X-linked hypophosphatemia (XLH) is characterized by high serum fibroblast growth factor 23 (FGF23) levels, resulting in impaired 1,25 dihydroxyvitamin D (1,25D) production. Adults with XLH develop a painful mineralization of the tendon-bone attachment site (enthesis), called enthesopathy. Treatment of mice with XLH (Hyp) with 1,25D or an anti-FGF23 antibody, both of which increase 1,25D signaling, prevents enthesopathy. Therefore, we undertook studies to determine a role for impaired 1,25D action in enthesopathy development. Entheses from mice lacking vitamin D 1-alpha-hydroxylase (Cyp27b1) (C-/-) had a similar enthesopathy to Hyp mice, while deletion of Fgf23 in Hyp mice prevented enthesopathy and deletion of both Cyp27b1 and Fgf23 in mice resulted in enthesopathy, demonstrating that the impaired 1,25D action due to high FGF23 levels underlied XLH enthesopathy development. Like Hyp mice, enthesopathy in C-/- mice was observed by P14 and was prevented, but not reversed, with 1,25D therapy. Deletion of the vitamin D receptor in scleraxis-expressing cells resulted in enthesopathy, indicating 1,25D acted directly on enthesis cells to regulate enthesopathy development. These results showed that 1,25D signaling was necessary for normal post-natal enthesis maturation and played a role in XLH enthesopathy development. Optimizing 1,25D replacement in pediatric XLH patients is necessary to prevent enthesopathy.
Rakshya Rana, Jiana T. Baker, Melissa Sorsby, Supriya Jagga, Shreya Venkat, Shaza Almardini, Eva S. Liu
Darier, Hailey-Hailey, and Grover’s diseases are rare acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell adhesion in the epidermis and desmosome organization. To better understand the underlying mechanisms leading to disease in these conditions we performed RNA-seq on lesional skin samples from patients. The transcriptomic profiles of Darier, Hailey-Hailey, and Grover’s disease were found to share a remarkable overlap, which did not extend to other common inflammatory skin diseases. Analysis of enriched pathways showed a shared upregulation in keratinocyte differentiation, and a decrease in cell adhesion and actin organization pathways in Darier, Hailey-Hailey, and Grover’s disease. Direct comparison to atopic dermatitis and psoriasis showed that the downregulation in actin organization pathways was a unique feature in the acantholytic skin diseases. Further, upstream regulator analysis suggested that a decrease in SRF/MRTF activity was responsible for the downregulation of actin organization pathways. Staining for MRTFA in lesional skin samples showed a decrease in nuclear MRTFA in patient skin compared to normal skin. These findings highlight the significant level of similarity in the transcriptome of Darier, Hailey-Hailey, and Grover’s disease, and identify decreases in actin organization pathways as a unique signature present in these conditions.
Quinn R. Roth-Carter, Hope E. Burks, Ziyou Ren, Jennifer L. Koetsier, Lam C. Tsoi, Paul W. Harms, Xianying Xing, Joseph Kirma, Robert M. Harmon, Lisa M. Godsel, Abbey L. Perl, Johann E. Gudjonsson, Kathleen J. Green
Rationale: Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. Objectives: We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 total patients undergoing femoral (N=23) or carotid (N=26) endarterectomy using single-cell ribonucleic acid sequencing (scRNA-seq; N=13), flow cytometry (N=24), and immunohistochemistry (N=12). Findings: Comparative scRNA-seq of CD45 positive-selected leukocytes from femoral (N=9; 35265 cells) and carotid (N=4; 30655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised 2.5- to 4-fold higher proportions of myeloid cells in carotid plaques, whereas non-inflammatory foam cell-like macrophages and LYVE1-overexpressing resident-like macrophages comprised 3.5- to 9-fold higher proportions of myeloid cells in femoral plaque (p<0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus femoral plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively anti-inflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. Conclusion: Human femoral plaques exhibit distinct macrophage profiles and diminished CD8+ T cell populations compared with carotid plaques. Experimental models elucidating determinants of plaque site-specific cell polarization cues are warranted.
