Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression, is the expansion of regulatory T-cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type-1 interferon (IFN). Blocking IFNAR1 (interferon alpha and beta receptor 1) on Tregs significantly decreases both, myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of multiple myeloma patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type-1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1 knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type-1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.
Yawara Kawano, Oksana Zavidij, Jihye Park, Michele Moschetta, Katsutoshi Kokubun, Tarek H. Mouhieddine, Salomon Manier, Yuji Mishima, Naoka Murakami, Mark Bustoros, Romanos Sklavenitis Pistofidis, Mairead Reidy, Yu J. Shen, Mahshid Rahmat, Pavlo Lukyanchykov, Esilida Sula Karreci, Shokichi Tsukamoto, Jiantao Shi, Satoshi Takagi, Daisy Huynh, Antonio Sacco, Yu-Tzu Tai, Marta Chesi, P. Leif Bergsagel, Aldo M. Roccaro, Jamil Azzi, Irene M. Ghobrial
Adult vascular smooth muscle cells (VSMCs) possess the peculiar ability to de-differentiate in response to extracellular cues, such as vascular damage and inflammation. De-differentiated VSMCs are proliferative, migratory, and have decreased contractile capacity. VSMC dedifferentiation contributes not only to vascular repair, but also to cardiovascular pathologies, such as intimal hyperplasia/restenosis in coronary artery or peripheral vascular diseases and arterial aneurysm. We here demonstrate the role of ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. The expression of UHRF1 correlates with the development of a wide array of vascular pathologies associated also with modulation of non-coding RNAs, such as microRNAs. Importantly, miR-145, a pivotal gene regulating VSMC plasticity, which is reduced in vascular diseases, was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggers VSMC proliferation by directly repressing the promoters of cell cycle inhibitor genes, such as p21 and p27, and of key pro-differentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm.Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and de-differentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies, modulating also the VSMC component.
Leonardo Elia, Paolo Kunderfranco, Pierluigi Carullo, Marco Vacchiano, Floriana Maria Farina, Ignacio Fernando Hall, Stefano Mantero, Cristina Panico, Roberto Papait, Gianluigi Condorelli, Manuela Quintavalle
Cell death is a key driver of disease progression and carcinogenesis in chronic liver disease (CLD), highlighted by the well-established clinical correlation between hepatocellular death and risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Moreover, hepatocellular death is sufficient to trigger fibrosis and HCC in mice. However, the pathways through which cell death drives CLD progression remain elusive. Here, we tested the hypothesis that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) with key roles in acute liver injury, may link cell death to injury responses and hepatocarcinogenesis in CLD. While liver-specific HMGB1 deficiency did not significantly affect chronic injury responses such as fibrosis, regeneration and inflammation, it inhibited ductular/progenitor cell expansion and hepatocyte metaplasia. HMGB1 promoted ductular expansion independently of active secretion in a non-autonomous fashion, consistent with its role as DAMP. Liver-specific HMGB1 deficiency reduced HCC development in three models with chronic injury but not in a model lacking chronic liver injury. Similar to CLD, HMGB1 ablation reduced the expression of progenitor and oncofetal markers, a key determinant of HCC aggressiveness, in tumors. In summary, HMGB1 links hepatocyte death to ductular reaction, progenitor signature and hepatocarcinogenesis in CLD.
Céline Hernandez, Peter Huebener, Jean-Philippe Pradere, Daniel J. Antoine, Richard A. Friedman, Robert F. Schwabe
Autophagy is important for liver homeostasis and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here we reveal the role of highmobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPC) implicated in liver repair and regeneration. DR caused by hepatic toxic diets (DDC or CDE) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor development in autophagy deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes, and could mediate HMGB1’s proliferative effects in isolated HPC. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury, but depending on NRF2 and inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone without disabling autophagy or causing injury was sufficient to cause inflammasomedependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under the autophagy deficient condition, which leads to HPC expansion but also tumor development.
