4. Discussion
Here, we showed that a substantial accumulation of pulmonary CD4+ TRM cells mediated the pathogenesis of silicosis, who exerted immunological memory and antigen-specific response to the invaded CS particles. The TRM cells, especially TRM-Tregs, rely on continual replenishment from circulating lymphocytes. The defined population by CD103 and CD69 displayed distinct functional phenotypes. Targeting the immunosuppressive CD103+TRM cells did not exert protective roles to silicosis. Whereas neutralizing IL-7 in the lung disrupted the maintenance of TRM-Teff cells during silicosis progression and exerted protective effects.
Since the invaded CS particles could not be cleared by the pulmonary immune system, it would lead to repeated and cycled antigen stimulation, in which our one-time intratracheal CS instillation gave rise to CD4+ TRM cells. Evidence from the Rag1–/– mice transferred CD4+ T cells that were previously exposed to CS revealed that new CS exposure induced fast TRM cell expansion and potent reaction, demonstrating the TRM cells exerted immunologic memory and antigen-specific characterization. Notably, the CS particles could induce T-cell proliferation by directly activating cells through T-cell antigen receptor (TCR) complexes but not rely on antigen-presenting cells [36]. While research about Be oxide-related toxicological effects demonstrated major histocompatibility complex class II (MHCII) alleles, particularly HLA-DPB1, are strongly associated with susceptibility of the particle-induced lung inflammation[37]. Further study about the structural or molecular mechanism of how CS particles activate T cells, for instance, exploring the CS binding site within the TCR footprint or the presenting MHC molecule and the peptide bound by the MHC, would provide insights into the disease mechanisms and targets for antigen-specific therapies.
By circulatory lymphocyte blockade and parabiosis experiment, we directly proved the circulating T cells contributed to expanded TRM cells, which complies with a general acknowledgment that lymphocytes migrating from lymphoid organs to tissues is likely to be the first step in the formation of TRM cells[12]. Analog to our results, the circulatory T cells were reported to contribute to CD4+TRM cells exacerbating asthma[38]. Significantly, we noticed circulating lymphocyte recruitment blockage by FTY720 decreased the percentage of Tregs in TRM, implying the TRM-Tregs were in high demand of replenishment from circulating lymphocytes. Additionally, the evidence that fewer Tregs with an impaired immunosuppressive function in the CS-exposed pulmonary CD4+ T cells transferred Rag1–/–mice indicated a critical role of circulating T cell to TRM-Tregs, as there was no circulating T cells replenishment in the Rag1–/–mice. Ultimately, our parabiosis experiment directly proved TRM-Tregs depended more on circulatory T cell replenishment. In supporting the notion that we previously reported the TRM-Tregs in the silicotic lung expressed high Nrp-1 (markers of thymus-derived Tregs)[25,39], further proving the TRMcells originally came from the thymus and recruited to peripheral tissue[39], but not the conversion of Teff cells to Tregs [40]. All the evidence supported the suggestion that circulating thymus-derived Tregs are required to replenish TRM-Tregs to ensure a tissue-local immunosuppressive environment that promotes tissue remodeling. Remarkably, even though FTY720 could attenuate silicosis by reducing TRM cells in the lung, the FTY720 treatment in silicosis should be cautious. A common adverse effect would be an increased incidence of respiratory infections. Given the association of tuberculosis with silicosis, such treatment would need to be given with caution.
Despite numerous studies exploring the role of Tregs in fibrotic disease, this topic is still debatable [41,42], which may be related to Tregs’ heterogeneity in peripheral tissues[43,44]. In pursuing the role of circulatory-derived CD103+ TRM-Tregs in silicosis, we applied FTY720 together with CD103 neutralizing antibody to the silicotic mice expecting to get an alleviated effect. Surprisingly, this treatment did not mitigate disease phenotype, but no aggregated fibrosis was observed, as research reported that depleting CD103+ TRM cells exacerbated pulmonary fibrosis [45]. While the restrained effector T proliferation was unleashed by CD103 neutralization, which was consistent with the notion that CD103+TRM-Tregs restrained pathogenic CD4+Teff cells in the lung [45]. Although in different models of pulmonary fibrosis, the role of CD103+ Tregs seems to be analog, which exerted an immunosuppressive but not pro-fibrotic phenotype. With the cell transfer method, we ultimately proved the appended CD103+ Tregs role in constraining CS-induced inflammatory response but not promoting CS-induced fibrosis. The subsided tissue local inflammation may be a previous step than tissue repair, during which CD103+Treg attenuated tissue inflammation by limiting Teff cell proliferation, however, it does not promote fibrosis progression. Co-expression of cell retention markers CD103 and CD69 endowed Tregs with stronger tissue resident characteristics since CD103 (αEβ7integrin) promotes T cell adhesion through the interaction with E-cadherin expressed by epithelial cells [46,47]. Additionally, the fact that high KLRG1 expression in CD103+Treg also indicated the high adhesive ability to epithelial since the binding site to E-cadherin of KLRG1 is distinct from the CD103 binding site [48]. Further, research about markers distinguishing pro-fibrotic or immunosuppressive Treg would provide insights into treating silicosis.
It should be noted that the CS-stimulated pulmonary CD4+ TRM cells expressed cell retention marker CD69, regardless of Teff cells or Tregs, implying a crucial role of this molecule in mediating silicosis. However, other groups illuminated that genetically deleting CD69 in mice results in a reduction of CD8+ TRM in the skin and lung but does not affect CD4+ TRMcells [49]. These findings established that although CD69 expression can identify TRM in tissues, CD69 per se is not necessarily sufficient for influencing TRM cell maintenance or function. While there is a recent report that CD69 expression on Tregs protects from inflammatory damage after myocardial infarction [50]. Thus, a comprehensive understanding of CD69 on silicosis and other disease models still needs dissection.
IL-2 and IL-7 are crucial cytokines in maintaining lymphocyte viability, TRM cells up-regulate receptors for the homeostatic cytokines [51,52]. The previous study indicated that targeting CD25 (IL-2Rα) aggravated lung inflammation, alleviating fibrogenesis due to Treg depletion [53]. Herein, we illuminated different IL-7R expressions between TRM-Teff cells and TRM-Tregs. Higher receptor levels may indicate high demands for the cytokine. In supporting the notion, a higher cytokine level of IL-7 in the serum was observed in silicosis patients [54], whose role in silicosis was elusive. We demonstrated that IL-7 neutralization in the lung was effective in alleviating CS-induced pulmonary fibrosis by decreasing pathogenic CD4+ TRM cells without affecting TRM-Tregs expressing low IL-7R. Neutralizing IL-7 locally in lung disrupted the maintenance of TRM-Teff cells, attenuated CS-induced pulmonary chronic inflammation, mitigated local inflammatory damage to lung tissue, and thus inhibited fibrosis progression (excessive tissue repair). We did not utilize neutralizing antibodies by intravenous treatment since it may affect immune responses in other organs. Expectedly, lung local IL-7 neutralization did not affect the immune response in the spleen. Besides, bronchoalveolar lavage is often used in silicosis treatment. Clinically, applying IL-7-neutralizing antibodies in this process may provide therapeutic potential. However, in view of the situation that IL-7 was universally required for lymphocyte maintenance, the side effect of neutralization is a problem to be illuminated in the future.
In conclusion, our results provided new insights into CS particle’s toxicological effects in the lung, all of which will be essential for the development of new therapeutic strategies for the intractable inflammation-associated fibrotic diseases.