The immune benefits of vitamin D3 supplementation beyond calcium and phosphate maintenance are highly clinically debated. Kidney expression of CYP27B1 is the source of endocrine, circulating 1,25(OH)2D3 (active form of vitamin D) that maintains serum calcium and phosphate. 1,25(OH)2D3 may also be made by the CYP27B1 enzyme in non-renal cells, like immune cells, in a process driven by cellular availability of 25(OH)D3 and inflammation. Due to the endocrine nature of 1,25(OH)2D3 in circulation, it is difficult to discern between these two sources. We recently created a regulatory deletion model of Cyp27b1 (M1/M21-DIKO) where mice have normal inflammatory-regulated Cyp27b1 expression in non-renal tissues (unlike global Cyp27b1-KO), but no expression within kidney. Here, utilizing on-tissue chemical derivatization and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI), we investigated the distribution of 1,25(OH)2D3 and 25(OH)D3 in the kidney, liver, spleen, and thymus. MALDI-MSI demonstrated increased 1,25(OH)2D3 in non-renal tissues such as the spleen after vitamin D3 supplementation in M1/M21-DIKO mice. Additionally, from this we found increased Il4 and decreased Tnfa in the spleen after vitamin D3 supplementation. Taken together, these data demonstrate non-renal production of 1,25(OH)2D3 in vivo and provide a consequence of vitamin D3 supplementation and non-renal 1,25(OH)2D3 production in cytokine changes.
Mark B. Meyer, Seong Min Lee, Shannon R. Cichanski, Diego F. Cobice, J. Wesley Pike
Despite epidermal turnover, the skin is host to a complex array of microbes including viruses, such as the human papillomavirus (HPV), which must infect and manipulate skin keratinocyte stem cells (KSC) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induces ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses (AK). Together these results define the “hit and run” mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lacks melanosome protection and is thus susceptible to sun-light-induced malignant transformation.
Huw J. Morgan, Carlotta Olivero, Boris Y. Shorning, Alex Gibbs, Alexandra L. Phillips, Lokapriya Ananthan, Annabelle Xiao Hui Lim, Licia Martuscelli, Cinzia Borgogna, Marco De Andrea, Martin Hufbauer, Richard G. Goodwin, Baki Akgül, Marisa Gariglio, Girish K. Patel
Autosomal dominant optic atrophy plus (ADOA+) is characterized by primary optic nerve atrophy accompanied by a spectrum of degenerative neurological symptoms. Despite ongoing research, no effective treatments are currently available for this condition. Our study provided evidence for the pathogenicity of an unreported c.1780T>C variant in the OPA1 gene through patient-derived skin fibroblasts and an engineered HEK293T cell line with OPA1 downregulation. We demonstrated that OPA1 insufficiency promoted mitochondrial fragmentation and increased DRP1 expression, disrupting mitochondrial dynamics. Consequently, this disruption enhanced mitophagy and caused mitochondrial dysfunction, contributing to the ADOA+ phenotype. Notably, the Drp1 inhibitor, mitochondrial division inhibitor-1 (Mdivi-1), effectively mitigated the adverse effects of OPA1 impairment. These effects included reduced Drp1 phosphorylation, decreased mitochondrial fragmentation, and balanced mitophagy. Thus, we propose that intervening in DRP1 with Mdivi-1 could correct mitochondrial abnormalities, offering a promising therapeutic approach for managing ADOA+.
