Cub domain-containing protein 1 (CDCP1) is a surface protein highly expressed on the surface of many cancer cells, however, the distribution of CDCP1 in normal tissues and its potential roles in non-tumor cells are poorly understood. We previously reported that CDCP1 interacts with CD6, a surface marker of T cells, suggesting that it is a novel immunoregulator, but the physiological significance of the newly discovered CDCP1-CD6 interaction remains unclear. In this report, we found that CDCP1 is present on both human and mouse retinal pigmented epithelial cells (RPEs), a component of the blood-retina barrier (BRB), using a new anti-CDCP1 monoclonal antibody that we developed. CDCP1 knockout (KO) mice on two different genetic backgrounds both developed significantly attenuated retinal T cell infiltration and uveitis after adoptive transfer of pre-activated pathogenic T cells in a model of autoimmune uveitis. We also found that tight junctions were severely disrupted with infiltrating T cells detected in the RPE flat mounts prepared from the WT but not CDCP1 KO mice during EAU development. Mechanistically, we discovered that CDCP1 on RPE was upregulated by IFNγ in vitro and after EAU induction in vivo. CD6 stimulation induced significantly increased RPE barrier permeability of WT, but not CDCP1 knockdown (KD) RPE, and activated T cells migrated through the WT RPE monolayes more efficiently than the CDCP1 KD RPE monolayers. In addition, CD6 stimulation of WT, but not the CDCP1 KD RPEs, induced massive stress fiber formation and focal adhesion disruption to reduce cell barrier tight junctions. These data suggest that CDCP1 on RPEs interacts with CD6 on T cells to induce RPE cytoskeleton remodeling and focal adhesion disruption, which open up the tight junctions to facilitate T cell infiltration for the development of uveitis.
Lingjun Zhang, Nozha Borjini, Yu Lun, Sweta Parab, Gospel Enyindah-Asonye, Rupesh Singh, Brent A. Bell, Vera L. Bonilha, Andrei I. Ivanov, David A. Fox, Rachel R. Caspi, Feng Lin
Chitinase 3-like 1 (CHI3L1) is the prototypic chitinase-like protein mediating inflammation, cell proliferation, and tissue remodeling. Limited data suggests CHI3L1 is elevated in human pulmonary arterial hypertension (PAH) and is associated with disease severity. Despite its importance as a regulator of injury/repair responses, the relationship between CHI3L1 and pulmonary vascular remodeling is not well understood. We hypothesize that CHI3L1 and its signaling pathways contribute to the vascular remodeling responses that occur in pulmonary hypertension (PH). We examined the relationship of plasma CHI3L1 levels and severity of PH in patients with various forms of PH, including Group 1 PAH and Group 3 PH, and found that circulating levels of serum CHI3L1 were associated with worse hemodynamics and correlated directly with mean pulmonary artery pressure and pulmonary vascular resistance. We also used transgenic mice with constitutive knockout and inducible overexpression of CHI3L1 to examine its role in hypoxia-, monocrotaline-, and bleomycin-induced models of pulmonary vascular disease. In all 3 mouse models of pulmonary vascular disease, pulmonary hypertensive responses were mitigated in CHI3L1 null mice and accentuated in transgenic mice that overexpress CHI3L1. Finally, CHI3L1 alone was sufficient to induce pulmonary arterial smooth muscle cell proliferation, inhibit pulmonary vascular endothelial cell apoptosis, induce the loss of endothelial barrier function, and induce endothelial-to-mesenchymal transition. These findings demonstrate that CHI3L1 and its receptors play an integral role in pulmonary vascular disease pathobiology and may offer a novel target for the treatment PAH and PH associated with fibrotic lung disease.
