1. INVOLVEMENT OF DIFFERENT SUBSETS OF REGULAOTRY T CELLS AND CYTOKINES IN THE SUPRESSION OF PRO-INFLAMMATORY RESPONCES DURING PROGRESSION OF VISCERAL LEISHMANIASIS IN BALB/C MICE.
Md Asad and Nahid Ali
Infectious Diseases and Immunology, Indian Institute of Chemical Biology, Jadavpur Kolkata, India
ABSTRACT:- Regulatory T cells (Treg cells) are considered as master of immune regulation that promotes bystander suppression of effector T cells and infectious tolerance. However, according to earlier report Tregs are also involved in controlling disease by facilitating timely homing of immune effector cells to the site of infection. Role of Tregs cells in human visceral leishmaniasis (VL), however is controversial. Infection of BALB/c mice with L. donovani strain AG83 leads to a progression of infection atleast till six months, mimicking human VL. Thus, investigation of these cells and their cytokines may elucidate the immunoregulatory mechanisms operating in VL.
We have observed earlier in mice that IL-4, IL-10 and TGF-β levels are enhanced and IL-12, a cytotoxic lymphocyte maturation factor and a central immunoregulator of initiation and maintenance of Th1 is down regulated during L. donovani infection. In the present study we have investigated the kinetics of different immune cells and their cytokines during disease. We have found that CD4+CD25+FoxP3+, CD4+CD25-FoxP3-, CD8+CD25-Foxp3- and CD8+CD25+FoxP3+ Treg cells are augmented with the progression of disease accompanied by enhanced secretion of immunoregulatory cytokines such as IL-10 and IL-4. Further, leishmanial antigen-stimulated culture supernatant of infected spleen show enhanced level of IL-10, IL-27 and TGF-β with negligible secretion of IL-17, IL-12 and IFN-γ as infection progresses. Thus the increase in parasite burden along with stimulation of IL-10, IL-27 and TGF-β may be responsible for the suppression of IL-12, IFN-γ and IL-17, which may ultimately help in progression of the disease.
Background:-Human VL is third most prevalent parasitic disease globally. It is an immunosuppressive disease. Several aspects of VL immunology have been exploited, but still it is poorly understood, like mechanism of regulation by the regulatory cells, their cytokines and paradox of Th1 and Th2 response in this disease, including their trafficking and mechanism of action. We have observed earlier in mice that IL-4, IL-10 and TGF-β levels are enhanced and IL-12 is down regulated during L. donovani infection. In the present study we have investigated the kinetics of different immune cells and their cytokine during the disease in mice model for better understanding the disease which may ultimately lead to better intervention.
Objective:- To investigate the correlation of parasite load with the dynamicity and evolution of immune regulation by different subsets of T cells and their cytokines with the development and progression of the visceral leishmaniasis in murine experimental model.
Fig. 1:- Liver and Spleen were excised from infected & Normal Control mice at different time point. Multiple impression smears were prepared and stained with Giemsa for organ parasite burdens, expressed as Leishman-Donovan units (LDU).
For Limiting Dilution Assay (LDA) weighed piece of spleen or liver was homogenized in Schneider’s medium to 1mg/ml, serially diluted in 96-well plates, examined for viable and motile promastigotes after 15 days.
Fig.2:- Profile of different cytokines at different time point.
Splenocytes from normal & infected mice were cultured & stimulated with Leishmania antigen. Supernatant was collected after 72 hours for cytokine ELISA.
CD4
IL-10
IFN-γ
IL-4
IL-17
CD8
IL-10
IL-4
IL-17
IFN-γ
Fig.3: Profile of different sub-sets of T cells & their cytokines through FACS.
Splenocytes from normal & infected mice were cultured overnight & stimulated with Leishmania antigen. Cells were collected for flow cytometry.
Conclusion:-
In the course of disease, parasite load increases showing peak at four month of infection, parasite burden decreases suggesting self curing nature of murine VL.
Development of the disease is accompanied by the increase in Th2 cytokines like, IL-10, TGF-β, IL-27 and IL-4, again showing peak at four month of infection. This enhanced level of Th2 cytokines with suppression of Th1 (IFN-γ) and Th17 (IL-17) responses suggest immune suppression during the disease progression.
Flow Cytometric studies show that with the advancement of the disease, different subsets of T cells are increasing. Among them, CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ cells are identified as major source of IL-10, IL-4 and IL-17 suggesting their involvement in immune regulation and development of murine VL.