Bilon Khambu, Nazmul Huda, Xiaoyun Chen, Daniel J. Antoine, Yong Li, Guoli Dai, Ulrike A. Köhler, Wei-Xing Zong, Satoshi Waguri, Sabine Werner, Tim D. Oury, Zheng Dong, Xiao-Ming Yin
Incidence of nonalcoholic steatohepatitis (NASH), which is considered a hepatic manifestation of metabolic syndrome, has been increasing worldwide with the rise in obesity; however, its pathological mechanism is poorly understood. Here, we demonstrate that the hepatic expression of aortic carboxypeptidase–like protein (ACLP), a glycosylated, secreted protein, increases in NASH in humans and mice. Furthermore, we elucidate that ACLP is a ligand, unrelated to WNT proteins, that activates the canonical WNT pathway and exacerbates NASH pathology. In the liver, ACLP is specifically expressed in hepatic stellate cells (HSCs). As fatty liver disease progresses, ACLP expression is enhanced via activation of STAT3 signaling by obesity-related factors in serum. ACLP specifically binds to frizzled-8 and low-density lipoprotein–related receptor 6 to form a ternary complex that activates canonical WNT signaling. Consequently, ACLP activates HSCs by inhibiting PPARγ signals. HSC-specific ACLP deficiency inhibits fibrosis progression in NASH by inhibiting canonical WNT signaling in HSCs. The present study elucidates the role of canonical WNT pathway activation by ACLP in NASH pathology, indicating that NASH can be treated by targeting ACLP-induced canonical WNT pathway activation in HSCs.
Toshiaki Teratani, Kengo Tomita, Takahiro Suzuki, Hirotaka Furuhashi, Rie Irie, Makoto Nishikawa, Junji Yamamoto, Toshifumi Hibi, Soichiro Miura, Tohru Minamino, Yuichi Oike, Ryota Hokari, Takanori Kanai
Apoptosis has been proposed as a key mechanism responsible for CD4+ T cell depletion and immune dysfunction during HIV infection. We demonstrated that Q-VD-OPH, a caspase inhibitor, inhibits spontaneous and activation-induced death of T cells from SIV-infected rhesus macaques (RMs). When administered during the acute phase of infection, Q-VD-OPH was associated with (a) reduced levels of T cell death, (b) preservation of CD4+/CD8+ T cell ratio in lymphoid organs and in the gut, (c) maintenance of memory CD4+ T cells, and (d) increased specific CD4+ T cell response associated with the expression of cytotoxic molecules. Although therapy was limited to the acute phase of infection, Q-VD-OPH–treated RMs showed lower levels of both viral load and cell-associated SIV DNA as compared with control SIV-infected RMs throughout the chronic phase of infection, and prevented the development of AIDS. Overall, our data demonstrate that Q-VD-OPH injection in SIV-infected RMs may represent an adjunctive therapeutic agent to control HIV infection and delaying disease progression to AIDS.
Mireille Laforge, Ricardo Silvestre, Vasco Rodrigues, Julie Garibal, Laure Campillo-Gimenez, Shahul Mouhamad, Valérie Monceaux, Marie-Christine Cumont, Henintsoa Rabezanahary, Alain Pruvost, Anabela Cordeiro-da-Silva, Bruno Hurtrel, Guido Silvestri, Anna Senik, Jérôme Estaquier
During development, Sox2 is indispensable for cell division and differentiation, yet its roles in regenerating tissues are less clear. Here, we used combinations of transgenic mouse models to reveal that Sox2 haploinsufficiency (Sox2haplo) increases rather than impairs cochlear regeneration in vivo. Sox2haplo cochleae had delayed terminal mitosis and ectopic sensory cells, yet normal auditory function. Sox2haplo amplified and expanded domains of damage-induced Atoh1+ transitional cell formation in neonatal cochlea. Wnt activation via β-catenin stabilization (β-cateninGOF) alone failed to induce proliferation or transitional cell formation. By contrast, β-cateninGOF caused proliferation when either Sox2haplo or damage was present, and transitional cell formation when both were present in neonatal, but not mature, cochlea. Mechanistically, Sox2haplo or damaged neonatal cochleae showed lower levels of Sox2 and Hes5, but not of Wnt target genes. Together, our study unveils an interplay between Sox2 and damage in directing tissue regeneration and Wnt responsiveness and thus provides a foundation for potential combinatorial therapies aimed at stimulating mammalian cochlear regeneration to reverse hearing loss in humans.
