The molecular mechanisms of sodium-glucose cotransporter-2 (SGLT2) inhibitors (SGLT2i) remain incompletely understood. Single-cell RNA sequencing and morphometric data were collected from research kidney biopsies donated by young persons with type 2 diabetes (T2D), aged 12-21 years, and healthy controls (HC). Participants with T2D were obese, had higher estimated glomerular filtration rates, mesangial and glomerular volumes than HC. Ten T2D participants had been prescribed SGLT2i (T2Di(+)) and 6 not (T2Di(-)). Transcriptional profiles showed SGLT2 expression exclusively in the proximal tubular (PT) cluster with highest expression in T2Di(-). However, transcriptional alterations with SGLT2i treatment were seen across nephron segments, particularly in the distal nephron. SGLT2i treatment was associated with suppression of transcripts in the glycolysis, gluconeogenesis, tricarboxylic acid cycle pathways in PT, but enhanced in thick ascending limb. Transcripts in the energy sensitive mammalian target of rapamycin complex1 (mTORC1) signaling pathway returned towards HC levels in all tubular segments in T2Di(+), consistent with a diabetes mouse model treated with SGLT2i. Decreased levels of phosphorylated S6 protein in proximal and distal tubules in T2Di(+) confirmed changes in mTORC1 pathway activity. We propose that SGLT2i treatment benefits the kidneys by mitigating diabetes-induced metabolic perturbations via suppression of mTORC1 signaling in kidney tubules.
Jennifer A. Schaub, Fadhl M. AlAkwaa, Phillip J. McCown, Abhijit S. Naik, Viji Nair, Sean Eddy, Rajasree Menon, Edgar A. Otto, Dawit Demeke, John Hartman, Damian Fermin, Christopher O'Connor, Lalita Subramanian, Markus Bitzer, Roger Harned, Patricia Ladd, Laura Pyle, Subramaniam Pennathur, Ken Inoki, Jeffrey B. Hodgin, Frank C. Brosius, Robert G. Nelson, Matthias Kretzler, Petter Bjornstad
Diabetic nephropathy (DN) is a polygenic disorder with few risk variants showing robust replication in large-scale genome-wide association studies. To understand the role of DNA methylation, it is important to have the prevailing genomic view to distinguish key sequence elements that influence gene expression. This is particularly challenging for DN because genome wide methylation patterns are poorly defined. While methylation is known to alter gene expression the importance of this causal relationship is obscured by array-based technologies since coverage outside promoter regions is low. To overcome these challenges, we performed methylation sequencing using leukocytes derived from participants of the Finnish Diabetic Nephropathy (FinnDiane) type 1 diabetes (T1D) study (n=39) that was subsequently replicated in a larger validation cohort (n=296). Gene body related regions made up >60% of the methylation differences and emphasised the importance of methylation sequencing. We observe differentially methylated genes associated with DN (DDN) in three independent T1D registries originating from Denmark (n=445), Hong Kong (n=107) and Thailand (n=130). Reduced DNA methylation at CTCF and Pol2B sites were tightly connected with DN pathways that include insulin signalling, lipid metabolism and fibrosis. To define the pathophysiological significance of these population findings, methylation indices were assessed in human renal cells such as podocytes and proximal convoluted tubules. The expression of core genes was associated with reduced methylation, elevated CTCF and Pol2B binding and the activation of insulin signalling phosphoproteins in hyperglycaemic cells. These experimental observations also closely parallel methylation-mediated regulation in human macrophage and vascular endothelial cells.
