Research
Abstract
Allelic heterogeneity (AH) has been noted in truncational TTN (TTNtv)-associated dilated cardiomyopathy (DCM), i.e., mutations affecting A-band-encoding exons are pathogenic, but those affecting Z-disc-encoding exons are likely benign. The lack of an in vivo animal model that recapitulates AH hinders the deciphering of the underlying mechanism. Here, we explored zebrafish as a candidate vertebrate model by phenotyping a collection of zebrafish ttntv alleles. We noted that cardiac function and sarcomere structure are more severely disrupted in ttntv-A than in ttntv-Z homozygous embryos. Consistently, cardiomyopathy-like phenotypes were presented in ttntv-A but not ttntv-Z adult heterozygous mutants. The phenotypes observed in ttntv-A alleles were recapitulated in null mutants with the entire titin-encoding sequences removed. Defective autophagic flux, largely due to impaired autophagosome-lysosome fusion, was also only noted in ttntv-A but not ttntv-Z models. Moreover, we found that genetic manipulation of ulk1a restored autophagy flux and rescued cardiac dysfunction in ttntv-A animals. Together, our findings presented adult zebrafish as an in vivo animal model for studying AH in TTNtv DCM, demonstrated TTN loss-of-function sufficient to trigger ttntv DCM in zebrafish, and uncovered ulk1a as a potential therapeutic target gene for TTNtv DCM.
Authors
Ping Zhu, Jiarong Li, Feixiang Yan, Shahidul Islam, Xueying Lin, Xiaolei Xu
Abstract
In autoimmunity, FOXP3+ regulatory T cells (Tregs) skew towards a pro-inflammatory, non-suppressive phenotype and are therefore unable to control the exaggerated autoimmune response. This largely impacts the success of autologous Treg therapy which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the central nervous system during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss-of-function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential, a pro-inflammatory Th1/17 signature and upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically-approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less suppressive CD49d+ Tregs in the cerebrospinal fluid of people with MS. In sum, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive pro-inflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.
Authors
Paulien Baeten, Ibrahim Hamad, Cindy Hoeks, Michael Hiltensperger, Bart Van Wijmeersch, Veronica Popescu, Lilian Aly, Veerle Somers, Thomas Korn, Markus Kleinewietfeld, Niels Hellings, Bieke Broux
Abstract
The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.
Authors
Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Takahide Nejo, Lauren S. Levine, Matthew H. Spitzer, Hideho Okada
Abstract
Radiotherapy induces a Type I interferon (T1IFN)-mediated anti-tumoral immune response that we hypothesized could be potentiated by a first-in-class ATM inhibitor leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound AZD0156 on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced anti-tumoral immune responses and sensitized to anti-PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells as therapeutic efficacy was blunted in CD8+ T cell-depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN leading to both innate and subsequent adaptive anti-tumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.
Authors
Qiang Zhang, Long Jiang, Weiwei Wang, Amanda K. Huber, Victoria M. Valvo, Kassidy M. Jungles, Erin A. Holcomb, Ashley N. Pearson, Stephanie The, Zhuwen Wang, Leslie A. Parsels, Joshua D. Parsels, Daniel R. Wahl, Arvind Rao, Vaibhav Sahai, Theodore S. Lawrence, Michael D. Green, Meredith A. Morgan
Abstract
Patients with mutations in the thyroid hormone (TH) cell transporter MCT8 gene develop severe neuro-psychomotor retardation known as the Allan-Herndon-Dudley syndrome (AHDS). It is assumed that this is caused by a reduction in TH signaling in the developing brain, and treatment remains understandably challenging. Given species differences in brain TH transporters and the limitations of studies in mice, we generated brain organoids (BOs) using human iPSCs from MCT8-deficient patients. We found that MCT8-deficient BOs exhibit (i) impaired T3 transport in developing neural cells, as assessed through deiodinase-3-mediated T3 catabolism, (ii) reduced expression of genes involved in neurogenesis and neuronal maturation, and (iii) reduced T3-inducibility of TH-regulated genes. In contrast, the TH-analogs 3,5-diiodothyropropionic acid and 3,3’,5-triiodothyroacetic acid triggered normal responses (induction/repression of T3-responsive genes) in MCT8-deficient BOs, constituting a proof-of-concept that lack of T3 transport underlies the pathophysiology of AHDS, demonstrating the clinical potential for TH analogues to be used in treating AHDS patients. MCT8-deficient BOs represent a species-specific relevant preclinical model that can be utilized to screen drugs with potential benefits as personalized therapeutics for AHDS patients.