Yan Lin, Dongdong Wang, Busu Li, Jiayin Wang, Ling Xu, Xiaohan Sun, Kunqian Ji, Chuanzhu Yan, Fuchen Liu, Yuying Zhao
Little is known about the expression patterns and functions of circular RNAs (circRNAs) in the heart of large mammals. In this study, we examined the expression profiles of circRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in neonatal pig hearts. Pig heart samples collected on postnatal days 1 (P1), 3 (P3), 7 (P7) and 28 (P28) were sent for total RNA sequencing. Our data revealed a total of 7000 circRNAs in the 24 pig hearts. Pathway enrichment analysis of hallmark gene sets demonstrated that differentially expressed circRNAs are engaged in different pathways. The most significant difference was observed between P1 and the other three groups (P3, P7 and P28) in pathways related to cell cycle and muscle development. Out of the ten circRNAs that were validated through real-time quantitative polymerase chain reaction (qRT-PCR) to confirm their expression, six exhibited significant effects on cell cycle activity in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) following small interfering RNA-mediated knockdown. The circRNA-miRNA-mRNA networks were constructed to understand the potential mechanisms of circRNAs in the heart. In conclusion, our study provided a dataset for exploring the roles of circRNAs in pig hearts. In addition, we identified several circRNAs that regulate cardiomyocyte cell cycle.
Ling Tang, Nyarige Verah, Pengsheng Li, Junwen Wang, Wuqiang Zhu
Acute kidney injury strongly upregulates the transcription factor Foxm1 in proximal tubule in vivo and Foxm1 drives epithelial proliferation in vitro. Here we report that deletion of Foxm1 either with a nephron specific Cre driver or by inducible global deletion reduces proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI-to-CKD transition with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report extracellular signal-regulated kinase (ERK) mediates FOXM1 induction downstream of the epidermal growth factor receptor (EGFR) in primary proximal tubule cells. We defined FOXM1 genomic binding sites by Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned datasets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identify two cis regulatory elements that bind FOXM1 and regulate CCNF expression, demonstrate that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK-FOXM1-CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.
Megan L. Noonan, Yoshiharu Muto, Yasuhiro Yoshimura, Aidan Leckie-Harre, Haojia Wu, Vladimir V. Kalinichenko, Benjamin D. Humphreys, Monica Chang-Panesso
TTK (MPS1) spindle assembly checkpoint kinase is an emerging cancer target. This preclinical study explored the anti-tumor mechanism of TTK inhibitor OSU13 to define a strategy for clinical development. We observed prominent anti-tumor activity of OSU13 in melanoma, colon, and breast cancer cells, melanoma patient-derived organoids, and mice bearing colon tumors associated with G2 cell cycle arrest, senescence, and apoptosis. OSU13-treated cells displayed DNA damage and micronuclei that triggered the cytosolic DNA-sensing cGAS-STING pathway. STING was required for the induction of several proteins involved in T cell recruitment and activity. Tumors from OSU13-treated mice showed an increased proportion of T and NK cells and evidence of PD-1/PD-L1 immune checkpoint activation. Combining a low-toxicity dose of OSU13 with anti-PD1 checkpoint blockade resulted in prominent STING- and CD8 T cell-dependent tumor inhibition and improved survival. These findings provide a rationale for utilizing TTK inhibitors in combination with immunotherapy in STING-proficient tumors.
Vijaya Bharti, Amrendra Kumar, Yinchong Wang, Nikhil Roychowdhury, Daniel de Lima Bellan, Beimnet B. Kassaye, Reese Watkins, Marina Capece, Catherine G. Chung, Gerard Hilinski, Anna E. Vilgelm
Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as anti-fibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photo-affinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-β1–activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress, affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-β1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct anti-fibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.
Hope A. Flaxman, Maria-Anna Chrysovergi, Hongwei Han, Farah Kabir, Rachael T. Lister, Chia-Fu Chang, Robert Yvon, Katharine E. Black, Andreas Weigert, Rajkumar Savai, Alejandro Egea-Zorrilla, Ana Pardo-Saganta, David Lagares, Christina M. Woo
Men who have sex with men (MSM) with HIV are at high risk for squamous intraepithelial lesion (SIL) and anal cancer. Identifying local immunological mechanisms involved in the development of anal dysplasia could aid treatment and diagnostics. Here we studied 111 anal biopsies obtained from 101 MSM with HIV, who participated in an anal screening program. We first assessed multiple immune subsets by flow cytometry, in addition to histological examination, in a discovery cohort (n = 54). Selected molecules were further evaluated by immunohistochemistry in a validation cohort (n = 47). Pathological samples were characterized by the presence of Resident Memory T cells with low expression of CD103 and by changes in Natural Killer cell subsets, affecting residency and activation. Furthermore, potentially immune suppressive subsets, including CD15+CD16+ mature neutrophils, gradually increased as the anal lesion progressed. Immunohistochemistry confirmed the association between the presence of CD15 in the epithelium and SIL diagnosis, with a sensitivity of 80% and specificity of 71% (AUC 0.762) for the correlation with high-grade SIL. A complex immunological environment with imbalanced proportions of resident effectors and immune suppressive subsets characterizes pathological samples. Neutrophil infiltration, determined by CD15 staining, may represent a valuable pathological marker associated with the grade of dysplasia.
