PMID- 38223200 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20240131 IS - 2573-9832 (Electronic) IS - 2573-9832 (Linking) VI - 6 IP - 1 DP - 2024 Jan TI - Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts. PG - 12-25 LID - 10.1096/fba.2023-00054 [doi] AB - Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFbeta) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFbeta and in the bleomycin model of experimental lung fibrosis using Tollip-/- mice. We hypothesize that if TOLLIP negatively regulates TGFbeta signaling, then Tollip-/- mouse lung fibroblasts (MLFs) would have enhanced response to TGFbeta treatment, and Tollip-/- mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFbeta (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFbeta-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the in vivo model of lung fibrosis, Tollip-/- mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFbeta signaling in vivo after bleomycin injury by SMAD2, ERK1/2, and TGFbetaR1 Western blot. In response to TGFbeta treatment, both WT and Tollip-/- MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip-/- MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip-/- MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFbeta signaling in Tollip-/- through SMAD2 in vitro and in vivo. Tollip-/- mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip-/- mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury. CI - (c)2023 The Authors FASEB BioAdvances published by The Federation of American Societies for Experimental Biology. FAU - Chow, Yu-Hua AU - Chow YH AD - Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine University of Washington Seattle Washington USA. AD - Center for Lung Biology University of Washington Seattle Washington USA. FAU - Lopez-Martinez, Cecilia AU - Lopez-Martinez C AD - Instituto de Investigacion Sanitaria del Principado de Asturias Oviedo Spain. AD - Centro de Investigacion Biomedica en Red (CIBER)-Enfermedades respiratorias Madrid Spain. AD - Instituto Universitario de Oncologia del Principado de Asturias Oviedo Spain. FAU - Liles, W Conrad AU - Liles WC AD - Center for Lung Biology University of Washington Seattle Washington USA. AD - Division of Allergy and Infectious Diseases, Department of Medicine University of Washington Seattle Washington USA. FAU - Altemeier, William A AU - Altemeier WA AD - Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine University of Washington Seattle Washington USA. AD - Center for Lung Biology University of Washington Seattle Washington USA. FAU - Gharib, Sina A AU - Gharib SA AD - Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine University of Washington Seattle Washington USA. AD - Center for Lung Biology University of Washington Seattle Washington USA. FAU - Hung, Chi F AU - Hung CF AD - Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine University of Washington Seattle Washington USA. AD - Center for Lung Biology University of Washington Seattle Washington USA. LA - eng GR - R01 HL166273/HL/NHLBI NIH HHS/United States GR - R03 HL155075/HL/NHLBI NIH HHS/United States PT - Journal Article DEP - 20231129 PL - United States TA - FASEB Bioadv JT - FASEB bioAdvances JID - 101733210 PMC - PMC10782472 OTO - NOTNLM OT - TGFbeta OT - TOLLIP OT - Toll-interacting protein OT - bleomycin OT - fibroblasts OT - lung injury OT - myofibroblasts OT - transforming growth factor beta COIS- The author(s) meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE). This was an independent, investigator-initiated study supported by Boehringer Ingelheim Pharmaceuticals, Inc. (BIPI). BIPI had no role in the design, analysis, or interpretation of the results in this study; BIPI was given the opportunity to review the manuscript for medical and scientific accuracy as it relates to BIPI substances, as well as intellectual property considerations. EDAT- 2024/01/15 06:42 MHDA- 2024/01/15 06:43 PMCR- 2023/11/29 CRDT- 2024/01/15 04:43 PHST- 2023/05/22 00:00 [received] PHST- 2023/11/20 00:00 [revised] PHST- 2023/11/22 00:00 [accepted] PHST- 2024/01/15 06:43 [medline] PHST- 2024/01/15 06:42 [pubmed] PHST- 2024/01/15 04:43 [entrez] PHST- 2023/11/29 00:00 [pmc-release] AID - FBA21421 [pii] AID - 10.1096/fba.2023-00054 [doi] PST - epublish SO - FASEB Bioadv. 2023 Nov 29;6(1):12-25. doi: 10.1096/fba.2023-00054. eCollection 2024 Jan.