Skip to main content
. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: Am J Transplant. 2021 Jan 4;21(7):2360–2371. doi: 10.1111/ajt.16417

Figure 5. Blockade IL-6 signaling reduces fibrosis in a murine model of CLAD.

Figure 5.

(A) Confluent primary MCs isolated from the lung of B6D2F1/J mice were grown in serum free media for 24 hours and then treated with vehicle, 50 ng/mL IL-6, 200 ng/mL sIL-6R or the combination of IL-6 and sIL-6R for 30 minutes. Only the combination of both IL-6 and sIL-6R induced STAT3 Tyr705 phosphorylation (n=4, representative image shown). (B) Adherent MNCs were isolated from the bone marrow of C57BL/6J and grown in coculture with primary pulmonary MCs for 48 hours and then mRNA was harvested from both compartments. MNC grown in coculture with MCs expressed significantly higher IL-6 mRNA compared to MNC grown alone (6.023±4.58 vs 23.13±4.34, n=3–8, ***p=0.007). (C) The presence of MNC causes MC to express 1.7-fold greater levels of collagen 1 mRNA (0.97±0.05 vs 1.63±0.27, n=4, **p<0.01) and 2.1-fold greater levels of ATX mRNA (0.68±0.31 vs 1.41±0.35, n=4, **p<0.01). Coculture had no effect on α-SMA mRNA expression. (D) The left lung of a B6D2F1/J donor mouse was transplanted into B6D2F1/J recipient (isograft) or a C57BL/6J recipient (allograft). Following transplantation, there was progressive increase in total lung homogenate IL-6 mRNA (n=3–6). At 28 and 40 days post-transplant, IL-6 mRNA expression was 3-fold and approximately 5-fold higher than baseline respectively (1.185±0.65 vs 3.629±1.59 vs 6.427±1.55, *** p=0.008 **** p<0.0001). (E) RNA isolation from CD45+ positive cells derived from either isografts or day 28 allografts showed a significant difference in IL-6 expression (5.211±1.618 vs 32.78±20.80, n=4–10, *p=0.0239). (F) There was also a significant increase in ADAM17 mRNA expression in CD45 positive cells isolated from allografts compared to matched isografts (0.8216±0.13 vs 1.673±0.27, ****p=0.0001). (G) Representative hematoxylin and eosin stained paraffin sections of isograft, allograft and allograft using an IL-6−/− recipient 28 days after transplantation. Allografts have extensive inflammation around the bronchovascular bundle (endothelitis and lymphocytic bronchitis) associated alveolar mononuclear cell infiltration. A mononuclear cell pleuritis was also present. Transplantation into an IL-6−/− recipient resulted in reduced parenchymal and pleural inflammation. (n=3, representative images shown, scale bar represents 500 μm) (H) Isograft, allograft and IL-6−/− allograft paraffin sections stained with trichrome. Compared to isografts, allografts show dense fibrosis around the bronchovascular bundle and along the pleura. This fibrosis was significantly reduced in allografts using an IL-6−/− recipient. (n=3, representative images shown, scale bar represents 500 μm) (I) Allografts had 4.5-fold greater hydroxyproline compared to isografts (20.1±2.1 vs. 90.3±34.7, n=6, **p<0.01). IL-6−/− recipient mice had 51% reduction in hydroxyproline when compared with wildtype allografts (46.3±15.0 vs 90.3±34.7, n=6, *p<0.01).