Recruitment of lymphocytes is a prominent feature of the inflammatory process in pulmonary tuberculosis. Here, we demonstrate that the expression of the chemokine, XCL1/lymphotactin, plays a role in the recruitment of T cells during the chronic stage of M. tuberculosis
infection. To demonstrate this, we describe, for the first time, the manipulation of target gene expression at a specific time during chronic pulmonary infection. Here, we have combine our recently published noninvasive local pulmonary therapy (12
) with the recently developed technology of gene silencing mediated by siRNA (31
) to study the role of the pulmonary XCL1/lymphotactin chemokine during chronic infection with M. tuberculosis
Intrapulmonary delivery of naked and aerosolized XCL1-targeting siRNA appeared confined to the lungs, as we did not find siRNA-tagged Alexa 488 or changes in XCL1 protein expression in any of the peripheral or distant tissues screened (lymph nodes, heart, spleen, or liver). Furthermore, the siRNA appeared to be associated with CD8 T cells and, to some extent, with CD4 T cells as well. Previous work (12
) identified activated CD8 T cells as the main producers of XCL1 and, to some extent, natural killer and mast cell populations. We did not test for colocalization of siRNA with natural killer and mast cell populations; therefore, total suppression of xcl1
transcript might not be limited to the CD8 T-cell population.
Local delivery of a single dose of XCL1-targeting siRNA was very efficient at reducing expression of both xcl1 transcripts and XCL1 protein. However, these changes were only transient, because the levels of xcl1 transcripts 5 days after treatment recovered to similar levels of expression found in control mice. As expected, a reduction in the expression of xcl1 transcript was associated with a reduction in the levels of XCL1 protein at 3 days after treatment, and remained low at 5 days after treatment, even when the levels of xcl1 transcript had already recovered.
The reduction in expression levels of XCL1 protein was associated with lower lymphocytic infiltration, both by histological analysis and by total lung CD4 and CD8 T-cell counts using flow cytometric analysis. These data suggest that XCL1 directly participates in the recruitment of T lymphocytes into the lung during chronic tuberculosis. Moreover, this also indicates that, during chronic infection, a continuous supply of XCL1 chemokine in the lungs is being provided, and that a temporary reduction of XCL1 chemokine at Day 60 of the infection is immediately reflected in a reduction in total T lymphocytes in the lungs. Furthermore, the focal lymphocytic aggregation in the granulomatous lesions was reduced in mice that had lower expression of XCL1. The data suggest that during the chronic stage of infection, pulmonary XCL1 also participates by driving/directing the T lymphocytes to the sites of inflammation. A similar finding was observed in our previous work (13
), where transgenic mice overexpressing pulmonary granulocyte-macrophage colony–stimulating factor had reduced expression of XCL1 and appeared unable to direct lymphocytes to the granulomatous lesions.
A temporary reduction in XCL1 also changed the expression levels of total IFN-γ. Activated CD4 and CD8 T cells are the main source of IFN-γ during chronic infection with M. tuberculosis
). Thus, the reduction in total IFN-γ levels was explained by decreased infiltration of total CD4 and CD8 T cells, which also resulted in reduction of numbers of CD4 and CD8 producing IFN-γ. Our previous studies (12
), using intrapulmonary delivery of recombinant XCL1, indicated that high levels of expression of this chemokine did not affect the total number of CD4 and CD8 T cells present in the lungs, but resulted in a decreased number of CD4 T cells (but not CD8 T cells) producing IFN-γ. At that time, we hypothesized that a reduction in the levels of XCL1 would increase the numbers of CD4 T cells producing IFN-γ. However, the results from this study demonstrate that such reduction did not affect the CD4 T cells producing IFN-γ specifically but did affect the total CD4 T cell population. Altogether, this suggests that, whereas there is a critical level of XCL1 needed to recruit CD4 and CD8 T cells in the lungs, only high levels of XCL1 control the production of IFN-γ in the CD4 T cells. Previous studies suggest that XCL1 and its receptor, XCR1, may also act as a negative regulator of CD4 T cell activation, which points to a possible autocrine or paracrine regulation mechanism (23
); however, the precise mechanism is unknown.
The decreased XCL1 levels and subsequent decrease in IFN-γ expression in the lungs also affected the expression of iNOS transcripts for a short period of time. iNOS is an essential enzyme required for activation of microbicidal activity via NO production during chronic tuberculosis (30
). The levels of iNOS transcripts after treatment followed a similar trend to that of the XCL1 transcripts. iNOS transcripts decreased at 3 days, but recovered rapidly at 5 days after treatment. We hypothesized that a significant drop in the expression of iNOS at 3 days after treatment should correlate with decreased antimicrobial activity and increased bacterial load. However, the bacterial load in the lungs of these mice did not change when compared with control mice at 3 or 5 days after treatment. These results may suggest that the timeframe of iNOS suppression was not sufficient to affect the bacterial load in the lungs, and that prolonged suppression of iNOS (and IFN-γ and XCL1) is needed to have an effect on the bacterial load.
Lower expression of XCL1 also affected lung fibrosis. In other studies, highly fibrotic areas correlated with increased TGF-β mRNA expression and with tissue colocalized with the expression of TGF-β and IL-4. In this study, we observed higher levels of TGF-β mRNA expression, but not IL-4 mRNA. However, the IHC staining demonstrated changes in the pattern of expression of both cytokines in the XCL1-targeting siRNA and control mice. These data, along with those of our early study, indicate an overall change in the inflammatory cell distribution associated with changes in the levels of XCL1 protein.
Finally, this transient therapy also demonstrates a much longer term effect on the immunopathological course of the chronic infection. After a transient but strong reduction of XCL1 around Day 60 of infection, the immune system appeared to overcompensate xcl1
expression over a long period of time (180 days). Furthermore, increased levels of XCL1 led to larger lymphocytic foci in the parenchyma, indicating a role for XCL1 in the accumulation of lymphocytes at specific sites. These data support the concept that XCL1 participates in driving/directing lymphocytes to the site of infection. Interestingly, when using intrapulmonary delivery of recombinant XCL1 in our previous study, we also found that increased levels of XCL1 increased the size of these lymphocytic foci (12
Immunotherapy is currently an important alternative in cancer and infectious diseases. As such, we demonstrate here, for the first time, in a murine tuberculosis model that it is possible to modulate the local environment and the lung immunopathology using siRNA. Currently, a variety of acute and chronic lung diseases have shown to have altered expression of cytokines/chemokines or other important factors, many of them could be directly involved in the lung pathology. Therefore, workers on many other respiratory diseases will clearly be able to use the approaches described here to elucidate the role of different molecules in pathogenesis (12
). These same approaches may be exploited as immunotherapeutic alternatives for respiratory diseases.