Although recent evidence has shown that IL‐6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL‐6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1‐XBP1 expansion pathway, IL‐4/IL‐13 and IL‐4/IL‐13/IL‐6‐mediated alternative programming of murine bone marrow‐derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase‐1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1‐XBP1 arm was achieved using STF‐083010 and was verified by RT‐PCR. The addition of IL‐6 to the conventional alternative programming cocktail IL‐4/IL‐13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response‐mediated induction of molecular chaperones. IRE1‐XBP1 inhibition substantially reduced the IL‐6‐mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL‐6 enhances ER expansion and the profibrotic capacity of IL‐4/IL‐13‐mediated activation of macrophages. Therapeutic strategies targeting IL‐6 or the IRE1‐XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages.
23Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and 24 excessive extracellular matrix deposition. Endoplasmic reticulum (ER) stress initiates the unfolded 25 protein response (UPR), a cellular stress response pathway that has been implicated in both 26 inflammatory and fibrotic processes. Here, we sought to investigate the role of the 13 kDa FK506-27 binding protein (FKBP13), an ER stress-inducible molecular chaperone, in various forms of pulmonary 28 fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy samples 29 from 24 patients with idiopathic pulmonary fibrosis (IPF) and 17 control subjects. FKBP13 expression 30 was found to be elevated in the fibrotic regions of IPF lung tissues, and within this cohort, was 31 correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also 32 increased in lung biopsies of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and 33 sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using 34 FKBP13 -/mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function 35 and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 36 21) and resolution (Day 50) phase. FKBP13 -/mice showed increased infiltration of inflammatory cells 37 and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of 38 fibrosis at Day 50. These changes were associated with an increased number of cells that stained 39 positive for TUNEL and cleaved caspase 3 in the FKBP13 -/lungs, indicating a heightened cellular 40 sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity or 41 progression of interstitial lung diseases, and that it has a biologically relevant role in protecting mice 42 against bleomycin-induced injury, inflammation and fibrosis. 43 following the description provided by Reyfman et al. (2018) [19] using Seurat [20] package in R. t-131 distributed stochastic neighbor embedding (tSNE plot), expression plot and violin plots were created 132 using Seurat package. Cell populations were defined by using the genes reported to be differentially 133 expressed between the cell populations [19]. 134 Animal Experiments: All animal work was approved by the Animal Research Ethics Board of 135 McMaster University (Hamilton, ON, Canada) under protocol number 12.02.06. Male C57BL6/J mice 136 aged 10-12 weeks were bred and housed at the McMaster University Central Animal Facility (CAF, 137Hamilton, ON, Canada). Animals were kept on a 12-hour light/dark cycle at a controlled temperature of 138 20-25°C and ambient humidity of ~50%. The animals were allowed access to food and water and were 139 fed ad libitum. 140
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