Joshua Slysz, Arjun Sinha, Matthew DeBerge, Shalini Singh, Harris Avgousti, Inhyeok Lee, Kristofor Glinton, Reina Nagasaka, Prarthana J. Dalal, Shaina J. Alexandria, Ching Man Wai, Ricardo Tellez, Mariavittoria Vescovo, Ashwin Sunderraj, Xinkun Wang, Matthew J. Schipma, Ryan K Sisk, Rishab Gulati, Jenifer Vallejo, Ryosuke Saigusa, Donald M. Lloyd-Jones, Jon Lomasney, Samuel E. Weinberg, Karen J. Ho, Klaus Ley, Chiara Giannarelli, Edward B. Thorp, Matthew J. Feinstein
Pansclerotic morphea (PSM) is a rare, devastating disease characterized by extensive soft tissue fibrosis, secondary contractions, and significant morbidity. PSM pathogenesis is unknown, and aggressive immunosuppressive treatments rarely slow disease progression. We aimed to characterize molecular mechanisms driving PSM and identify therapeutically targetable pathways by performing single-cell and spatial RNA-sequencing on lesional and non-lesional skin biopsies of a PSM patient 12-months apart and 6 healthy controls. We then validated using immunostaining and in vitro approaches.Fibrotic skin was characterized by prominent type-II IFN response, accompanied by infiltrating myeloid, B-cells, and T-cells, which were the main IFN-γ source. We identified unique CXCL9+ fibroblasts enriched in PSM, characterized by increased chemokine expression, including CXCL9, CXCL10, and CCL2. CXCL9+ fibroblasts were related to profibrotic COL8A1+ myofibroblasts, which had enriched TGF-β response. In vitro, TGF-β and IFN-γ synergistically increased CXCL9 and CXCL10 expression, contributing to the perpetuation of IFN-γ responses. Further, cell-cell interaction analyses revealed cDC2B dendritic cells as a key communication hub between CXCL9+ fibroblasts and COL8A1+ myofibroblasts. These results define PSM as an inflammation-driven condition centered on type-II IFN responses. This work identified key pathogenic circuits between T-cells, cDC2Bs, and myofibroblasts, and suggests JAK1/2 inhibition is a potential therapeutic option in PSM.
Enze Xing, Feiyang Ma, Rachael Wasikowski, Allison C. Billi, Mehrnaz Gharaee-Kermani, Jennifer Fox, Craig Dobry, Amanda Victory, Mrinal Sarkar, Xianying Xing, Olesya Plazyo, Henry W. Chen, Grant C. Barber, Heidi Jacobe, Pei-Suen Tsou, Robert L. Modlin, John Varga, J. Michelle Kahlenberg, Lam C. Tsoi, Johann E. Gudjonsson, Dinesh Khanna
Intestinal mucins play an essential role in the defense against bacterial invasion and the maintenance of gut microbiota, which is instrumental in the regulation of host immune systems; hence, its dysregulation is a hallmark of metabolic disease and intestinal inflammation. However, the mechanism by which intestinal mucins control the gut microbiota as well as disease phenotypes remains nebulous. Herein, we report that N-acetylglucosamine (GlcNAc)-6-O-sulfation of O-glycans on intestinal mucins performs a protective role against obesity and intestinal inflammation. Chst4-/- mice, lacking GlcNAc-6-O-sulfation of the mucin O-glycans, showed significant weight gain and increased susceptibility to dextran sodium sulfate-induced colitis as well as colitis-associated cancer accompanied by significantly reduced immunoglobulin A (IgA) production caused by impaired T follicular helper cell-mediated IgA response. Interestingly, the protective effects of GlcNAc-6-O-sulfation against obesity and intestinal inflammation depend on the gut microbiota, evidenced by the modulation of the gut microbiota by co-housing or microbiota transplantation reversing disease phenotypes and IgA production. Collectively, our findings provide novel insight into the significance of host glycosylation, more specifically GlcNAc-6-O-sulfation on intestinal mucins, in protecting against obesity and intestinal inflammation via regulation of the gut microbiota.
Hirohito Abo, Aoi Muraki, Akihito Harusato, Tetsuya Imura, Maki Suzuki, Kohta Takahashi, Timothy L. Denning, Hiroto Kawashima
Optimal lung repair and regeneration is essential for recovery from viral infections including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known if the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections including the need for hospitalization and post-discharge mortality; this persists even after adjusting for common confounding factors. Further, we use a combination of lung organoid assays, single cell RNA sequencing (Sc-seq) and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show for the first time that lung organoids have a functional circadian clock, and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration- in tracheal cells via the Wnt/β-catenin pathway and through IL1β in alveolar epithelial cells. We speculate, that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.