Authors
Federico Salas-Lucia, Sergio Escamilla, Antonio C. Bianco, Alexandra Dumitrescu, Samuel Refetoff
Abstract
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to cystic fibrosis (CF), a life-threating autosomal recessive genetic disease. While recently approved Trikafta dramatically ameliorates CF lung diseases, there is still a lack of effective medicine to treat CF-associated liver disease (CFLD). To address this medical need, we used a recently established CF rabbit model to test if Sotagliflozin, a Sodium-Glucose cotransporter 1 and 2 (SGLT1/2) inhibitor drug that is approved to treat diabetes, can be repurposed to treat CFLD. Sotagliflozin treatment led to systemic benefits to CF rabbits, evidenced by increased appetite and weight gain as well as prolonged lifespan. For CF liver related phenotypes, the animals benefited from normalized blood chemistry and bile acid parameters. Further, Sotagliflozin alleviated non-alcoholic steatohepatitis (NASH)-like phenotypes including liver fibrosis. Intriguingly, Sotagliflozin treatment markedly reduced the otherwise elevated endoplasmic reticulum (ER) stress responses in the liver and other affected organs of CF rabbits. In summary, our work demonstrates that Sotagliflozin attenuates liver disorders in CF rabbits, and merits Sotagliflozin as a potential drug to treat CFLD.
Authors
Xiubin Liang, Xia Hou, Mohamad Bouhamdan, Yifei Sun, Zhenfeng Song, Carthic Rajagopalan, Hong Jiang, Hong-Guang Wei, Jun Song, Dongshan Yang, Yanhong Guo, Yihan Zhang, Hongmei Mou, Jifeng Zhang, Y. Eugene Chen, Fei Sun, Jian-Ping Jin, Kezhong Zhang, Jie Xu
Abstract
In humans, Type 2 Diabetes Mellitus (T2DM) shows a higher prevalence in men compared to women, phenotype that has been attributed to a lower peripheral insulin sensitivity in men. Whether sex-specific differences in pancreatic β-cell function also contribute is largely unknown. Here we characterized the electrophysiological properties of β-cells in intact mouse male and female islets. Elevation of glucose concentration above 5 mM triggers an electrical activity with a similar glucose dependence in β-cells of both sexes. However, female β-cells have a more depolarized membrane potential and increased firing frequency compared to males. The higher membrane depolarization in female β-cells is caused by ~50% smaller Kv2.1 K+ currents compared to males but otherwise unchanged KATP, Ca2+-activated BK and SK, and background TASK1/TALK1 K+ current densities. In female β-cells the higher depolarization causes a membrane potential-dependent inactivation of the voltage-gated Ca2+ channels (CaV) resulting in reduced Ca2+ entry. Nevertheless, this reduced Ca2+ influx is offset by the higher action potential firing frequency. Since exocytosis of insulin granules does not show a sex-specific difference we conclude that the higher electrical activity promotes insulin release in females improving glucose tolerance.