Ishant Khurana, Harikrishnan Kaipananickal, Scott Maxwell, Sørine Birkelund, Anna Syreeni, Carol Forsblom, Jun Okabe, Mark Ziemann, Antony Kaspi, Haloom Rafehi, Anne Jørgensen, Keith Al-Hasani, Merlin C. Thomas, Guozhi Jiang, Andrea O.Y. Luk, Heung Man Lee, Yu Huang, Yotsapon Thewjitcharoen, Soontaree Nakasatien, Thep Himathongkam, Christopher Fogarty, Rachel Njeim, Assaad Eid, Tine Willum Hansen, Nete Tofte, Evy Connie Ottesen, Ronald C.W. Ma, Juliana C.N. Chan, Mark Emmanuel Cooper, Peter Rossing, Per-Henrik Groop, Assam El-Osta
SIPRα on macrophages binds with CD47 to resist pro-engulfment signals, but how the downstream signal of SIPRα controls tumor-infiltrating macrophages (TIMs) is still poorly clarified. Here we reported that the CD47/SIRPα axis requires the deneddylation of tyrosine phosphatase SHP2. Mechanistically, SHP2 is constitutively neddylated on K358 and K364 sites, thus its auto-inhibited conformation is maintained. In response to CD47-liganded SIRPα, SHP2 is deneddylated by SENP8, which leads to the dephosphorylation of relevant substrates at the phagocytic cup and subsequent inhibition of macrophage phagocytosis. Furthermore, neddylation inactivated myeloid-SHP2 and greatly boosted the efficacy of colorectal cancer (CRC) immunotherapy. Importantly, we observed that the supplementation with SHP2 allosteric inhibitors sensitized the immune treatment-resistant CRC to immunotherapy. Our results emphasized that the CRC subtype which is unresponsive to immunotherapy relies on SIRPαhiSHP2hiNEDD8lo TIMs, and highlighted the need to further combine the strategy of SHP2 targeting in colorectal cancer therapy.
Yiqing Li, Hui Zhou, Pan Liu, Dandan Lv, Yichun Shi, Bufu Tang, Jiaqi Xu, Tingting Zhong, Wangting Xu, Jie Zhang, Jianying Zhou, Kejing Ying, Yongchao Zhao, YI Sun, Zhinong Jiang, Hongqiang Cheng, Xue Zhang, Yue-Hai Ke
The role of tumor-associated macrophages (TAMs) along with the regulatory mechanisms underlying distinct macrophage activation states remain poorly understood in prostate cancer (PCa). Herein, we reported that PCa growth in macrophage-specific Ubc9 deficient mice is substantially suppressed compared to their wild-type littermates, an effect partially ascribed to the augmented CD8+ T cell response. Biochemical and molecular analyses revealed that the signal transducer and activator of transcription 4 (STAT4) is a crucial UBC9-mediated SUMOylation target, with lysine residue 350 (K350) as the major modification site. Site-directed mutation of STAT4 (K350R) enhanced its nuclear translocation and stability, thereby facilitating the proinflammatory activation of macrophages. Importantly, administration of UBC9 inhibitor, 2-D08, promoted the antitumor effect of TAMs and increased the expression of PD-1 on CD8+ T cells, supporting a synergistic antitumor efficacy once it combined with the immune checkpoint blockade (ICB) therapy. Together, our results demonstrated that ablation of UBC9 could reverse the immunosuppressive phenotype of TAMs via promoting STAT4 mediated macrophage activation and macrophage-CD8+ T cell crosstalk, which provides valuable insights to halt the pathogenic process of tumorigenesis.
Jun Xiao, Fei Sun, Ya-Nan Wang, Bo Liu, Peng Zhou, Fa-Xi Wang, Hai-Feng Zhou, Yue Ge, Tian-Tian Yue, Jia-Hui Luo, Chun-Liang Yang, Shan-Jie Rong, Ze-Zhong Xiong, Sheng Ma, Qi Zhang, Yang Xun, Chun-Guang Yang, Yang Luan, Shao-Gang Wang, Cong-Yi Wang, Zhi-Hua Wang
Aberrant immune responses to resident microbes promote inflammatory bowel disease and other chronic inflammatory conditions. However, how microbiota-specific immunity is controlled in mucosal tissues remains poorly understood. Here, we find that mice lacking epithelial expression of microbiota-sensitive histone deacetylase 3 (HDAC3) exhibit increased accumulation of commensal-specific CD4+ T cells in the intestine, provoking the hypothesis that epithelial HDAC3 may instruct local microbiota-specific immunity. Consistent with this, microbiota-specific CD4+ T cells and epithelial HDAC3 expression were concurrently induced following early-life microbiota colonization. Further, epithelial-intrinsic ablation of HDAC3 decreased commensal-specific Tregs, increased commensal-specific Th17 cells, and promoted T cell-driven colitis. Mechanistically, HDAC3 was essential for NFκB-dependent regulation of epithelial MHC class II (MHCII). Epithelial-intrinsic MHCII dampened local accumulation of commensal-specific Th17 cells in adult mice, and protected against microbiota-triggered inflammation. Remarkably, HDAC3 enabled the microbiota to induce MHCII on epithelial cells and limit the number of commensal-specific T cells in the intestine. Collectively, these data reveal a central role for an epithelial histone deacetylase in directing the dynamic balance of tissue-intrinsic CD4+ T cell subsets that recognize commensal microbes and control inflammation.
