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Bimodal fibrosis in a novel mouse model of bleomycin-induced usual interstitial pneumonia

ABSTRACT: A novel mouse model of induced-usual interstitial pneumonia shows bimodal fibrosis with honeycombing, similar to chronic lung disease seen with idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis is pathologically represented by usual interstitial pneumonia (UIP). Conventional bleomycin models used to study pathogenic mechanisms of pulmonary fibrosis display transient inflammation and fibrosis, so their relevance to UIP is limited. We developed a novel chronic induced-UIP (iUIP) model, inducing fibrosis in D1CC×D1BC transgenic mice by intra-tracheal instillation of bleomycin mixed with microbubbles followed by sonoporation (BMS). A bimodal fibrotic lung disease was observed over 14 wk, with an acute phase similar to nonspecific interstitial pneumonia (NSIP), followed by partial remission and a chronic fibrotic phase with honeycombing similar to UIP. In this secondary phase, we observed poor vascularization despite elevated PDGFRβ expression. γ2PF- and MMP7-positive epithelial cells, consistent with an invasive phenotype, were predominantly adjacent to fibrotic areas. Most invasive cells were Scgb1a1 and/or Krt5 positive. This iUIP mouse model displays key features of idiopathic pulmonary fibrosis and has identified potential mechanisms contributing to the onset of NSIP and progression to UIP. The model will provide a useful tool for the assessment of therapeutic interventions to oppose acute and chronic fibrosis.

SUBMITTER: Miura Y

PROVIDER: S-EPMC8572746 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionIn lung fibrosis, alveolar epithelium degenerates progressively. The goal of regenerative medicine is to aid repair and regeneration of the lost tissues in parenchyma and airways for which mobilization of tissue-resident endogenous or bone marrow-derived exogenous stem cells niches is a critical step. We used a lung injury model in mice to identify and characterize functional lung stem cells to clarify how stem cell niches counteract this degenerative process.MethodsShort term assay (STA) - Bleomycin-induced lung inflammation and fibrosis were assessed in a model of idiopathic pulmonary fibrosis in wild-type (WT), gp91phox-/- (NOX-/-), and gp91phoxMMP-12 double knockout (DKO) mice on C57Bl/6 background and Hoechst 33322 dye effluxing side population (SP) cells characterized. Long term assay (LTA) - In a bleomycin induced lung fibrosis model in C57Bl6 mice, the number of mature cells were quantified over 7, 14, and 21 days in bone marrow (BM), peripheral blood (PB), lung parenchyma (LP) and bronchoalveolar lavage (BAL) fluid by FACS. BrdU pulse chase experiment (10 weeks) was used to identify label retaining cells (LRC). BrdU+ and BrdU- cells were characterized by hematopoietic (CD45+), pluripotency (TTF1+, Oct3/4+, SSEA-3+, SSEA-4+, Sca1+, Lin-, CD34+, CD31+), and lung lineage-specific (SPC+, AQP-5+, CC-10+) markers. Clonogenic potential of LRCs were measured by CFU-c assays.ResultsSTA- In lung, cellularity increased by 5-fold in WT and 6-fold in NOX-/- by d7. Lung epithelial markers were very low in expression in all SP flow sorted from lung of all three genotypes cultured ex vivo. (p < 0.01). Post-bleomycin, the SP in NOX-/- lung increased by 3.6-fold over WT where it increased by 20-fold over controls. Type I and II alveolar epithelial cells progressively diminished in all three genotypes by d21 post-bleomycin. D7 post-bleomycin, CD45+ cells in BALf in NOX-/- was 1.7-fold > WT, 57% of which were Mf that decreased by 67% in WT and 83% in NOX-/- by d21.LTA- Cellularity as a factor of time remained unchanged in BM, PB, LP and BAL fluid. BrdU+ (LRC) were the putative stem cells. BrdU+CD45+ cells increased by 0.7-fold and SPC+CC10+ bronchoalveolar stem cells (BASC), decreased by ~40-fold post-bleomycin. BrdU+VEGF+ cells decreased by 1.8-fold while BrdU-VEGF+ cells increased 4.6-fold. Most BrdU- cells were CD45-. BrdU- BASCs remained unchanged post-bleomycin. CFU-c of the flow-sorted BrdU+ cells remained similar in control and bleomycin-treated lungs.ConclusionSTA- Inflammation is a pre-requisite for fibrosis; SP cells, being the putative stem cells in the lungs, were increased (either by self renewal or by recruitment from the exogenous bone marrow pool) post-bleomycin in NOX-/- but not in DKO indicating the necessity of cross-talk between gp91phox and MMP-12 in this process; ex vivo cultured SP progressively lose pluripotent markers, notably BASC (SPC+CC10+) - significance is unknown. LTA- The increase in the hematopoietic progenitor pool in lung indicated that exogenous progenitors from circulation contribute to lung regeneration. Most non-stem cells were non-hematopoietic in origin indicating that despite tissue turnover, BASCs are drastically depleted possibly necessitating recruitment of progenitors from the hematopoietic pool. Loss of VEGF+ LRC may indicate a signal for progenitor mobilization from niches. BrdU- BASC population may be a small quiescent population that remains as a reserve for more severe lung injury. Increase in VEGF+ non-LRC may indicate a checkpoint to counterbalance the mobilization of VEGF+ cells from the stem cell niche.

Project description:Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the two FDA-approved drugs for IPF treatment; however, the exact pathophysiological mechanisms of fibrosis progression and response to therapy are still poorly understood. In this work, the molecular fingerprint of fibrosis progression and response to nintedanib treatment have been investigated by mass spectrometry-based bottom-up proteomics in paraffin-embedded lung tissues from bleomycin-induced (BLM) pulmonary fibrosis mice. Our proteomics results unveiled that (i) samples clustered depending on the tissue fibrotic grade (mild, moderate, and severe) and not on the time course after BLM treatment; (ii) the dysregulation of different pathways involved in fibrosis progression such as the complement coagulation cascades, advanced glycation end products (AGEs) and their receptors (RAGEs) signaling, the extracellular matrix-receptor interaction, the regulation of actin cytoskeleton, and ribosomes; (iii) Coronin 1A (Coro1a) as the protein with the highest correlation when evaluating the progression of fibrosis, with an increased expression from mild to severe fibrosis; and (iv) a total of 10 differentially expressed proteins (padj-value ≤ 0.05 and Fold change ≤-1.5 or ≥1.5), whose abundance varied in the base of the severity of fibrosis (mild and moderate), were modulated by the antifibrotic treatment with nintedanib, reverting their trend. Notably, nintedanib significantly restored lactate dehydrogenase B (Ldhb) expression but not lactate dehydrogenase A (Ldha). Notwithstanding the need for further investigations to validate the roles of both Coro1a and Ldhb, our findings provide an extensive proteomic characterization with a strong relationship with histomorphometric measurements. These results unveil some biological processes in pulmonary fibrosis and drug-mediated fibrosis therapy.

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Bimodal fibrosis in a novel mouse model of bleomycin-induced usual interstitial pneumonia

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