Transmembrane and tetratricopeptide repeat 4 (Tmtc4) is a recently described novel deafness gene in mice. Tmtc4-knockout mice have rapidly progressive postnatal hearing loss due to overactivation of the unfolded protein response (UPR); however, the cellular basis and human relevance of Tmtc4-associated hearing loss in the cochlea was not heretofore appreciated. We created a hair-cell-specific conditional knockout mouse that phenocopies the constitutive knockout with postnatal onset deafness, demonstrating that Tmtc4 is a hair-cell specific deafness gene. Furthermore, we identified a human family in which Tmtc4 variants segregate with adult-onset progressive hearing loss. Lymphoblastoid cells derived from multiple affected and unaffected family members, as well as human embryonic kidney cells engineered to harbor each of the variants, demonstrated that the human Tmtc4 variants confer hypersensitivity of the UPR towards apoptosis. These findings provide evidence that TMTC4 is a deafness gene in humans and further implicate the UPR in progressive hearing loss.
Jiang Li, Byung Yoon Choi, Yasmin Eltawil, Noura Ismail Mohamad, Yesai Park, Ian R. Matthews, Jin-Hee Han, Bong Jik Kim, Elliott H. Sherr, Dylan K. Chan
HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-MODY) and hypomorphic HNF1A variants confer type 2 diabetes risk, but a lack of experimental systems for interrogating mature human islets has limited our understanding of how the transcription factor HNF1α regulates adult islet function. Here, we combined conditional genetic targeting in human islet cells, RNA sequencing, chromatin mapping with Cleavage Under Targets & Release Using Nuclease (CUT&RUN), and transplantation-based assays to determine HNF1α-regulated mechanisms in adult human pancreatic α and β cells. Short hairpin RNA-mediated (shRNA) suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion after transplantation in mice, recapitulating phenotypes observed in diabetic patients. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATPase transporters and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional de-repression through HNF1α. CUT&RUN mapping of HNF1α DNA-binding sites in primary human islets imputed a subset of HNF1α-regulated genes as direct targets. These data elucidate mechanistic links between HNF1A loss and diabetic phenotypes in mature human α and β cells.
Mollie F. Qian, Romina J. Bevacqua, Vy M.N. Coykendall, Xiong Liu, Weichen Zhao, Charles A. Chang, Xueying Gu, Xiao-Qing Dai, Patrick E. MacDonald, Seung K. Kim
Germline APC mutation in familial adenomatous polyposis (FAP) patients promotes gastrointestinal polyposis, including the formation of frequent gastric fundic gland polyps (FGPs). In this study, we investigated how dysregulated Wnt signaling promotes FGPs and why they localize to the corpus region of the stomach. We developed a biobank of FGP and surrounding non-polyp corpus biopsies and organoids from FAP patients for comparative studies. Polyp biopsies and polyp-derived organoids exhibited enhanced Wnt target gene expression. Polyp-derived organoids with intrinsically upregulated Wnt signaling showed poor tolerance to further induction, suggesting that high Wnt restricts growth. Targeted genomic sequencing revealed that most gastric polyps did not arise via APC loss-of-heterozygosity. Studies in genetic mouse models demonstrated that heterozygous Apc loss increased epithelial cell proliferation in the corpus but not the antrum, while homozygous Apc loss was not maintained in the corpus yet induced hyperproliferation in antrum. Our findings suggest that heterozygous APC mutation in FAP patients may be sufficient to drive polyp formation in the corpus region while subsequent loss-of-heterozygosity to further enhance Wnt signaling is not tolerated. This finding contextualizes the abundant yet benign nature of gastric polyps in FAP patient corpus compared to the rare, yet adenomatous polyps in the antrum.
Kevin P. McGowan, Elizabeth Delgado, Theresa M. Keeley, Elise S. Hibdon, Danielle Kim Turgeon, Elena M. Stoffel, Linda C. Samuelson
Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This library was validated using seven genetic mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2–/– and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn–/– IgD+/– mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire–/– mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.