Joaquín Burgos, Aleix Benítez-Martínez, Cristina Mancebo-Pérez, Nuria Massana, Antonio Astorga-Gamaza, Josep Castellvi, Stefania Landolfi, Adrià Curran, Jorge N. Garcia-Perez, Vicenç Falcó, María J. Buzón, Meritxell Genescà
Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation (AF). The mechanisms underlying DM-associated AF are unclear. AF and DM are both related to inflammation. We investigated whether DM-associated inflammation contributed to AF risk. Mice were fed with high fat diet to induce type II DM and were subjected to IL-1β antibodies, macrophage depletion by Clodronate liposomes, a mitochondrial antioxidant (mitoTEMPO), or a cardiac ryanodine receptor (RyR2) stabilizer (S107). All tests were performed at 36-38 weeks of age. DM mice presented with increased AF inducibility, enhanced mitochondrial reactive oxygen species (mitoROS) generation, and activated innate immunity in the atria as evidenced by enhanced monocyte chemoattractant protein-1 (MCP-1) expression, macrophage infiltration, and IL-1β levels. Signs of aberrant RyR2 Ca2+ leak were observed in the atria of DM mice. IL-1β neutralization, macrophage depletion, mitoTEMPO, and S107 significantly ameliorated the AF vulnerability in DM mice. Atrial overexpression of MCP-1 increased AF occurrence in normal mice through the same mechanistic signaling cascade as observed in DM mice. In conclusion, macrophage-mediated IL-1β contributed to DM-associated AF risk through mitoROS modulation of RyR2 Ca2+ leak.
Xiaoxu Zhou, Hong Liu, Feng Feng, Gyeoung-Jin Kang, Man Liu, Yugene Guo, Samuel C. Dudley Jr.
Loss-of-function mutations of the gene encoding the trafficking protein particle complex subunit 9 (trappc9) cause autosomal recessive intellectual disability and obesity by unknown mechanisms. Genome-wide analysis links trappc9 to non-alcoholic fatty liver disease (NAFLD). Trappc9-deficient mice have been shown to appear overweight shortly after weaning. Here, we analyzed serum biochemistry and histology of adipose and liver tissues to determine the incidence of obesity and NAFLD in trappc9-deficient mice and combined transcriptomic and proteomic analyses, pharmacological studies, and biochemical and histological examinations of postmortem mouse brains to unveil mechanisms involved. We found that trappc9-deficient mice presented with systemic glucose homeostatic disturbance, obesity and NAFLD, which were relieved upon chronic treatment combining dopamine receptor D2 (DRD2) agonist quinpirole and DRD1 antagonist SCH23390. Blood glucose homeostasis in trappc9-deficient mice was restored upon administrating quinpirole alone. RNA-sequencing analysis of DRD2-containing neurons and proteomic study of brain synaptosomes revealed signs of impaired neurotransmitter secretion in trappc9-deficient mice. Biochemical and histological studies of mouse brains showed that trappc9-deficient mice synthesized dopamine normally, but their dopamine-secreting neurons had a lower abundance of structures for releasing dopamine in the striatum. Our study suggests that trappc9 loss-of-function causes obesity and NAFLD by constraining dopamine synapse formation.
Yan Li, Usman Muhammad, Ellen Sapp, Yuting Ke, Zejian Wang, Adel Boudi, Marian DiFiglia, Xueyi Li
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