-Negron, Hehai Pan, Hoogeun Song, Ann DeVine, Rohan S. Katti, Samir Devalaraja, Gabrielle E. Ciotti, Michael V. Gonzalez, Erik F. Williams, Ileana Murazzi, Dimitris Ntekoumes, Nicolas Skuli, Hakon Hakonarson, Daniel J. Zabransky, Jose G. Trevino, Ashani Weeraratna, Kristy Weber, Malay Haldar, Joseph A. Fraietta, Sharon Gerecht, T.S. Karin Eisinger-Mathason
While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNAseq data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different than those observed in Nr2e3 rodent models. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.
Nathaniel K. Mullin, Laura R. Bohrer, Andrew P. Voigt, Lola P. Lozano, Allison T. Wright, Vera L. Bonilha, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker
The immune system can control cancer progression. However, even though some innate immune sensors of cellular stress are expressed intrinsically in epithelial cells, their potential role in cancer aggressiveness and subsequent overall survival in humans is mainly unknown. Here, we show that NLR family CARD Domain Containing 4 (NLRC4) is downregulated in epithelial tumor cells of colorectal cancer (CRC) patients by using spatial tissue imaging. Strikingly, only the loss of tumor NLRC4 but not stromal is associated with poor immune infiltration (mainly dendritic and CD4+/CD8+ T cells) and accurately predicts progression to metastatic Stage IV and decrease of overall survival. By combining multi-omics approaches, we show that restoring NLRC4 expression in human colorectal cancer cells triggers a broad inflammasome-independent immune reprogramming consisting of Type-I IFN signaling genes and the release of chemokines and myeloid growth factors involved in the tumor infiltration and activation of dendritic cells (DCs) and T cells. Consistently, such reprogramming in cancer cells is sufficient to directly mature human DCs towards a Th1 antitumor immune response through IL-12 production in vitro. In multiple human carcinomas (colorectal, lung, and skin), we confirmed that NLRC4 expression in patient tumors is strongly associated with Type-I IFN genes, immune infiltrates and high microsatellite instability. Thus, we shed light on the epithelial innate immune sensor NLRC4 as a novel therapeutic target to promote an efficient antitumor immune response against the aggressiveness of various carcinomas.
Charlotte Domblides, Steven Crampton, Hong Liu, Juliet M. Bartleson, Annie Nguyen, Claudia Champagne, Emily E. Landy, Lindsey Spiker, Christopher Proffitt, Sunil Bhattarai, Anissa P. Grawe, Matias Fuentealba Valenzuela, Lydia Lartigue, Isabelle Mahouche, Jeremy Dupaul-Chicoine, Kazuho Nishimura, Félix Lefort, Marie Decraecker, Valérie Velasco, Sonia Netzer, Vincent Pitard, Christian Roy, Isabelle Soubeyran, Victor Racine, Patrick Blanco, Julie Déchanet-Merville, Maya Saleh, Scott W. Canna, David Furman, Benjamin Faustin
Molecular profiling of clear cell RCC (ccRCC) tumors of clinical trial patients has identified distinct transcriptomic signatures with predictive value, yet data in non-clear cell variants (nccRCC) are lacking. We examined the transcriptional profiles of RCC tumors representing key molecular pathways, from a multi-institutional, real-world patient cohort, including ccRCC (n = 508) and centrally-reviewed nccRCC (n = 149) samples. ccRCC had increased angiogenesis signature scores compared to the heterogeneous group of nccRCC tumors (mean z-score 0.37 vs –0.99, P < 0.001), while cell cycle, fatty acid oxidation (FAO)/AMPK signaling, fatty acid synthesis (FAS)/pentose phosphate signature scores were increased in one or more nccRCC subtypes. Among both ccRCC and nccRCC tumors, T-effector scores statistically correlated with increased immune cell infiltration and were more commonly associated with immunotherapy-related markers (PD-L1+/TMB-High/MSI-High). In conclusion, this study provides evidence of differential gene transcriptional profiles among ccRCC vs nccRCC tumors, providing new insights for optimizing personalized and histology-specific therapeutic strategies for patients with advanced RCC.
Pedro Barata, Shuchi Gulati, Andrew Elliott, Hans J. Hammers, Earle F. Burgess, Benjamin A. Gartrell, Sourat Darabi, Mehmet A. Bilen, Arnab Basu, Daniel M. Geynisman, Nancy A. Dawson, Matthew R. Zibelman, Tian Zhang, Shuanzeng Wei, Charles J. Ryan, Elisabeth I. Heath, Kelsey A. Poorman, Chadi Nabhan, Rana R. McKay
BACKGROUND. Early antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAb) after acute/early ARTi is relevant, but poorly understood. METHODS. We characterize antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after infection) and early HIV (60-128 days after infection). RESULTS. Plasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 Envs representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In two of the three acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter. CONCLUSIONS. Results indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants. TRIAL REGISTRATION. NCT02656511 FUNDING. National Institutes of Health grants U01AI169767; R01AI162646; UM1AI164570; UM1AI164560; U19AI096109; K23GM112526; T32AI118684, P30-AI-045008, P30 AI027763, R24 AI067039. Gilead Sciences grant INUS2361354; Viiv healthcare grant A126326.