Emily M. Eshleman, Tzu-Yu Shao, Vivienne Woo, Taylor Rice, Laura Engleman, Bailey J. Didriksen, Jordan Whitt, David B. Haslam, Sing Sing Way, Theresa Alenghat
Genetic variants in the third intron of the PRDM6 gene have been associated with blood pressure traits in multiple genome-wide association studies (GWAS). By combining fine mapping, massive-ly parallel reporter assays, and gene editing we identified the causal variants for hypertension as super-enhancers that drive the expression of PRDM6 and are partly regulated by STAT1. The het-erozygous SMC-specific Prdm6 knockout mice (Prdm6fl/+ Sm22Cre) exhibited a markedly high-er number of renin-producing cells in the kidneys at embryonic day 18.5 compared to wild-type littermates and developed salt-induced systemic hypertension that was completely responsive to the renin inhibitor aliskiren. Strikingly, RNA-seq analysis of the mice aorta identified a network of PRDM6-regulated genes that are located in GWAS-associated loci for blood pressure, most nota-bly Sox6, which modulates renin-expression in the kidney. Accordingly, the smooth muscle cell-specific disruption of Sox6 in Prdm6fl/+ Sm22Cre mice resulted in a dramatic reduction of renin. Fate mapping and histological studies also showed increased numbers of neural crest-derived cells accompanied by increased collagen deposition in the kidneys of Prdm6fl/+ Wnt1Cre-ZsGreen1Cre compared to wild-type mice. These findings establish the role of PRDM6 as a regulator of renin-producing cells and an attractive target for the development of antihypertensive drugs.
Kushan L. Gunawardhana, Lingjuan Hong, Trojan Rugira, Severin Uebbing, Joanna Kucharczak, Sameet Mehta, Dineth R. Karunamuni, Brenda Cabrera-Mendoza, Renato Polimanti, James P. Noonan, Arya Mani
Mutations in the human Progranulin (GRN) gene are a leading cause of frontotemporal lobar degeneration (FTLD). While previous studies implicate aberrant microglial activation as a disease-driving factor in neurodegeneration in the thalamocortical circuit in Grn-/- mice, the exact mechanism for neurodegeneration in FTLD-GRN remains unclear. By performing comparative single-cell transcriptomics in the thalamus and frontal cortex of Grn-/- mice and patients with FTLD-GRN, we have uncovered a highly conserved astroglial pathology characterized by upregulation of gap junction protein GJA1, water channel AQP4, and lipid-binding protein APOE, and downregulation of glutamate transporter SLC1A2 that promoted profound synaptic degeneration across the two species. This astroglial toxicity could be recapitulated in mouse astrocyte-neuron cocultures and by transplanting induced pluripotent stem cell-derived astrocytes to cortical organoids, where Progranulin-deficient astrocytes promoted synaptic degeneration, neuronal stress, and TDP-43 proteinopathy. Together, these results reveal previously unappreciated astroglial pathology as a key mechanism in neurodegeneration in FTLD-GRN.
