We have identified an immunomodulatory role for a novel population of resident lung MSC during lung injury. We have identified ABCG2 as a marker appropriate to localize and study these cells in vivo in both murine and human tissue. We documented a decrease in endogenous luMSC following bleomycin injury, suggesting that endogenous luMSC fail to effectively participate in lung repair. Our work definitively demonstrates that rescue of luMSC with exogenous cells attenuates the inflammation, fibrosis and pulmonary arterial hypertension associated with bleomycin induced lung injury. One potential mechanism we explored to explain the decrease in inflammation is a decreased T-effector cell proliferation mediated by luMSC. This inhibitory effect is limited to the MSC and not lung fibroblasts. The studies described herein also highlight a resident lung origin for a population of luMSC distinct in function and transcriptome from lung fibroblasts. Our work suggests that “rescuing” endogenous luMSCs to initiate repair in the setting of lung injury could mitigate the need for cell transplant. The ability to study these cells in vivo in both murine and human tissue provides a novel tool for the study of lung MSC.
Multipotent MSC have been isolated and characterized from various tissues (38
). MSC with a phenotype similar to BM-MSC, but associated with pulmonary tissue have also been identified in the SP (23
), BAL from human lung allografts (41
) and as a Sca-1 positive fraction of adult mouse lung (42
). More recently we have demonstrated that over the course of a lifetime hematopoietic cells from myeloid lineage take up residence in stroma of adipose tissue, assume a phenotype indistinguishable from tissue resident MSC and differentiate to fat (17
). Taking these data into consideration we analyzed the origin of the luMSC by bone marrow transplantation analysis. After 20 weeks in the presence or absence of injury we found the Hoechstlow
cells were consistently GFP negative, therefore derived from adult lung tissue. It is interesting to note that Lama et al. (41
) also identified the lung tissue origin of BAL derived MSC in patient allograft tissue following transplantation; however, an initial BM origin cannot be excluded at this time.
Given the phenotypic similarities of luMSC to BM-MSC and their resident tissue origin we hypothesized that luMSC would participate in the repair process of lung following bleomycin injury. LuMSC administered intravenously at the time of injury decreased the degree of fibrosis and collagen deposition in lungs as determined by Ashcroft scoring 14 days following intratracheal bleomycin exposure. Measurement of right ventricular systolic pressures demonstrated the attenuation of PAH and associated vascular remodeling. A caveat to these studies was the ability of luFB treatment to decrease muscularization in response to bleomycin treatment to levels similar to luMSC. While the positive effects of luMSC may be attributed to the regulation of effector T cell proliferation, the effect of luFB observed in these studies may be explained by differences in vessel SMC thickness, as well as differences in immunomodulation or differences in additional paracrine factors produced between the FB and MSC resulting in SMC contraction or proliferation. Essentially lung FB were included in these studies as a lung derived mesenchymal or stromal cell type lacking the stem cell properties which would characterize them as MSC. Further characterization of differences between luFB and luMSC is necessary in order to elucidate the factors that distinguish therapeutic effects of lung MSC. Surprisingly, similar to BM-MSC, protective effects occurred in the absence of lung engraftment. One may hypothesize that a luMSC would differentiate into lung tissue types during repair. However the issue of niche for these cells to reside during injury may be an obstacle to their engraftment or differentiation. Additionally, the possibility for isolation and culture procedures altering these cells' function cannot be discounted.
Another important observation of our studies was the immunosuppressive effect of luMSC. LuMSC administration down-regulated the appearance of inflammatory cells in the BAL and was associated with an anti-proliferative effect on effector T cells in vitro. In patients with IPF, and in animal models of fibrosis, the response of the lung environment has been reported to be profibrotic, antifibrotic or have no effect depending on the phenotype of T cells present (4
). To determine whether the anti-inflammatory effect of luMSC was related to the regulation of T cell proliferation previously reported for BM-MSC and BAL derived MSC (33
) we performed co-culture analysis. We demonstrate that luMSC significantly decreased effector T cell proliferation while luFB had no effect. Interestingly, with increasing concentrations of T cells the inhibition of proliferation was more pronounced suggesting that the T cells were effectively communicating with each other. MSC have also been shown to induce apoptosis of activated T cells (43
), however, we did not identify apoptosis as a mechanism of regulation in our assays. A paracrine anti-inflammatory role for BM-MSC has been described for the decrease in macrophage secretion of TNF (7
) and T cell proliferation (33
) via the soluble mediators IL-1RN, AIP6 and PGE2
, respectively. However, these and other previous reports have characterized a lack of T cell costimulatory molecules on the MSC, whereas we have identified the presence of CD80 receptor protein and mRNA in these luMSC and luFB under basal conditions. The presence of CD80 suggests that although a paracrine role has been described for MSC, cell-cell contact with T cells may also mediate important effects by these populations. It is also important to note that T cell proliferation was not observed in lung tissue following bleomycin injury on days 7 or 14, suggesting that the luMSC likely regulates the T cell response in an extrapulmonary site. LuMSC likely represent a resident pulmonary stromal cell type similar to BM-MSC which exhibit anti-inflammatory effects via regulation of T effector proliferation. Both luMSC and luFB may also regulate additional T cell subpopulations which merits further study.
In light of our data demonstrating that luMSC but not luFB attenuate bleomycin induced lung injury, it is interesting to consider differences between these stromal cell populations within the pulmonary tissue. Transcriptional profiling indicated that the luMSC differ from lung fibroblasts in decreased expression of genes involved in inflammation and fibrosis. LuFB express increased levels of selected genes such as Cxcl14, and fibrosis related genes periostin (44
), type III collagen as well as the wnt/βcatenin targets Tbx20 (45
) and sfrp1(46
). Both luMSC and luFB express AIP6/TSG6 which has anti-inflammatory properties (47
). MSC present in lung tissue can be activated following cardiac injury to increase the secretion of AIP6 decreasing inflammation, infarct size and improving cardiac function (47
). The expression of IL1RN, another key mediator in the anti-inflammatory repertoire of BM-MSC(15
), is limited to luMSC and not FB. Interestingly, when compared to published BM-MSC gene lists luMSC and luFB exhibited similar expression patterns (34
). It is therefore likely that the differences between the lung mesenchymal cell types, luMSC and luFB, are a key in our understanding of their functional differences.
Characteristics of luMSC define their similarity to BM-MSC and their ability to protect from injury via regulation of T cell proliferation. Taken together these results suggest that resident luMSC may function to regulate pulmonary tissue repair. If one accepts this hypothesis, questions that remain are the true function of these cells within the context of the lung, and identification of their niche which presumably dictates function (49
). The luMSC present in the recipient lung do not appear sufficient for protection or repair following bleomycin injury. This deficiency may be due to an impaired function, a decrease in their number following injury, or a combination of both. Rescuing the deficient endogenous luMSC population via the administration of exogenous luMSC attenuated injury. Bleomycin injured lung tissue is known to stimulate the proliferation and recruitment of BM-MSC both in vitro and in vivo (10
). Injury may therefore affect administered luMSC differently than endogenous cells. Administered luMSC may also function outside the lung to suppress the injury, and the number of cells administered is likely an important factor where a “threshold dose” of luMSC may be required for sufficient repair. Our results demonstrate that exogenously administered luMSCs decrease lung injury while endogenous cell function is compromised and suggest a therapeutic strategy for rescue of endogenous cells to facilitate lung repair during injury. The repeatability of isolation of this well-characterized population of cells and the ability to identify them in vivo allows this venue of study. The existence of this cell population in human lung tissue also suggests the use of mouse models to study luMSC may have an impact on understanding their role in human disease processes.