In the present study, the effect of PM2.5 on the formation of granuloma and the integrity of already established granuloma was studied in the in vitro model of human granuloma using PBMCs and Mycobacterium bovis BCG. An attempt was also made to examine the change in expression of the genes responsible for maintaining dormancy and reactivation and to correlate these findings with CFU, histopathology, and finally with Th1, Th2 and Th17 cytokine levels.
Extraction of PM2.5
PM2.5 was extracted from both teflon (PTFE) and quartz filters and suspended at a 1mg/ml concentration. This suspension was visualized under a light microscope to examine the contamination with filter material. The light microscope image of the lyophilized extracts of PM2.5 extracted from quartz filter showed the presence of filter fiber materials; however, it was not observed in the case of teflon filter. The PM2.5 extract from both quartz and teflon filters was then passed through a 0.45μm nylon filter, which led to the removal of fibre material. No loss of PM2.5 mass was observed during filtering process based on the weight determination before and after filtration (data not shown). Based on the various parameters like the volume of the solvent required for sonication, duration of centrifugation and yield of the pollutant, the PTFE filter was found to be advantageous over quartz filter (Online Resource 1) and was selected for further studies.
Effect of PM2.5 on PBMCs viability
To determine the maximum concentration of PM2.5 that does not affect the in vitro PBMCs viability, MTT assay was performed. In this, PBMCs in RPMI media were treated with different concentrations of PM2.5, followed by MTT assay at 24hr and 48 hr of treatment. Amongst three different concentrations used, the % viability of PBMCs was affected at the highest concentration of PM2.5, i.e., 50μg/ml after both 24hr and 48h (Fig. 1). At 24 hr, the percent viability was observed to be 31% at 50 μg/ml of PM2.5, 95% at 0.1μg/ml, 96% at 1 μg/ml and 94% at 10μg/ml of PM2.5. A similar trend was also seen at 48 hr of PM2.5 treatment. Therefore, the concentrations 0.1μg/ml (lowest) and 10μg/ml (highest) of PM2.5 were used for further experiments.
Development of granuloma
To obtain a well-defined granuloma, optimal MOI of M. bovis BCG was required and was determined based on histopathological examination (H&E and ZN staining). Two MOIs (0.1 and 0.2 comprising of 1 bacillus per 10 PBMCs and 2 bacilli per 10 PBMCs, respectively) were used. At MOI 0.1, a well-defined granuloma was formed, however at MOI 0.2, the lymphoid cells were scattered and scanty clusters resembling granuloma were observed as compared to MOI 0.1. Further, ZN staining for AFB showed that at MOI 0.2, the bacilli were observed in large clumps indicating an excess of bacilli (data not shown). Thus, well-defined granuloma formation at MOI 0.1, it was used for further experiments (Online Resource 3).
Effect of PM2.5 on the formation of PBMC granuloma using Mycobacterium bovis BCG
i) Effect on granuloma structure
After day 10 of granuloma induction in the presence and absence of two different concentrations of PM2.5 (0.1 and 10ug/ml), the ECM containing PBMCs and bacilli were subjected to H&E and ZN staining. The histopathology showed images of granuloma induction in the presence and absence of PM2.5 (Fig. 2). In the presence of 0.1μg/ml PM2.5 concentration, granuloma's appearance was similar to that of the control group with cluster of lymphoid cells. However, in the presence of 10μg/ml PM2.5 concentration, histopathology revealed loosely clustered macrophages and lymphocytes, suggestive of ill-defined granuloma (Fig. 2). Thus, it was observed that as the concentration of PM2.5 increases, the granuloma formation by M. bovis BCG gets disrupted, forming a loose ill-defined granuloma.
