Myofibroblast de-differentiation proceeds via distinct transcriptomic and phenotypic transitions

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Abstract

Myofibroblasts are the major cellular source of collagen, and their accumulation – via differentiation from fibroblasts and resistance to apoptosis – is a hallmark of tissue fibrosis. Clearance of myofibroblasts by de-differentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to de-differentiate myofibroblasts, but the resultant cellular phenotypes have neither been comprehensively characterized nor compared. Here we show that PGE2 elicited de-differentiation of human lung myofibroblasts via cAMP/PKA while FGF2 utilized MEK/ERK. The two mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast de-differentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with that occurring in vivo most closely resembling the former.

Authors

Sean M. Fortier, Loka R. Penke, Dana M. King, Tho X. Pham, Giovanni Ligresti, Marc Peters-Golden

TLR-2-mediated activation of innate responses in the upper airways confers anti-viral protection of the lungs

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Abstract

The impact of respiratory virus infections on global health is felt not just during a pandemic but for many, endemic seasonal infections pose an equal and ongoing risk of severe disease. Moreover, vaccines and antiviral drugs are not always effective or available for many respiratory viruses. We investigated how induction of effective and appropriate antigen independent innate immunity in the upper airways can prevent spread of respiratory virus infection to the vulnerable lower airways. Activation of Toll-like receptor-2 (TLR2), when restricted to the nasal turbinates results in prompt induction of innate immune-driven anti-viral responses through action of cytokines, chemokines and cellular activity in the upper but not the lower airways. We define how nasal epithelial cells and recruitment of macrophages work in concert and play pivotal roles to limit progression of influenza virus to the lungs and sustain protection for up to seven days. These results reveal underlying mechanisms of how control of viral infection in the upper airways can occur and also support the implementation of strategies that can activate TLR2 in nasal passages to provide rapid protection, especially for at-risk populations, against severe respiratory infection when vaccines and antiviral drugs are not always effective or available.

Authors

Georgia Deliyannis, Chinn Yi Wong, Hayley A. McQuilten, Annabell Bachem, Michele V. Clarke, Xiaoxiao Jia, Kylie Horrocks, Weiguang Zeng, Jason Girkin, Nichollas E. Scott, Sarah L. Londrigan, Patrick C. Reading, Nathan W. Bartlett, Katherine Kedzierska, Lorena E. Brown, Francesca A. Mercuri, Christophe Demaison, David C. Jackson, Brendon Y. Chua

PDLIM2 repression by ROS in alveolar macrophages promotes lung tumorigenesis

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Abstract

One of the most fundamental and challenging questions in the cancer field is how immunity is transformed from tumor immunosurveillance to tumor-promoting inflammation. Here, we identify the tumor suppressor PDZ-LIM domain-containing protein 2 (PDLIM2) as a checkpoint of alveolar macrophages (AMs) important for lung tumor suppression. During lung tumorigenesis, PDLIM2 expression in AMs is down-regulated by reactive oxygen species (ROS)-activated transcription repressor BTB and CNC homology 1 (BACH1). PDLIM2 down-regulation leads to constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3), driving AM pro-tumorigenic polarization/activation and differentiation from monocytes attracted from the circulation to suppress cytotoxic T lymphocytes (CTLs) and promote lung cancer. PDLIM2 down-regulation also decreases AM phagocytosis. These findings establish ROS/BACH1/PDLIM2/STAT3 as a signaling pathway driving AMs for lung tumor promotion.

Authors

Liwen Li, Fan Sun, Lei Han, Xujie Liu, Yadong Xiao, Alyssa D. Gregory, Steven D. Shapiro, Gutian Xiao, Zhaoxia Qu

Autophagy stimulation reduces ocular hypertension in murine glaucoma model via autophagic degradation of mutant myocilin

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Abstract

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with Primary Open Angle Glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic cause of POAG. We have previously shown that mutant myocilin accumulates in the endoplasmic reticulum (ER) and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activates autophagy but it is functionally impairecd in cultured human trabecular meshwork (TM) cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsens mutant myocilin accumulation and exacerbates IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy is associated with chronic ER stress-induced transcriptional factor, CHOP. Deletion of CHOP corrects impaired autophagy, enhances recognition and degradation of mutant myocilin by autophagy,and reduces glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via Tat-beclin 1 peptide or torin 2, promotes autophagic degradation of mutant myocilin and reduces elevated IOP in Tg-MYOCY437H mice. Together, our studies provide a novel treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.