Amruta Naik, Kaitlyn M. Forrest, Oindrila Paul, Yasmine Issah, Utham Kashyap Valekunja, Soon Yew Tang, Akhilesh B. Reddy, Elizabeth J. Hennessy, Thomas G. Brooks, Fatima N. Chaudhry, Apoorva Babu, Michael P. Morley, Jarod A. Zepp, Gregory R. Grant, Garret FitzGerald, Amita Sehgal, G. Scott Worthen, David B. Frank, Edward E. Morrisey, Shaon Sengupta
Gene therapy is under advanced clinical development for several lysosomal storage disorders. Pompe disease, a debilitating neuromuscular illness that affects infants, children, and adults with different degrees of severity, is caused by a deficiency of lysosomal glycogen-degrading enzyme acid alpha-glucosidase (GAA). Here, we demonstrated that adeno-associated virus (AAV9)-mediated systemic gene transfer fully reversed glycogen storage in all key therapeutic targets - skeletal and cardiac muscles, the diaphragm, and the central nervous system (CNS) - in both young and severely affected old Gaa knockout mice. Furthermore, the therapy reversed secondary cellular abnormalities in skeletal muscle, such as autophagy and mTORC1/AMPK signaling. We used a newly developed AAV9 vector encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues. These results lay the groundwork for future clinical development strategy in Pompe disease.
Naresh K Meena, Davide Randazzo, Nina Raben, Rosa Puertollano
Lysine-specific demethylase 1 (LSD1) is a histone demethylase that promotes stemness and cancer cell survival, including in prostate cancer. Most prostate malignancies are adenocarcinomas with luminal differentiation. However, some tumors undergo cellular reprogramming to a more lethal subset termed neuroendocrine prostate cancer (NEPC) with neuronal differentiation. The frequency of NEPC is increasing since widespread use of potent androgen receptor signaling inhibitors. Currently, there are no effective treatments for NEPC. We previously determined that LSD1 promotes survival of prostate adenocarcinoma tumors. However, the role of LSD1 in NEPC is unknown. Here, we determined that LSD1 is highly upregulated in NEPC vs. adenocarcinoma patient tumors. LSD1 suppression with RNAi or allosteric LSD1 inhibitors—but not catalytic inhibitors—reduced NEPC cell survival. RNA-seq analysis revealed that LSD1 represses pathways linked to luminal differentiation, and TP53 was the top reactivated pathway. We confirmed that LSD1 suppressed the TP53 pathway by reducing TP53 occupancy at target genes while LSD1’s catalytic function was dispensable for this effect. Mechanistically, LSD1 inhibition disrupted LSD1-HDAC interactions, increasing histone acetylation at TP53 targets. Finally, LSD1 inhibition suppressed NEPC tumor growth in vivo. These findings suggest that blocking LSD1’s non-catalytic function may be a promising new treatment strategy for NEPC.
Anbarasu Kumaraswamy, Zhi Duan, Diana Flores, Chao Zhang, Archana Sehrawat, Ya-Mei Hu, Olivia A. Swaim, Eva Rodansky, William K. Storck, Joshua A. Kuleape, Karan Bedi, Rahul Mannan, Xiao-Ming Wang, Aaron M. Udager, Visweswaran Ravikumar, Armand Bankhead III, Ilsa Coleman, John K. Lee, Colm Morrissey, Peter S. Nelson, Arul Chinnaiyan, Arvind Rao, Zheng Xia, Joel A. Yates, Joshi J. Alumkal
Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled as elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underly many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential to neutralize ROS and establish a pro-osteogenic REDOX environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis leads to acute degradation of RUNX2, impaired osteoblast differentiation and reduced bone formation. Conversely, reducing ROS using Catalase enhances RUNX2 stability and promotes osteoblast differentiation and bone formation when GSH biosynthesis is limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilizes RUNX2 and improves bone development in the Runx2+/- haploinsufficient mouse model of human Cleidocranial Dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarifies how ROS negatively impacts osteoblast differentiation and bone formation.
Guoli Hu, Yilin Yu, Deepika Sharma, Shondra M. Pruett-Miller, Yinshi Ren, Guo-Fang Zhang, Courtney M. Karner
Low-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of response remain unclear. Here, we characterized the post-hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production). We assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n=29), ATG plus granulocyte-colony stimulating factor (ATG/G-CSF, n=28), or placebo (n=31). Treatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (p<0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNFα (p<0.05 for all) 2 weeks post-treatment and a durable CD4 exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [p=0.011] and PD1+CD4 TEMRA MFI [p<0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG non-responders displayed higher proportions of senescent T cells (at baseline and post-treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker). Altogether in these exploratory analyses, Th1 inflammation-associated serum, CD4 exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.
Laura M. Jacobsen, Kirsten Diggins, Lori Blanchfield, James A. McNichols, Daniel J. Perry, Jason Brant, Xiaoru Dong, Rhonda Bacher, Vivian H. Gersuk, Desmond A. Schatz, Mark A. Atkinson, Clayton E. Mathews, Michael J. Haller, S. Alice Long, Peter S. Linsley, Todd M. Brusko
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