An ST2‐dependent role of bone marrow‐derived group 2 innate lymphoid cells in pulmonary fibrosis

Recent evidence supports that bone marrow (BM)‐derived hematopoietic progenitor cells play an important role in lung injury and fibrosis. While these cells give rise to multiple cell types, the ST2 (Il1rl1)‐expressing group 2 innate lymphoid cells (ILC2s) derived from BM progenitors have been implicated in tissue repair and remodeling, including in lung fibrosis. To further investigate the precise role of BM‐derived ILC2s in the pathogenesis of fibrotic lung disease, their importance in the bleomycin‐induced lung fibrosis model was evaluated by analyzing the effects of selective ST2 deficiency in the BM compartment. The results showed that while ST2‐sufficient control mice exhibited activation of lung IL‐33/ST2 signaling, ILC2 recruitment, IL‐13 induction, and fibrosis, these responses were significantly diminished in ST2‐deficient‐BM chimera mice, with selective loss of ST2 expression only in the BM. This diminished response to bleomycin was similar to that seen in ST2 global knockout mice, suggesting the predominant importance of ST2 from the BM compartment. In wild‐type mice, ILC2 recruitment to the lung was accompanied by a concomitant decrease in ST2+ BM cells. ST2‐deficient BM cells were unresponsive to IL‐33‐induced ILC2 maturation. Finally, lineage‐negative wild‐type, but not ST2‐deficient BM cells from bleomycin‐treated mice stimulated lung fibroblast type I collagen expression, which was associated with elevated TGFβ expression in the BM cells. Taken together, these findings suggested that the BM‐derived ILC2s were recruited to fibrotic lung through the IL‐33/ST2 pathway, and contributed to fibroblast activation to promote lung fibrosis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145267/1/path5092.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145267/2/path5092_am.pd

B7H3-dependent myeloid-derived suppressor cell recruitment and activation in pulmonary fibrosis

http://deepblue.lib.umich.edu/bitstream/2027.42/175995/2/2022_MDSC-B7H3_TJ_Frontiers in Immunol.pdfPublished versionDescription of 2022_MDSC-B7H3_TJ_Frontiers in Immunol.pdf : Published versio

Conditional Knockout of Telomerase Reverse Transcriptase in Mesenchymal Cells Impairs Mouse Pulmonary Fibrosis.

Telomerase is typically expressed in cellular populations capable of extended replication, such as germ cells, tumor cells, and stem cells, but is also induced in tissue injury, repair and fibrosis. Its catalytic component, telomerase reverse transcriptase (TERT) is induced in lung fibroblasts from patients with fibrotic interstitial lung disease and in rodents with bleomycin-induced pulmonary fibrosis. To evaluate the fibroblast specific role of TERT in pulmonary fibrosis, transgenic mice bearing a floxed TERT allele were generated, and then crossed with an inducible collagen α2(I)-Cre mouse line to generate fibroblast specific TERT conditional knockout mice. TERT-specific deficiency in mesenchymal cells caused attenuation of pulmonary fibrosis as manifested by reduced lung hydroxyproline content, type I collagen and α-smooth muscle actin mRNA levels. The TERT-deficient mouse lung fibroblasts displayed decreased cell proliferative capacity and higher susceptibility to induced apoptosis compared with control cells. Additionally TERT deficiency was associated with heightened α-smooth muscle actin expression indicative of myofibroblast differentiation. However the impairment of cell proliferation and increased susceptibility to apoptosis would cause a reduction in the myofibroblast progenitor population necessary to mount a successful myofibroblast-dependent fibrotic response. These findings identified a key role for TERT in fibroblast proliferation and survival essential for pulmonary fibrosis

TERT and telomerase in floxed TERT vs WT mice.

<p>Total RNA or protein lysates were prepared from the lung tissues and MLF from BLM or PBS-injected TERT <i>fl/fl</i> or WT mice. TERT gene expression was analyzed by qRT-PCR and expressed as 2^-ΔΔCT (n = 3) in (A), and the telomerase activities were detected by TRAP-ELISA kit and expressed as fold change over their PBS control, respectively (B). n = 3. *, P < 0.05.</p

The impairment of pulmonary fibrosis in TERT CKO mice.

<p>(A) The MLF were isolated from TERT CKO or WT mice at 21 days after BLM injection, and analyzed for TERT mRNA by qRT-PCR. The expression was expressed as the fold change of the level in PBS-treated WT MLF. n = 3–5 mice per group. (B) The lungs from TERT CKO and control mice were homogenized at day 21 after BLM treatment, and measured for whole lung collagen content by HYP assay. n = 3–5 mice per group. (C) Lung tissue RNA extracted from the indicated murine strains was also analyzed for type I collagen mRNA by qRT-PCR. n = 3–5 mice per group. (D) Lung tissue lysates were prepared by RIPA buffer, and analyzed for α-SMA protein in the indicated murine strain by Western blotting (top panel). Quantitative data was normalized by the internal control GAPDH, and shown as the percentage of the GAPDH signals (bottom panel). n = 3–4 mice per group. (E) Representative H & E stained lung tissue sections at day 21after BLM treatment are shown. Original magnification × 20. *, P < 0.05.</p

Generation and verification of the TERT CKO mice.

<p>(A) Schematic gene-targeting map of TERT gene. The construction of TERT floxed and TERT CKO alleles are shown before and after tamoxifen treatment. TERT gene exon 1–2 was floxed after recombination between WT and targeted alleles. Primers pairs P1/P2 and P3/P5 were used to detect floxed TERT alleles. Primer pair P1/P5 was used to detected the TERT gene excision by Cre. (B) Representative Southern blot analysis. The ES clone with homologous recombination was digested with the restriction enzyme Bsr GI followed by Southern blotting with Dig-labeled 5’ probe as shown in (A). The detected WT and targeted alleles are 14.58 and 7.3 kb, respectively. (C) PCR genotyping using genomic DNA from mouse tails. The PCR fragments for WT was 154bp, for TERT fl/+ they were 154 and 343 bp, for TERT <i>fl/fl</i> it was 343 bp, and for TERT CKO it was 215bp after tamoxifen-induced Cre excision. “Ladder” referred to 100 bp DNA ladder.</p

The in vitro excision of MLF TERT by Cre activation.

<p>The MLF were isolated from TERT <i>fl/fl</i> mice, and then transduced with 100 MOI of AdCre vector or AdGFP control vector. Six days after transduction, TERT mRNA (A) and telomerase (B) were analyzed, respectively, as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142547#pone.0142547.g002" target="_blank">Fig 2</a>. n = 3. (C) The MLF from TERT <i>fl/fl</i> or TERT <i>fl/fl</i>/,Cre+/- mice were treated with 5 μM of 4-OHT in vitro at the same time for 6 days, and TERT mRNA was analyzed. n = 3. *, P < 0.05.</p

Effect of TERT overexpression on α-SMA expression.

<p>BJ and BJ 5ta fibroblasts were plated in 6-well plates. The cells were starved with DMEM supplemented with 0.5% FBS for 4 hours before TGF-β1 treatment for an additional 24 for mRNA or 48 hours for protein analysis. (A) BJ and BJ5ta cells were analyzed for TERT mRNA by qRT-PCR. n = 3. *, P < 0.05. (B) The cell lysates were harvested in RIPA buffer, and analyzed for α-SMA protein expression by Western blotting. A representative blot was shown. The GAPDH was used as internal control.</p