Authors

Ramesh B. Kasetti, Prabhavathi Maddineni, Charles C. Kiehlbauch, Shruti Patil, Charles C. Searby, Beth Levine, Val C. Sheffield, Gulab S. Zode

Adipocyte-derived extracellular vesicles regulate survival and function of pancreatic β-cells

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Abstract

Extracellular vesicles (EVs) are implicated in the crosstalk between adipocytes and other metabolic organs, and an altered biological cargo has been observed in EVs from human obese adipose tissue (AT). Yet, the role of adipocyte-derived EVs in pancreatic β-cells remains to be determined. Here, we explored the effects of EVs, released from both rodent and human isolated adipocytes and human AT explants, on survival and function of pancreatic β-cells and human pancreatic islets. EVs from healthy 3T3-L1 adipocytes increased survival and proliferation and promoted insulin secretion in INS-1E β-cells and human pancreatic islets, both untreated or exposed to cytokines or glucolipotoxicity, while EVs from inflamed adipocytes caused β-cell death and dysfunction. Human lean adipocyte-derived EVs produced similar beneficial effects, while EVs from obese AT explants were harmful for human EndoC-βH3 β-cells. We observed differential expression of microRNAs in EVs from healthy and inflamed adipocytes, as well as alteration in signaling pathways and expression of β-cell genes, adipokines and CKs in recipient β-cells. These in vitro results suggest that, depending on the physiopathological state of AT, adipocyte-derived EVs may influence β-cell fate and function.

Authors

Iacopo Gesmundo, Barbara Pardini, Eleonora Gargantini, Giacomo Gamba, Giovanni Birolo, Alessandro Fanciulli, Dana Banfi, Noemi Congiusta, Enrica Favaro, Maria Chiara Deregibus, Gabriele Togliatto, Gaia Zocaro, Maria Felice Brizzi, Raul M. Luque, Justo P. Castaño, Maria Alessandra Bocchiotti, Simone Arolfo, Stefania Bruno, Rita Nano, Mario Morino, Lorenzo Piemonti, Huy Ong, Giuseppe Matullo, Juan M. Falcón-Pérez, Ezio Ghigo, Giovanni Camussi, Riccarda Granata

Complex consequences of Cantu Syndrome SUR2 variant R1154Q in genetically modified mice

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Abstract

Cantu Syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by ~30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of human SUR2[R1154Q] mutation to the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed an underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed divergent transcripts, one encoding full length SUR2 protein, and the other with in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in ~40% and ~90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in non-functional channels. SUR2[R1154Q] CS patient tissue and iPSC-derived cardiomyocytes showed only full length SUR2 transcripts, although further studies will be required to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.

Authors

Haixia Zhang, Alex M. Hanson, Tobias U. Scherf de Almeida, Christopher H. Emfinger, Conor McClenaghan, Theresa Harter, Zihan Yan, Paige E. Cooper, G. Schuyler Brown, Eric C. Arakel, Robert P. Mecham, Attila Kovacs, Carmen M. Halabi, Blanche Schwappach, Maria S. Remedi, Colin G. Nichols

Dromedary camels as a natural source of neutralizing nanobodies against SARS-CoV-2

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Abstract

The development of prophylactic and therapeutic agents for coronavirus disease 2019 (COVID-19) is a current global health priority. Here, we investigated the presence of cross-neutralizing antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in dromedary camels that were Middle East respiratory syndrome (MERS)-CoV-seropositive but MERS-CoV-free. The tested 229 dromedaries had anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins, including the S trimer, M, N, and E proteins. Using SARS-CoV-2 competitive immunofluorescence immunoassays and pseudovirus neutralization assays, we found medium-to-high titers of cross-neutralizing antibodies against SARS-CoV-2 in these animals. Through linear B cell epitope mapping using phage immunoprecipitation sequencing and a SARS-CoV-2 peptide/proteome microarray, we identified a large repertoire of betacoronavirus cross-reactive antibody specificities in these dromedaries and demonstrated that the SARS-CoV-2-specific VHH antibody repertoire is qualitatively diverse. This analysis revealed not only several SARS-CoV-2 epitopes that are highly immunogenic in humans, including a neutralizing epitope, but also epitopes exclusively targeted by camel antibodies. The identified SARS-CoV-2 cross-neutralizing camel antibodies are not proposed as a potential treatment for COVID-19. Rather, their presence in non-immunized camels supports the development of SARS-CoV-2 hyperimmune camels, which could be a prominent source of therapeutic agents for the prevention and treatment of COVID-19.