Elze Rackaityte, Irina Proekt, Haleigh S. Miller, Akshaya Ramesh, Jeremy F. Brooks, Andrew F. Kung, Caleigh Mandel-Brehm, David J.L. Yu, Colin R. Zamecnik, Rebecca Bair, Sara E. Vazquez, Sara Sunshine, Clare L. Abram, Clifford A. Lowell, Gabrielle Rizzuto, Michael R. Wilson, Julie Zikherman, Mark S. Anderson, Joseph L. DeRisi
Idiopathic Pulmonary Fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2a, and its cognate receptor FPr (Ptfgr) are implicated as a TGFβ1 independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen treated IER-SftpcI73T mice develop an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr null (FPr-/-) line showed attenuated weight loss and gene dosage dependent rescue of mortality compared to FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single cell RNA sequencing, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts which were reprogrammed to an “inflammatory/transitional” cell state in a PGF2a/ FPr dependent manner. Collectively, the findings provide evidence for a role for PGF2a signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.
Luis R. Rodriguez, Soon Yew Tang, Willy Roque Barboza, Aditi Murthy, Yaniv Tomer, Tian-Quan Cai, Swati Iyer, Katrina Chavez, Ujjalkumar Subhash Das, Soumita Ghosh, Charlotte Cooper, Thalia T. Dimopoulos, Apoorva Babu, Caitlin F. Connelly, Garret A. FitzGerald, Michael F. Beers
The widely used chemotherapy cisplatin causes permanent hearing loss in 40-60% of cancer patients. One drug, sodium thiosulfate, is approved by the FDA for use in pediatric patients with localized solid tumors for preventing cisplatin-induced hearing loss, but more drugs are desperately needed. Here, we tested dabrafenib, an FDA-approved BRAF kinase inhibitor and anticancer drug, in a clinically relevant multi-dose cisplatin mouse model. The protective effects of dabrafenib, given orally twice daily with cisplatin, were determined by functional hearing tests and cochlear outer hair cells counts. Toxicity of the drugs co-treatment was evaluated, and levels of pERK were measured. Dabrafenib, in dose of 3 mg/kg/bw, twice daily, in mice, was determined to be the minimum effective dose and it is equivalent to one tenth of the daily FDA-approved dose for human cancer treatment. The levels of hearing protection acquired, 20-25 dB at the three frequencies tested, in both female and male mice, persisted for four months after completion of treatments. Moreover, dabrafenib exhibited a good in vivo therapeutic index (> 25), hearing protection in two different mouse strains, and diminished cisplatin-induced weight loss. Altogether, this study demonstrates that dabrafenib is a promising candidate drug for protection from cisplatin-induced hearing loss.
Matthew A. Ingersoll, Richard D. Lutze, Chithra K. Pushpan, Regina G. Kelmann, Huizhan Liu, Mark T. May, William J. Hunter, David Z.Z. He, Tal Teitz
The selective targeting of pathogenic T cells is a “holy grail” in the development of new therapeutics for T cell-mediated disorders including many autoimmune diseases and graft-versus-host disease. We describe the development of a CD6-targeted antibody-drug conjugate (CD6-ADC) by conjugating an inactive form of monomethyl auristatin E (MMAE), a potent mitotic toxin, onto a monoclonal antibody (mAb) against CD6, an established T cell surface marker. Even though CD6 is present on all T cells, only the activated (pathogenic) T cells vigorously divide and thus are susceptible to the anti-mitotic MMAE-mediated killing via the CD6-ADC. We found CD6-ADC selectively killed activated proliferating human T cells and antigen-specific mouse T cells in vitro. Furthermore, in vivo, whereas the CD6-ADC had no significant detrimental effect on normal T cells in naïve CD6 humanized mice, the same dose of CD6-ADC, but not the controls, efficiently treated two pre-clinical models of autoimmune uveitis and a model of graft-versus-host disease. These results provide evidence suggesting that CD6-ADC could be further developed as a novel therapeutic agent for the selective elimination of pathogenic T cells and treatment of many T cell-mediated disorders.