Authors
Noelia Jacobo-Piqueras, Tamara Theiner, Stefanie M. Geisler, Petronel Tuluc
Abstract
Fibroblasts are stromal cells known to regulate local immune responses important for wound healing and scar formation; however, the cellular mechanisms driving damage and scarring in cutaneous lupus erythematosus (CLE) patients remain poorly understood. Dermal fibroblasts in systemic lupus erythematosus (SLE) patients are abnormally exposed to cytokines, but the impact of inflammatory mediators on fibroblast responses in non-scarring versus scarring CLE subtypes is unclear. Here, we examined responses to cytokines in dermal fibroblasts from non-lesional skin of 22 SLE patients with CLE and 34 healthy controls. Notably, inflammatory cytokine responses were exaggerated in SLE fibroblasts compared to healthy controls. In lesional CLE biopsies, these same inflammatory profiles were reflected in single cell RNA sequencing of SFRP2+ and inflammatory fibroblast subsets, and TGF-β was identified as a critical upstream regulator for inflammatory fibroblasts in scarring discoid lupus lesions. In vitro cytokine stimulation of non-lesional fibroblasts from patients who scar from CLE identified an upregulation of collagens, particularly in response to TGF-β, whereas inflammatory pathways were more prominent in non-scarring patients. Our study revealed that SLE fibroblasts are poised to hyper-respond to inflammation, with differential responses among scarring versus non-scarring disease, providing a potential skin-specific target for mitigating damage.
Authors
Suzanne K. Shoffner-Beck, Lisa Abernathy-Close, Stephanie Lazar, Feiyang Ma, Mehrnaz Gharaee-Kermani, Amy Hurst, Craig Dobry, Deepika Pandian, Rachael Wasikowski, Amanda Victory, Kelly Arnold, Johann E. Gudjonsson, Lam C. Tsoi, J. Michelle Kahlenberg
Abstract
Suppressor of Fused (SUFU) is widely regarded as a key negative regulator of the Sonic Hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public dataset containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused pre-axial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted medulloblastoma tumorigenesis. These data reveal multi-faceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counter-intuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.
Authors
Boang Han, Yu Wang, Shen Yue, Yun-hao Zhang, Lun Kuang, Bin-bin Gao, Yue Wang, Ziyu Zhang, Xiaohong Pu, Xin-fa Wang, Chi-chung Hui, Ting-ting Yu, Chen Liu, Steven Y. Cheng
Abstract
Despite effective antibiotic therapy, brain destructive inflammation often cannot be avoided in pneumococcal meningitis. The causative signals are mediated predominantly through TLR recruited myeloid differentiation primary response adaptor (MyD) 88 as indicated by a dramatic pneumococcal meningitis phenotype of Myd88-/- mice. Because lipoproteins and ssRNA are crucial for recognition of Gram-positive bacteria such as Streptococcus pneumoniae by the host immune system, we comparatively analyzed the disease courses of Myd88-/- and Tlr2/13-/- mice. Their phenotype resemblance indicated TLR2 and -13 as master sensors of Streptococcus pneumoniae in the cerebrospinal fluid. The neutralizing anti-TLR2 antibody (T2.5) and chloroquine (CQ) – the latter applied here as an inhibitor for murine TLR13 and its human orthologue TLR8 – abrogated activation of murine and human primary immune cells exposed to antibiotic-treated Streptococcus pneumoniae. The inhibitory effect of the T2.5/CQ cocktail was stronger than that of dexamethasone, the current standard adjunctive drug for pneumococcal meningitis. Accordingly, TLR2/13 blockade concomitant with ceftriaxone application significantly improved the clinical course of pneumococcal meningitis compared to treatment with ceftriaxone alone or in combination with dexamethasone. Our study implicates the importance of murine TLR13 and human TLR8, besides TLR2, in pneumococcal meningitis pathology, and suggests their blockade as a promising antibiotic therapy adjunct.
Authors
Susanne Dyckhoff-Shen, Ilias Masouris, Heba Islam, Sven Hammerschmidt, Barbara Angele, Veena Marathe, Jan Buer, Stefanie Völk, Hans-Walter Pfister, Matthias Klein, Uwe Koedel, Carsten J. Kirschning
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