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.