Lingjun Zhang, Liping Luo, Jin Y. Chen, Rupesh Singh, William M. Baldwin III, David A. Fox, Daniel J. Lindner, Daniel F. Martin, Rachel R. Caspi, Feng Lin
Control of visceral leishmaniasis (VL) depends on pro-inflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, pro-inflammatory cytokines produced by Th1 cells can damage tissues, and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type I regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target to alter the balance between Th1 and Tr1 cells for improved anti-parasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6 mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important co-inhibitory receptor in VL patients and experimental VL, and reveal tissue-specific heterogeneity of co-inhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.
Chelsea L. Edwards, Jessica A. Engel, Fabian De Labastida Rivera, Susanna S. Ng, Dillon Corvino, Marcela Montes de Oca, Teija C.M. Frame, Shashi Bhushan Chauhan, Siddharth Sanker Singh, Awnish Kumar, Yulin Wang, Jinrui Na, Pamela Mukhopadhyay, Jason S. Lee, Susanne Nylén, Shyam Sundar, Rajiv Kumar, Christian R. Engwerda
Hair loss is a debilitating condition associated with the depletion of dermal papilla cells (DPCs), which can be replenished by dermal sheath cells (DSCs). Hence, strategies aimed at increasing the population of DPCs and DSCs hold great promise for the treatment of hair loss. In this study, we demonstrated that introducing exogenous DPCs and DSCs (hair follicle mesenchymal stem cells) could effectively migrate and integrate into the dermal papilla and dermal sheath niches, leading to enhanced hair growth and prolonged anagen phases. However, the homing rates of DPCs and DSCs were influenced by various factors, including recipient mouse depilation, cell passage number, cell dose, and immune rejection. Through in vitro and in vivo experiments, we further discovered that the CXCL13/CXCR5 pathway mediated the homing of DPCs and DSCs into hair follicle niches. This study underscores the potential of cell-based therapies for hair loss by targeted delivery of DPCs and DSCs to their respective niches, and sheds light on the intriguing concept that isolated mesenchymal stem cells can home back to their original niche microenvironment.
Kaitao Li, Fang Liu, Ye He, Qian Qu, Pingping Sun, Lijuan Du, Jin Wang, Ruosi Chen, Yuyang Gan, Danlan Fu, Zhexiang Fan, Bingcheng Liu, Zhiqi Hu, Yong Miao
Osteoclasts specialize in bone resorption and are critical for bone remodeling. Previous studies have shown that osteoclasts possess abundant mitochondria and derive most energy through oxidative phosphorylation (OXPHOS). However, the energy substrates fueling OXPHOS in osteoclasts remain to be fully defined. Here, we showed that osteoclast differentiation was coupled with increased oxidation of glucose, glutamine and oleate. Transcriptomic analyses with RNA sequencing revealed marked upregulation of genes participating in OXPHOS and mitochondrial fatty acids oxidation, during osteoclast differentiation. Increased mitochondrial oxidation of long-chain fatty acids was required for osteoclast differentiation in vitro. However, blocking fatty acid oxidation in vivo, by deletion of Cpt1a in osteoclast progenitors, impaired osteoclast formation only in the female mice. The Cpt1a-deficient females were further protected from osteoclast activation by a high fat diet. The males, on the contrary, exhibited normal bone resorption despite Cpt1a deletion, regardless of the dietary fat content. Moreover, concurrent deletion of Mpc1 and Cpt1a, blocking mitochondrial oxidation of both glucose and fatty acids in the osteoclast lineage, failed to impede bone resorption in the males. The study therefore uncovers a female-specific dependence on mitochondrial oxidation of fatty acids and glucose in osteoclasts in vivo.
Chao Song, Arianna Valeri, Fangfang Song, Xing Ji, Xueyang Liao, Tyler Marmo, Rebecca A. Seeley, Jared Rutter, Fanxin Long
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