Patrick J. Atkinson, Yaodong Dong, Shuping Gu, Wenwen Liu, Elvis Huarcaya Najarro, Tomokatsu Udagawa, Alan G. Cheng
Single cancer cell sequencing studies currently use randomly-selected cells, limiting correlations between genomic aberrations, morphology and spatial localization. We laser-captured microdissected single cells from morphologically-distinct areas of primary breast cancer and corresponding lymph node metastasis and performed whole-exome or deep-target sequencing of greater than 100 such cells. Two major subclones co-existed in different areas of the primary tumor, and the lymph node metastasis originated from a minor subclone in the invasive front of the primary tumor with additional copy number changes including 8q gain, but no additional point mutations in driver genes. Lack of metastasis-specific driver events lead us to assess whether other clonal and subclonal genomic aberrations pre-existing in primary tumors contribute to lymph node metastasis. Gene mutations and copy number variations analyzed in five breast cancer tissue sample sets revealed that copy number variations in several genomic regions, including areas within chromosome 1p, 8q, 9p, 12q and 20q, harboring several metastasis-associated genes, were consistently associated with lymph node metastasis. Moreover, clonal expansion was observed in an area of morphologically-normal breast epithelia, likely driven by a driver mutation and a subsequent amplification in chromosome 1q. Our study illuminates the molecular evolution of breast cancer and genomic aberrations contributing to metastases.
Li Bao, Zhaoyang Qian, Maria B. Lyng, Ling Wang, Yuan Yu, Ting Wang, Xiuqing Zhang, Huanming Yang, Nils Brünner, Jun Wang, Henrik J. Ditzel
Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the protein ATXN1, which is involved in transcriptional regulation. Although symptoms appear relatively late in life, primarily from cerebellar dysfunction, pathogenesis begins early, with brain-wide transcriptional changes detectable as early as a week after birth in SCA1 knock-in mice. Given the importance of this postnatal period for cerebellar development, we asked whether this region might be developmentally altered by mutant ATXN1. We found that expanded ATXN1 stimulates the proliferation of postnatal cerebellar stem cells in SCA1 mice. These hyper-proliferating stem cells tended to differentiate into GABAergic inhibitory interneurons rather than astrocytes; this significantly increased the GABAergic inhibitory interneuron synaptic connections, disrupting cerebellar Purkinje cell function in a non-cell autonomous manner. We confirmed the increased basket cell-Purkinje cell connectivity in human SCA1 patients. Mutant ATXN1 thus alters the neural circuitry of the developing cerebellum, setting the stage for the later vulnerability of Purkinje cells to SCA1. We propose that other late-onset degenerative diseases may also be rooted in subtle developmental derailments.
Chandrakanth Reddy Edamakanti, Jeehaeh Do, Alessandro Didonna, Marco Martina, Puneet Opal
Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERB as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid, and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous pro-inflammatory mechanism in un-challenged cells. However, REV-ERBα plays the dominant role as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation and used this to reveal how pro-inflammatory cytokines trigger rapid degradation of REV-ERα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERα protein couple the core clock to innate immunity.
Marie Pariollaud, Julie Gibbs, Thomas Hopwood, Sheila Brown, Nicola Begley, Ryan Vonslow, Toryn Poolman, Baoqiang Guo, Ben Saer, D. Heulyn Jones, James P. Tellam, Stefano Bresciani, Nicholas C.O. Tomkinson, Justyna Wojno-Picon, Anthony W.J. Cooper, Dion A. Daniels, Ryan P. Trump, Daniel Grant, William Zuercher, Timothy M. Willson, Andrew S. MacDonald, Brian Bolognese, Patricia L. Podolin, Yolanda Sanchez, Andrew S.I. Loudon, David W. Ray