Gregory D. Whitehill, Jaimy Joy, Francesco E. Marino, Ryan J. Krause, Suvadip Mallick, Hunter M. Courtney, Kyewon Park, John W. Carey, Rebecca Hoh, Heather Hartig, Vivian Pae, Sannidhi Sarvadhavabhatla, Maria Sophia B. Donaire, Steven G. Deeks, Rebecca M. Lynch, Sulggi A. Lee, Katharine J. Bar
BACKGROUND. Features of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM. METHODS. Plasma from children with uncomplicated malaria (UM, n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-Platelet Factor 4/polyanion (anti-PF4/P), anti-Phospholipid, anti-Phosphatidylserine, anti-Myeloperoxidase, anti-Proteinase 3, anti-dsDNA, anti-Beta-2-Glycoprotein I (β2GPI), and anti-Cardiolipin. Non-malaria coma (NMC, n = 49) and healthy controls (HC, n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses. RESULTS. Median anti-PF4/P and anti-PS IgG levels were elevated with malaria infection relative to HC (P < 0.001) and NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in CM (median = 0.27, IQR: 0.19–0.41) compared to UM (median = 0.19, IQR: 0.14–0.22, P ≤ 0.0001). Anti-PS IgG levels did not differ between UM and CM (P = 0.39). When CM cases were stratified by malaria retinopathy (Ret) status, levels of anti-PF4/P IgG correlated negatively with peripheral platelet count in Ret+ CM cases (Rs = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02–275, P = 0.048). Plasma from CM patients induced a greater platelet activation capacity in an ex-vivo assay relative to plasma from UM patients (P = 0.02). Platelet activation was associated with anti-PF4/P IgG levels (Rs = 0.293, P = 0.035). CONCLUSIONS. Thrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.
Iset M. Vera, Anne Kessler, Visopo Harawa, Ajisa Ahmadu, Thomas E. Keller, Stephen T.J. Ray, Terrie E. Taylor, Stephen J. Rogerson, Wilson L. Mandala, Morayma Reyes Gil, Karl B. Seydel, Kami Kim
Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation (OXPHOS). Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC) that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux towards lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice.
Piyarat Siripoksup, Guoshen Cao, Ahmad A. Cluntun, J. Alan Maschek, Quentinn Pearce, Marisa J. Lang, Mi-Young Jeong, Hiroaki Eshima, Patrick J. Ferrara, Precious C. Opurum, Ziad S. Mahmassani, Alek D. Peterlin, Shinya Watanabe, Maureen A. Walsh, Eric B. Taylor, James E. Cox, Micah J. Drummond, Jared Rutter, Katsuhiko Funai
Renal interstitial fibrosis is an important mechanism in the progression of chronic kidney disease (CKD) to end-stage kidney disease. However, we lack specific treatments to slow or halt renal fibrosis. Ribosome profiling identified upregulation of a secreted micropeptide, C4orf48 (Cf48), in mouse diabetic nephropathy. Cf48 RNA and protein levels were upregulated in tubular epithelial cells in human and experimental CKD. Serum Cf48 levels were increased in human CKD and correlated with loss of kidney function, increasing CKD stage, and the degree of active interstitial fibrosis. Cf48 overexpression in mice accelerated renal fibrosis, while Cf48 gene deletion or knockdown by antisense oligonucleotides significantly reduced renal fibrosis in CKD models. In vitro, recombinant Cf48 (rCf48) enhanced TGF-β1-induced fibrotic responses in renal fibroblasts and epithelial cells independent of Smad3 phosphorylation. Cellular uptake of Cf48 and its pro-fibrotic response in fibroblasts operated via the transferrin receptor. RNA immunoprecipitation-sequencing identified Cf48 binding to mRNA of genes involved in the fibrotic response, including Serpine1, Acta2, Ccn2, and Col4a1. rCf48 binds to the 3′-untranslated region of Serpine1 and increases mRNA half-life. We identify the secreted Cf48 micropeptide as a potential enhancer of renal fibrosis which operates as an RNA-binding peptide to promote the production of extracellular matrix.
Jiayi Yang, Hongjie Zhuang, Jinhua Li, Ana B. Nunez-Nescolarde, Ning Luo, Huiting Chen, Andy Li, Xinli Qu, Qing Wang, Jinjin Fan, Xiaoyan Bai, Zhiming Ye, Bing Gu, Yue Meng, Xingyuan Zhang, Di Wu, Youyang Sia, Xiaoyun Jiang, Wei Chen, Alexander N. Combes, David J. Nikolic-Paterson, Xueqing Yu
Just as the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet, the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using two human PCa tissue microarray cohorts, we first demonstrated that nuclear ERα expression was heterogeneous among patients, being only detected in half of tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimic the androgen transcriptional response and induce specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprograms PCa metabolism, is associated with disease progression, and could be targeted for therapeutic purposes.
Camille Lafront, Lucas Germain, Gabriel H. Campolina-Silva, Cindy Weidmann, Line Berthiaume, Hélène Hovington, Hervé Brisson, Cynthia Jobin, Lilianne Frégeau-Proulx, Raul Cotau, Kevin Gonthier, Aurélie Lacouture, Patrick Caron, Claire Ménard, Chantal Atallah, Julie Riopel, Éva Latulippe, Alain Bergeron, Paul Toren, Chantal Guillemette, Martin Pelletier, Yves Fradet, Clémence Belleannée, Frédéric Pouliot, Louis Lacombe, Éric Lévesque, Étienne Audet-Walsh
Tissue regeneration is limited in several organs including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here we identified neuronal differentiation features of MD cells that sense the local and systemic environment, secrete angiogenic, growth and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors including CCN1 as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ-specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue regenerative therapeutic strategies.
Georgina Gyarmati, Urvi Nikhil Shroff, Anne Riquier-Brison, Dorinne Desposito, Wenjun Ju, Sean D. Stocker, Audrey Izuhara, Sachin Deepak, Alejandra Becerra Calderon, James L. Burford, Hiroyuki Kadoya, Ju-Young Moon, Yibu Chen, Markus M. Rinschen, Nariman Ahmadi, Lester Lau, Daniel Biemesderfer, Aaron W. James, Liliana Minichiello, Berislav Zlokovic, Inderbir S. Gill, Matthias Kretzler, János Peti-Peterdi
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