Further, the treatment of already established granuloma with PM2.5 demonstrated that at 0.1μg/ml PM2.5 concentration, granuloma's appearance was similar to that of the control group with a cluster of cells comprising lymphocytes and macrophages (Fig. 2). However, treatment with 10μg/ml PM2.5 concentration resulted in the disruption of granuloma as indicated by loosely packed macrophages and lymphocytes (Fig. 2). Therefore, it was observed that in in-vitro experiments, as the concentration of PM2.5 increases, the granuloma formed by M. bovis BCG gets disrupted. Further, acid-fast staining of control and PM2.5 treated granulomas, although showed the presence of acid-fast bacilli, however, no evident changes were observed in the bacterial load in the stained granulomas (Fig. 3).
ii) Effect of pollutant on bacterial load in the granuloma
To see the effect of the pollutant on the bacterial load in the granuloma formed in PM2.5, pellets obtained from granuloma were plated on 7H11 agar and observed for the appearance of colonies. There was no significant difference in the CFUs in control granulomas and those formed in the presence of 0.1μg/ml and 10 μg/ml of PM2.5, respectively (Fig. 4a). Further the treatment of already formed granuloma with PM2.5 also did not show any significant difference in the CFUs in control and pollutant treated granulomas (Fig. 4b)
iii) Effect of PM2.5 on the expression of dormancy and reactivation associated mycobacterial genes
The PBMCs granuloma formed with M.bovis BCG in the presence and absence of PM2.5 were harvested and processed for qRT-PCR to study the gene expression changes. Relative expression of four mycobacterial genes related to dormancy (tgs1, icl, hspx) and reactivation (rpfB) was studied. As it is evident from Fig5a, there was a significant upregulation in the expression of icl gene (p<0.001) and rpfB gene(p<0.05) in granuloma formed in presence of PM2.5 as compared to control granuloma. On the other hand, tgs1 along with hspX genes were significantly downregulated (p<0.001) in the mycobacterial cells recovered from granulomas formed in the presence of pollutant as compared to control granulomas. Overall, these results indicate downregulation of dormancy associated genes and upregulation of reactivation associated rpfB gene in the granuloma formed in the presence of PM2.5. Alternatively, experiments were carried out to study the mycobacterial gene expression in the already established PBMCs granuloma using M. bovis BCG following treatment with PM2.5 for 72hr. As shown in Fig. 5b, there was a significant upregulation in the expression of the icl gene (p<0.05) in PM2.5 treated granuloma compared to untreated control granuloma. On the other hand, considerable downregulation was observed in tgs1 and hspx genes (p<0.01) in pollutant treated granulomas with respect to the control group. However, the rpfB gene though was significantly downregulated at 0.1µg/ml of PM2.5 (p<0.001) but was non-significantly upregulated (1.7-fold) at 10 µg/ml of PM2.5. Overall, these results indicate the downregulation of dormancy associated genes along with reactivation associated rpfB gene in the granuloma treated with 0.1 µg/ml of PM2.5 and a non significant upregulation of rpfB gene at 10 µg/ml of PM2.5 as compared to control untreated granuloma (Fig. 5b).
iv) Effect on cytokines level
The levels of different cytokines were measured in the culture supernatants that were preserved during the time of granuloma harvesting. The cytokines of Th1 cells (TNFα, IFNγ and IL-2), Th2 cells (IL-10, IL-6 and IL-4) and Th17 cells (IL-17A) were measured in the supernatants from PBMCs granuloma formed with M. bovis BCG in the presence and absence of PM2.5 using flow cytometry as well as in the already established granuloma treated with PM2.5. Fig6 shows the levels above-mentioned cytokine in all the above mentioned three groups (control, 0.1μg/ml PM2.5, 10μg/ml PM2.5, respectively). Among all the cytokines tested, a significant increase in TNF-α level (p < 0.01) was obtained in the granuloma formed in the presence of pollutant only at a high concentration of 10 μg/ml compared to control granuloma, whereas, there was no significant difference in all other cytokines tested. On the other hand, in the case of treatment of established granuloma with PM2.5, although there was an increase in IFNγ, TNFα, IL-17 and IL-10, these changes were statistically non-significant compared to control untreated granuloma (data not shown).