L Chelluri, P CE., P Vennila, A Gokhale, V Adavi, R KS, R K
L Chelluri, P CE., P Vennila, A Gokhale, V Adavi, R KS, R K. Preliminary report on immunomodulation of mesenchymal stem cells in M.tb infection. The Internet Journal of Infectious Diseases. 2009 Volume 8 Number 1.
Tumor necrosis factor-alpha and interferon-gamma are two important pleiotropic inflammatory cytokines that perpetuate tissue damage after increased stress and in the presence of excess glucocorticoids with respect to tuberculosis (TB) infection. Activated macrophage dysfunction leads to granuloma formation, cavities, and fibrosis. Current treatment strategies are not amenable to long-term compliance, contraindications, increased relapse rates, and compounding drug resistance. A natural supplement devoid of the abovementioned complications needs to be developed and checked for healthy immune reconstitution targeting
TB and the inflammatory cytokines
Cytokines are soluble, extracellular proteins released by cells that regulate the immune response. Cytokine actions include the generation of inflammation and the activation of phagocytes and B cells. In pulmonary Tb infection, the secretion of the pro-inflammatory cytokines include IL-12, IL-18 and TNF-alpha during early stage of infection, which is essential for induction of Th1 response. TNF-alpha, IL-12 , and IL-18 promote clearance of the infection by enhancing the levels of IFN-gamma produced by different cell populations, including T cells, NK cells and gamma-delta T cells. In contrast, early secretion of IL-4, IL-10 has been to polarize the immune response toward Th2 (1,2).
Protective immunity requires a Th1 response, and tumour necrosis factor (TNF-alpha ) is needed as an additional, macrophage activating factor (3). With a mixed Th1-Th2 cytokine response, TNF becomes toxic and mediates the gross tissue destruction characteristic of active, progressive tuberculosis (4,5). Stress and corticosteroids tend to drive newly recruited T cells towards Th2 responses. The protective Th1 immunity, a characteristic feature of granuloma is regulated by two mechanisms i.e., IFN-gamma itself, while essential for granuloma formation, may also limit the uncontrolled expansion of mycobacteria-reactive T cells (3,6,7). Secondly, endogenous IL-10, produced during mycobacterial infections, can reduce IL-12 production from mycobacteria infected DC’s, migration of DC’s to draining lymph nodes and the expansion of IFN-gamma secreting T-cells. It is to be seen if these dendritic cells play any role in skewing the immune responses and reduce the inflammation, particularly the TNF-alpha and IFN-gamma responses in mycobacterium infection (8).
Material & Method
The prospective study has IRB and IEC clearance. The subjects recruited into the study were new sputum positive cases without prior treatment for TB. The subjects had proven case history of TB with sputum positivity and X-ray findings and culture proven. Our analysis included data from 5 subjects all of whom were new sputum positive(+3) and culture proven. The venous blood was drawn prior to the initiation of DOTS treatment.
Bone marrow aspirates were collected from iliac crest in 6%ACD solution after obtaining high risk informed consent. The aspirates were diluted with equal volume of normal saline. Bone marrow mononuclear fraction was obtained using Ficoll gradient. The mononuclear fraction was plated at 25,000 cells in T25 flask. Non-adherent component was removed on day 3 and cultured further with DMEM (Gibco,USA) supplemented with 10% FBS (Gibco, USA), FGF2 at 10ng/ml (Sigma, USA) Streptomycin and Pencillin. The confluent cultures on day 14 were trypsinised and expanded up until Passage 2.
Isolation of PBMC’s
Peripheral blood (5.0 ml) and Plain blood (2.0ml) was drawn from 5 diagnosed TB subjects in heparin vacutainer with informed consent. The mononuclear fraction was obtained the same way as from bonemarrow on ficoll gradient. The PBMC’s count was adjusted to 1.0 x 106 cells/ml. Serum was separated for cytokine analysis.
Mixed Lymphocyte cultures
The PBMC’s were cultured at a concentration of 1.0x106 cells/ml as against three different doses of mesenchymal stem cells i.e., 10 X103 cells/ml, 10 X104 cells/ml, 10 X105 cells/ml in triplicate. PHA (Sigma, USA) was used as a positive control for PBMC’s stimulation. The cultures were incubated at 5% CO2 for 5 days. The culture supernatants were collected on day 5 for cytokine analysis.
The serum and culture supernatants were processed by ELISA for TNF-alpha and Interferon-Gamma levels. (Invitrogen, USA). Briefly, the serum and the culture supernatants were incubated with respective antibodies followed by rigorous washing and removal of excess non-specific antibodies. The secondary antibody IgG type was conjugated to horseradish peroxidase enzyme and incubated with the antigen-antibody complex. The captured antibody was quantified with the aid of chromogen substrate and readings of the optical density at 450nm. The values were converted to unit measurement using the standard plot.
Student’s ‘t’ test was performed for statistical significance.
TNF-α and IFN-γ levels were gauged in serum from untreated TB cases and compared with cytokine levels obtained from culture supernatants of their peripheral blood MNCs when co-cultured with three different doses (103,104and 105 cells/1μl) of MSCs from healthy individuals. Figure 1 demonstrates the TNF-α levels of the 5 subjects prior to treatment and when 1 million MNCs from each of them was treated with three different doses of allogeneic MSCs.The figures demonstrate a peak at 103 cells/1μl and a significant trough at 105 cells/1μl of MSC. Figures 2 represents the IFN-γ levels obtained when left untreated and their corresponding values when co cultured with variable doses MSCs.The levels of IFN-γ increased with an increase in the dosage of MSCs. Also a paired t –test was performed to check if there was any significant difference in the cytokine values of the patients prior to treatment and after treatment with varying doses of MSCs. At concentrations of 104 and 105 cells/1μl of MSCs, there was a significant difference observed (p=.20, at 80% confidence level, t< 0.90) in the TNF-α levels. But no such results were obtained when this test was performed to check the difference in IFN-γ levels.
The present study was conducted with an aim to study the
MSC’s have been recently reported to have an anti-inflammatory role in experimental models of acute pulmonary and renal injury(11). MSCs have been shown to exert a profound inhibition on activated T cells when stimulated by allogeneic or mitogenic stimuli(12,13,14).They could some how skew the immune system towards an anti inflammatory tolerant phenotype preventing further tissue damage. MSC recruitment would entail the suppression of T cell mediated cytotoxicity which would otherwise lead to necrosis, cavities, a consequence of M.Tb infection (10,13,15).The subjects thus selected were all new sputum positive and were not given any treatment. There was a significant difference (t <.90, 80% confidence level, p=.20) between the means of untreated samples and the samples cocultured with 105 and 104 cells/1μl of MSC.
At 103cells/1μl there was no significant difference. This outcome demonstrates that MSCs invitro could have an impact in skewing a shift in the immune system making it more tolerant and less prone to tissue damage. This is a novel finding and the data generated can be used to conduct an in vivo study. The results of the study are promising and a large studies are warranted to be able to use this natural treatment instead of medication which has side effects. The chief limitation of the present study is to perform in an equal number of normal individuals in comparison with TB subjects, to conclusively report that the MSC’s have a critical role in regenerating healthy lung epithelium to resist further damage from infection mediated inflammatory diseases.