Xiuna Sun, Erika Nakajima, Carmelissa Norbrun, Parand Sorkhdini, Alina Xiaoyu Yang, Dongqin Yang, Corey E. Ventetuolo, Julie Braza, Alexander Vang, Jason Aliotta, Debasree Banerjee, Mandy Pereira, Grayson Baird, Qing Lu, Elizabeth O. Harrington, Sharon Rounds, Chun Geun Lee, Hongwei Yao, Gaurav Choudhary, James R. Klinger, Yang Zhou
BACKGROUND. Systemic sclerosis (SSc) is an autoimmune, connective tissue disease characterized by vasculopathy and fibrosis of the skin and internal organs. METHODS. We randomized 15 participants with early diffuse cutaneous SSc to tofacitinib 5 mg twice a day or matching placebo in a Phase I/II double-blind, placebo-controlled trial. The primary outcome measure was safety and tolerability at or before Week 24. In order to understand the changes in gene expression associated with tofacitinib treatment in each skin cell populations, we compared single cell gene expression in punch skin biopsies obtained at baseline and 6 weeks following the initiation of treatment. RESULTS. Tofacitinib was well tolerated; there were no participants, who experienced Grade 3 or higher adverse effects (AEs) before or at Week 24. Trends in efficacy outcome measures favored tofacitnib. Baseline gene expression in fibroblast and keratinocyte subpopulations indicates interferon (IFN) activated gene expression. Tofacitinib inhibited IFN-regulated gene expression in the SFRP2/DPP4 fibroblasts (progenitors of myofibroblasts) and MYOC and CCL19, representing adventitial fibroblasts (p< 0.05), as well as in the basal and keratinized layers of the epidermis. Gene expression in macrophages and dendritic cells indicated inhibition of STAT3 by tofacitinib (p<0.05). No clinically meaningful inhibition of T cells and endothelial cells in the skin tissue was observed. CONCLUSION. These results indicate that mesenchymal and epithelial cells of a target organ in SSc, not the infiltrating lymphocytes, may be the primary focus for therapeutic effects of a janus kinase inhibitor. TRIAL REGISTRATION. clinicaltrials.gov NCT03274076. FUNDING SOURCE. This was an investigator-initiated trial designed by the Sponsor and the steering committee. The industry funder, Pfizer, had no role in collecting, analyzing, and interpreting the data. The manuscript was drafted by the authors and was reviewed by Pfizer Inc. before final submission. No medical writer was involved in creating the manuscript. DK was supported by NIH/NIAMS R01 AR070470 and NIH/NIAMS K24 AR063120. JMK, JEG, LCT are supported by the Taubman Medical Research Institute and NIH-P30 AR075043. LCT was also supported by NIH/NIAMS K01AR072129. The corresponding author had full access to all data congregates in the study and made the final decision to submit the manuscript for publication.
Dinesh Khanna, Cristina M. Padilla, Lam C. Tsoi, Vivek Nagaraja, Puja Khanna, Tracy Tabib, J. Michelle Kahlenberg, Amber Young, Suiyuan Huang, Johann e. Gudjonsson, David A. Fox, Robert Lafyatis
B-lymphocytes have long been recognized for their critical contributions to adaptive immunity, providing defense against pathogens through cognate antigen presentation to T cells and antibody production. More recently appreciated is that B cells are also integral in securing self-tolerance; this has led to interest in their therapeutic application to down-regulate unwanted immune responses such as transplant rejection. In this study, we found that PMA and ionomycin-activated mouse B cells acquire regulatory properties following stimulation through Toll-like receptor (TLR)4/TLR9 receptors (Bregs-TLR). Bregs-TLR efficiently inhibited T cell proliferation in vitro and prevented allograft rejection. Unlike most reported Breg activities, the inhibition of alloimmune responses by Bregs-TLR relied on the expression of TGF-βand not IL-10. In vivo, Bregs-TLR interrupted donor-specific T cell expansion and induced regulatory T cells in a TGF-β dependent manner. RNA-seq analyses corroborated the involvement of TGF-β pathways in Breg-TLR function, identified potential gene pathways implicated in preventing graft rejection, and suggested new targets to foster Breg regulation.
Kang Mi Lee, Qiang Fu, Guoli Huai, Kevin Deng, Ji Lei, Lisa Kojima, Divyansh Agarwal, Peter Van Galen, Shoko Kimura, Naoki Tanimine, Laura Washburn, Heidi Yeh, Ali Naji, Charles G. Rickert, Christian LeGuern, James F. Markmann
Junctional adhesion molecule-A (JAM-A) is expressed in several cell types including epithelial and endothelial cells as well as some leukocytes. In intestinal epithelial cells (IEC), JAM-A localizes to cell junctions and plays a role in regulating barrier function. In vitro studies with model cell lines have shown that JAM-A contributes to IEC migration, however in vivo studies investigating the role of JAM-A in cell migration-dependent processes such as mucosal wound repair have not been performed. In this study, we developed an inducible intestinal epithelial-specific JAM-A knockdown mouse model (Jam-aER-ΔIEC). While acute induction of IEC-specific loss of JAM-A did not result in spontaneous colitis, such mice had significantly impaired mucosal healing after chemically induced colitis and after biopsy colonic wounding. In vitro primary cultures of JAM-A deficient IEC demonstrated impaired migration in wound healing assays. Mechanistic studies revealed that JAM-A stabilizes formation of protein signaling complexes containing Rap1A/Talin/β1-integrin at focal adhesions of migrating IECs. Loss of JAM-A in primary IEC led to decreased Rap1A activity and protein levels of Talin and β1-integrin and a reduction in focal adhesion structures. These findings suggest that epithelial JAM-A plays a critical role in controlling mucosal repair in vivo through dynamic regulation of focal adhesions
Shuling Fan, Kevin Boerner, Chithra K. Muraleedharan, Asma Nusrat, Miguel Quiros, Charles A. Parkos
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