Authors

Lotfi Chouchane, Jean-Charles Grivel, Elmoubasher Farag, Igor Pavlovski, Selma Maacha, Abbirami Sathappan, Hamad Al-Romaihi, Sirin Abuaqel, Manar Ata, Aouatef Ismail Chouchane, Sami Remadi, Najeeb M. Halabi, Arash Rafii, Mohammed Al-Thani, Nico Marr, Murugan Subramanian, Jingxuan Shan

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size

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Abstract

Limitations in cell proliferation are important for normal function of differentiated tissues, and essential for the safty of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion and determined growth potential, differentiation and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S-phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Authors

Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V. Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S. Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli

Epithelial IL-33 appropriates exosome trafficking for secretion in chronic airway disease

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Abstract

Interleukin-33 (IL-33) is a key mediator of chronic airway disease driven by type-2 immune pathways, yet the non-classical secretory mechanism for this cytokine remains undefined. We performed a comprehensive analysis in human airway epithelial cells, which revealed that tonic IL-33 secretion is dependent on the ceramide biosynthetic enzyme neutral sphingomyelinase 2 (nSMase2). IL-33 is co-secreted with exosomes by the nSMase2-regulated multivesicular endosome (MVE) pathway as surface-bound cargo. In support of these findings, human chronic obstructive pulmonary disease (COPD) specimens exhibited increased epithelial expression of the abundantly secreted IL33Δ34 isoform and augmented nSMase2 expression compared to non-COPD specimens. Using an Alternaria-induced airway disease model, we found the nSMase2 inhibitor GW4869 abrogated both IL-33 and exosome secretion as well as downstream inflammatory pathways. This work elucidates a novel aspect of IL-33 biology that may be targeted for therapeutic benefit in chronic airway diseases driven by type-2 inflammation.

Authors

Ella Katz-Kiriakos, Deborah F. Steinberg, Colin E. Kluender, Omar A. Osorio, Catie Newsom-Stewart, Arjun Baronia, Derek E. Byers, Michael J. Holtzman, Dawn Katafiasz, Kristina L. Bailey, Steven L. Brody, Mark J. Miller, Jennifer Alexander-Brett

Shared recognition of citrullinated tenascin-C peptides by T and B cells in rheumatoid arthritis

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Abstract

Tenascin-C, an extracellular matrix protein that has proinflammatory properties, is a recently described antibody target in rheumatoid arthritis. In this study, we utilized a systematic discovery process and identified five novel citrullinated tenascin-C (cit-TNC) T cell epitopes. CD4+ T cells specific for these epitopes were elevated in the peripheral blood of subjects with rheumatoid arthritis and showed signs of activation. Cit-TNC-specific T cells were also present among synovial fluid T cells and secreted interferon-γ. Two of these cit-TNC peptides were recognized by antibodies within the serum and synovial fluid of individuals with RA. Detectable serum levels of cit-TNC reactive antibodies were prevalent among subjects with RA and positively associated with cyclic citrullinated peptide (CCP) reactivity and the HLA shared epitope. Furthermore, cit-TNC reactive antibodies were correlated with rheumatoid factor and elevated in subjects with a history of smoking. Taken together this work confirms cit-TNC as an autoantigen that is targeted by autoreactive CD4+ T cells and autoantibodies in patients with RA. Furthermore, our findings suggest that a unique set of epitopes recognized by both CD4+ T cells and B cells have the potential to amplify autoimmunity and promote the development and progression of rheumatoid arthritis.

Authors

Jing Song, Anja Schwenzer, Alicia Wong, Sara Turcinov, Cliff Rims, Lorena Rodríguez-Martínez, David Arribas-Layton, Christina Gerstner, Virginia S. Muir, Kim S. Midwood, Vivianne Malmström, Eddie A. James, Jane H. Buckner