Elise Marsan, Dmitry Velmeshev, Arren Ramsey, Ravi K. Patel, Jiasheng Zhang, Mark Koontz, Madeline G. Andrews, Martina de Majo, Cristina Mora, Jessica Blumenfeld, Alissa N. Li, Salvatore Spina, Lea T. Grinberg, William Seeley, Bruce L. Miller, Erik M. Ullian, Matthew F. Krummel, Arnold Kriegstein, Eric J. Huang
Cortical neural dynamics mediate information processing for the cerebral cortex, implicated in fundamental biological processes, such as vision and olfaction, in addition to neurological and psychiatric diseases. Spontaneous pain is a key feature of human neuropathic pain. Whether spontaneous pain pushes cortical network into an aberrant state, and if so, whether it can be brought back to a ‘normal’ operating range to ameliorate pain are unknown. Using a clinically relevant mouse model of neuropathic pain with spontaneous pain-like behavior, we report that orofacial spontaneous pain activated a specific area within the primary somatosensory cortex (S1), displaying synchronized neural dynamics revealed by intravital two-photon calcium imaging. This synchronization was underpinned by local GABAergic interneuron hypoactivity. Pain-induced cortical synchronization could be attenuated by manipulating local S1 networks or clinically effective pain therapies. Specifically, both chemogenetic inhibition of pain-related c-Fos-expressing neurons, and selective activation of GABAergic interneurons, significantly attenuated S1 synchronization. Clinically effective pain therapies including carbamazepine and nerve root decompression could also dampen S1 synchronization. More importantly, restoring a ‘normal’ range of neural dynamics, through attenuating pain-induced S1 synchronization, alleviated pain-like behavior. These results suggest spontaneous pain pushes S1 regional network into a synchronized state, whereas reversal of this synchronization alleviates pain.
Weihua Ding, Lukas Fischer, Qian Chen, Ziyi Li, Liuyue Yang, Zerong You, Kun Hu, Xinbo Wu, Xue Zhou, Wei Chao, Peter Hu, Tewodros Mulugeta Dagnew, Daniel M. DuBreuil, Shiyu Wang, Suyun Xia, Caroline Bao, Shengmei Zhu, Lucy Chen, Changning Wang, Brian Wainger, Peng Jin, Jianren Mao, Guoping Feng, Mark T. Harnett, Shiqian Shen
BACKGROUND. Antiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown. METHODS. We carried an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5’-Leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets. RESULTS. Clones carrying proviruses with 5’-Leader defects can cause persistent NSV up to ~103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor site (MSD) and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced non-infectious virions containing viral RNA but lacking Envelope. CONCLUSION. These findings show that proviruses with 5’-Leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5’-Leader can help understanding failure to completely suppress viremia. FUNDING. Office of the NIH Director and National Institute of Dental & Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases, NIH, to the PAVE, BEAT-HIV and DARE Martin Delaney collaboratories.
Jennifer A. White, Fengting Wu, Saif Yasin, Milica Moskovljevic, Joseph Varriale, Filippo Dragoni, Angelica Camilo Contreras, Jiayi Duan, Mei Y. Zheng, Ndeh F. Tadzong, Heer B. Patel, Jeanelle Mae C. Quiambao, Kyle Rhodehouse, Hao Zhang, Jun Lai, Subul A. Beg, Michael Delannoy, Christin Kilcrease, Christopher J. Hoffmann, Sébastien Poulin, Frédéric Chano, Cecile Tremblay, Jerald Cherian, Patricia Barditch-Crovo, Natasha Chida, Richard D. Moore, Michael F. Summers, Robert F. Siliciano, Janet D. Siliciano, Francesco R. Simonetti
Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we reveal that intestinal GUCY2C is selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2CHigh neuropod cells associate with co-cultured dorsal root ganglia neurons and induce hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient, but not GUCY2C-deficient, neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling and VP that was comparable to chemically-induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.
Joshua R. Barton, Annie K. Londregan, Tyler D. Alexander, Ariana A. Entezari, Shely Bar-Ad, Lan Cheng, Angelo C. Lepore, Adam E. Snook, Manuel Covarrubias, Scott A. Waldman
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