3.6 Neutralizing IL-7 in lung retarded silicosis progression through disrupting the pathogenic TRM-Teff cell maintenance.
Now that the CS-induced pathogenic CD4+TRM cells expandedin situ , we next explored interventions targeting their maintenance in the lung, in which IL-7 was reported to be essential[34]. Hence, we first examined the IL-7R (CD127) levels on the CD4+ TRM cells in silicotic lung. Expectedly, CD4+ TRMcells expressed higher levels of IL-7R compared to those of the naïve T cells (CD44). Strikingly, within CD4+ TRM cells, TRM- Teff cells but not Tregs expressed a higher level of IL-7R indicating a high demand for IL-7 of those Teff cells (Fig 6A). We then treated the silicotic mice with IL-7-neutralizing antibody through intratracheal (i.t. ) instillation (Fig 6B). To gain insights, we sorted CD4+ T cells from the lungs and spleens and did qPCR analysis. Significantly, in the treated pulmonary CD4+T cells, we observed alleviated Th1 and Th17-related transcripts (Tbx21 , Ifng , Il2 ,Rorc and Il17a ), as well as decreased mRNA levels associated with cell activation (Icos , Ctla4 ,Klrg1 , and Pdcd1 ). Additionally, the cell retention markers (Cxcr6 and Itgae ) were decreased, implying a reduction of TRM cells (Fig 6C). Strikingly, the anti-IL-7 treatment augmented Il10 transcripts, suggesting suppressive inflammatory responses, while markers related to Treg cells,Foxp3, and Areg were unaltered (Fig 6C). However, these effects were diminished in the splenic CD4+ T cells (Fig 6D), suggesting that pulmonary local IL-7 neutralization did not affect immune response in other organs.
To confirm the transcriptional changes, we further did flow cytometric analysis to the pulmonary CD4+ TRMcells but did not observe reduced TRM cells in the lung (Fig 6E), while the ratio of CD103 to CD103+ in CD69+CD4+ TRM cells was decreased (Fig 6F), suggesting TRM-Teff cells were restrained. However, we did not observe a significantly altered ratio of TRM-Tregs within CD4+TRM (Fig 6G). To ascertain the effects on TRM-Teff cells, we scrutinized the composition of T cell subsets. Consistent with the transcript data, pulmonary IL-7 neutralization blunted T-bet+ Th1-type TRM, but not the ROR-γt+ Th17-type TRM cells albeit with an increasing trend (Fig 6H). Additionally, pulmonary IL-7 neutralization decreased Ki67 levels of TRM-Teff cells (Fig 6I), confirming that IL-7 plays a positive role in TRM cell proliferation[35].
Last, to gain insight into the profound effect of IL-7 neutralization on silicosis, lung histological analysis was performed. Expectedly, local anti-IL-7 treatment in the lung mitigated disease phenotype with reduced cell accumulation and cellular nodule formation (Fig 6J), as well as attenuated collagen deposition revealed by Masson’s trichrome staining (Fig 6K). Consistent with previous sorted cells transcript results, in the lung tissue, the Th1-related cytokine (Ifng ) and transcriptional factors (Tbx21 ) transcripts were blunted (Fig 6L). The neutralization decreased the transcripts of Il17adespite exerting no effects on those of Rorc in the lung (Fig 6L), implying that pulmonary IL-7 neutralization alleviated CS particles-induced inflammatory response. In accordance with alleviated fibrosis, the declined Col1a1 and Fibronectin transcripts in the lung further validated the conclusion (Fig 6M). Collectively, these data demonstrated that the maintenance of TRM-Teff cells depended on IL-7 in the lung. Targeting IL-7 would be a potential intervention against CS-induced pulmonary fibrosis.