Histopathological Correlations between Mediastinal Fat-Associated Lymphoid Clusters and the Development of Lung Inflammation and Fibrosis following Bleomycin Administration in Mice

Abstract

Bleomycin (BLM) has been reported to induce lung inflammation and fibrosis in human and mice and showed genetic susceptibility. Interestingly, the C57BL/6 (B6) mice had prominent mediastinal fat-associated lymphoid cluster (MFALCs) under healthy condition, and showed susceptibility to development of lung fibrosis following BLM administration. However, the pathogenesis of lung lesion progression, and their correlation with MFALC morphologies, remain to be clarified. To investigate the correlations between MFALC structures and lung injuries in B6 mice, histopathological examination of mediastinal fat tissues and lungs was examined at 7 and 21 days (d) following a single 50 μL intranasal (i.n.) instillation of either BLM sulfate (5 mg/kg) (BLM group) or phosphate-buffered saline (control group). The lung fibrosis was examined by Masson’s trichrome (MT) stain of paraffin sections and mRNA expression levels of Col1a1, Col3a1, and Acta2 in different frozen lung samples. Furthermore, immunohistochemistry for CD3, B220, Iba1, Gr1, BrdU, LYVE-1, and peripheral node addressin (PNAd) was performed to detect T- and B-cells, macrophages, granulocytes, proliferating cells, lymph vessels (LVs), and high endothelial venules (HEVs). We found that MFALCs were more abundant in the BLM group as compared to the control group. The lung of BLM group developed pneumonitis with severe cellular infiltrations at 7 days and significant collagen deposition (MT) and higher expression of Col1a1, and Col3a1 at 21 days post-administration. Numerous immune cells, proliferating cells, HEVs, and LVs were observed in both MFALCs and lungs of the BLM group. Interestingly, PNAd + HEVs were observed in the lungs of the BLM group, but not the control group. Moreover, numerous Gr1 + polymorphonuclear and mononuclear-like ring cells were found in the MFALCs and lungs of the BLM group. Interestingly, flow cytometric analysis revealed a significant increase of B-cell populations within the MFALCs of BLM group suggesting a potential proliferative induction of B-cells following inflammation. Furthermore, significant positive correlations were observed between quantitative parameters of these immune cells in both the lungs and MFALCs. Thus, we suggest a potentially important role for MFALCs and HEVs in the progression of lung disease, especially in inflammatory lung disease.

IL-25 contributes to lung fibrosis by directly acting on alveolar epithelial cells and fibroblasts

“…We also assessed whether IN delivery with IL-25 contributes to lung fibrosis in mice. The delivery methods for developing murine lung fibrosis model include intratracheal route, 19,20 IN route, [21][22][23] oropharyngeal aspiration route, 24 and subcutaneous route. 25 We chose IN route because its simplicity, speed, and noninvasive nature.…”

Pathological Alternations of Mediastinal Fat-Associated Lymphoid Cluster and Lung in a Streptozotocin-Induced Diabetic Mouse Model

“…ab6671, Abcam) and IL-5 (LS-C177535-100, Life Span Bioscience) antibodies (at dilution of 1:200 and 1:500, respectively). The immunohistochemical staining was performed as per the author's previous report (Elewa et al, 2014(Elewa et al, , 2016(Elewa et al, , 2018. Except for the immunohistochemical staining of Gr-1, CD3, and CD31, the antigen retrieval was performed by heating the section at 105°C for 15 min in 10 mM CB (pH 6.0) solution.…”

“…It has previously been revealed that the histopathological features of lung injury in several mice models include accumulations of mononuclear cells, thickening of the interalveolar septa, collapsed alveoli, and increased collagen fiber deposition in the interalveolar septa, which may even lead to severe lung fibrosis replacing the normal lung architectures (Elewa et al, 2016(Elewa et al, , 2018. Furthermore, a significantly positive correlation was observed between MFALCs development and lung injury pathogenesis in several mouse models.…”

“…They are named mesenteric FALCs (Moro et al, 2010) and mediastinal FALCs "MFALCs" (Elewa et al, 2014), respectively. Furthermore, the development of such clusters has been demonstrated to be associated with inflammation (Benezech et al, 2015;Elewa et al, 2018). Yet, the literature does not report any study concerning the effect of metabolic diseases on the development of MFALCs.…”

“…Furthermore, a significantly positive correlation was observed between MFALCs development and lung injury pathogenesis in several mouse models. These included genetic autoimmune diseases (Elewa et al, 2016(Elewa et al, , 2017; septic condition such as bleomycin induced-pneumonitis (Elewa et al, 2018); and aseptic infections such as Mycoplasma pulmonis (Boonyarattanasoonthorn et al, 2019). However, the pathological alternations of MFALCs and their possible role in lung injury pathogenesis in DM are not clarified.…”