Galectin-1 inhibition attenuates profibrotic signaling in hypoxia-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by lung remodeling arising from epithelial injury, aberrant fibroblast growth, and excessive deposition of extracellular matrix. Repeated epithelial injury elicits abnormal wound repair and lung remodeling, often associated with alveolar collapse and edema, leading to focal hypoxia. Here, we demonstrate that hypoxia is a physiological insult that contributes to pulmonary fibrosis (PF) and define its molecular roles in profibrotic activation of lung epithelial cells. Hypoxia increased transcription of profibrotic genes and altered the proteomic signatures of lung epithelial cells. Network analysis of the hypoxic epithelial proteome revealed a crosstalk between transforming growth factor-ß1 and FAK1 (focal adhesion kinase-1) signaling, which regulated transcription of galectin-1, a profibrotic molecule. Galectin-1 physically interacted with and activated FAK1 in lung epithelial cells. We developed a novel model of exacerbated PF wherein hypoxia, as a secondary insult, caused PF in mice injured with subclinical levels of bleomycin. Hypoxia elevated expression of phosphorylated FAK1, galectin-1, and α-smooth muscle actin and reduced caspase-3 activation, suggesting aberrant injury repair. Galectin-1 inhibition caused apoptosis in the lung parenchyma and reduced FAK1 activation, preventing the development of hypoxia-induced PF. Galectin-1 inhibition also attenuated fibrosis-associated lung function decline. Further, galectin-1 transcript levels were increased in the lungs of IPF patients. In summary, we have identified a profibrotic role of galectin-1 in hypoxia signaling driving PF.

Hematopoietic not systemic impairment of Roquin expression accounts for intestinal inflammation in Roquin-deficient mice.

Roquin, an E3 ligase, is involved in curtailing autoimmune pathology as seen from studies using mice with mutated (Rc3h1(san/san)) or disrupted (Rc3h1(gt/gt)) Rc3h1 gene. The extent to which intestinal immunopathology is caused by insufficient Roquin expression in the immune system, or by Roquin impairment in non-hematopoietic cells, has not been determined. Using bone marrow cells from Rc3h1(gt/gt) mice transferred into irradiated normal mice (Rc3h1(gt/gt) → NL chimeras), we show that inflammation developed in the small intestine, kidney, lung, liver, and spleen. Proinflammatory cytokine levels were elevated in lamina propria lymphocytes (LPLs). Inflammation in the liver was accompanied by areas of hepatocyte apoptosis. Lung inflammation consisted of an influx of both T cells and B cells. Small intestinal LPLs had increased numbers of CD44(hi), CD62L(lo), KLRG1(+), ICOS(+) short-lived effector cells, indicating an influx of activated T cells. Following oral infection with L. monocytogenes, Rc3h1(gt/gt) → NL chimeras had more liver pathology and greater numbers of bacteria in the Peyer's patches than NL → NL chimeras. These findings demonstrate that small intestinal inflammation in Rc3h1(san/san) and Rc3h1(gt/gt) mice is due to a failure of Roquin expression in the immune system and not to insufficient systemic Roquin expression.

Small intestine inflammation in Roquin-mutant and Roquin-deficient mice.

Roquin, an E3 ubiquitin ligase that localizes to cytosolic RNA granules, is involved in regulating mRNA stability and translation. Mice that have a M199R mutation in the Roquin protein (referred to as sanroque or Roquin(san/san) mice) develop autoimmune pathologies, although the extent to which these occur in the intestinal mucosa has not been determined. Here, we demonstrate that Roquin(san/san) mice reproducibly develop intestinal inflammation in the small intestine but not the colon. Similarly, mice generated in our laboratory in which the Roquin gene was disrupted by insertion of a gene trap cassette (Roquin(gt/gt) mice) had small intestinal inflammation that mimicked that of Roquin(san/san) mice. MLN cells in Roquin(san/san) mice consisted of activated proliferating T cells, and had increased numbers of CD44(hi) CD62L(lo) KLRG1(+) short-lived effector cells. Proportionally more small intestinal intraepithelial lymphocytes in Roquin(san/san) mice expressed the ICOS T cell activation marker. Of particular interest, small intestinal lamina propria lymphocytes in Roquin(san/san) mice consisted of a high proportion of Gr-1(+) T cells that included IL-17A(+) cells and CD8(+) IFN-γ(+) cells. Extensive cytokine dysregulation resulting in both over-expression and under-expression of chemotactic cytokines occurred in the ileum of Roquin(san/san) mice, the region most prone to the development of inflammation. These findings demonstrate that chronic inflammation ensues in the intestine following Roquin alteration either as a consequence of protein mutation or gene disruption, and they have implications for understanding how small intestinal inflammation is perpetuated in Crohn's disease (CD). Due to the paucity of animal models of CD-like pathophysiology in the small intestine, and because the primary gene/protein defects of the Roquin animal systems used here are well-defined, it will be possible to further elucidate the underlying genetic and molecular mechanisms that drive the disease process.