Differential immune regulation of activated T cells between cutaneous and mucosal leishmaniasis as a model for pathogenesis

Abstract

Cutaneous (CL) and mucosal leishmaniasis (ML) are characterized by a predominant type 1 immune response (IFN-gamma and TNF-alpha production) and strong inflammatory response in the lesions with few parasites. This exacerbated type 1 response is more evident in ML as compared to CL. Our main hypothesis is that a differential immune regulation of T cell activation leads to over reactive T cells in ML. In the present study, we investigated immunological factors that could explain the mechanisms behind it by comparing some immune regulatory mechanisms between ML and CL patients: frequency of cells expressing co-stimulatory molecules, apoptotic markers, T cell activation markers; and ability of neutralizing antibodies to IL-2, IL-12 and IL-15 do down-regulate IFN-gamma production in leishmania antigen-stimulated peripheral blood mononuclear cells (PBMC). Interestingly, in CL anti-IL-2 and anti-IL-15 significantly suppressed antigen-specific IFN-gamma production, while in ML only anti-IL-2 suppressed IFN-gamma production. Finally, higher frequency of CD4+ T cells expressing CD28-, CD69+ and CD62L(low) were observed in ML as compared to CL. These data indicate that an exacerbated type 1 response in ML is differentially regulated and not appropriately down modulated, with increased frequencies of activated effectors T cells, maintaining the persistent inflammatory response and tissue damage observed in ML.

Figure 1

IFN-γ production on SLA-stimulated PBMC supernatants from CL and ML patients. PBMC cultures from 22 CL and 22 ML patients were incubated for 72 h in the absence or in the presence of SLA (10 μg/mL). IFN-γ levels were measured in PBMC culture supernatants by ELISA sandwich technique and results expressed in pg/mL based on a standard curve with recombinant IFN-γ. The results represent the levels of IFN-γ from SLA-stimulated cultures subtracted by levels on unstimulated cultures. Statistical comparisons between CL and ML groups were done by Mann–Whitney U test.

Figure 2

Ability of anti-IL-2 (20 μg/mL), anti-IL-12 (20 μg/mL) or anti-IL-15 (20 μg/mL) in suppress SLA (10 μg/mL)-induced IFN-γ production in CL and ML patients. Anti-IL-2 and anti-IL-15 were added to SLA-stimulated PBMC cultures from 16 each CL and ML patients and anti-IL-12 was added to SLA-stimulated PBMC of eight CL and nine ML patients. IFN-γ levels were determined by ELISA on PBMC supernatants. a, c and e show mean ± SD of results using cultured PBMCs from CL patients; b, d and f show results using PBMCs from ML patients. All conditions were tested in triplicate in each experiment. In all these experiments, a control with antibody of the same isotype was done, and they do not inhibit IFN-γ production. Statistical comparisons were done using the Wilcoxon matched-pairs test.

Figure 3

Down-modulation of SLA-induced IFN-γ production by anti-IL-2 (20 μg/mL) and anti-IL-15 (20 μg/mL) is more evident in CL (n = 16) than in ML (n = 16) patients. A comparative analysis of the percentage of suppression modulated by anti-IL-2 or anti-IL-15 is shown as the mean ± SD suppression in SLA-stimulated PBMC from CL vs. ML patients. Data are compiled from the raw ELISA data shown in Figure 2(a,b,e,f ). Ability of anti-IL-2 (20 μg/mL) or anti-IL-15 (20 μg/mL) to suppress PPD (10 μg/mL)-induced IFN-γ production from PBMC cultures from six CL and six ML patients. Shown here is the mean ± SD suppression. IFN-γ levels were measured by ELISA on supernatants of PPD-stimulated PBMC cultures with or without addition of anti-IL-2 or anti-IL-15 (4b). All conditions were tested in triplicate in each experiment. In all these experiments, a control with antibody of the same isotype was done, and they do not inhibit IFN-γ production. Statistical comparisons between the degrees of suppression in CL vs. ML groups were done by Mann–Whitney U test.

Figure 4

Suppression of SLA-induced IFN-γ in PBMC from CL (n = 9) and ML (n = 6) patients by addition of CTLA-4 (5 μg/mL). The bars represents mean ± SD of the suppression levels of the groups. Differences between CL and ML are not statistically significant. Control experiments were performed with PPD-stimulated PBMC of six PPD positive healthy subjects, and show a suppression of 56 ± 43.3%.

Figure 5

Frequency of CD4+ T cells expressing cell activation markers ex vivo. The mean ± SD of the frequency of CD4+ cells in PBMC from eight CL vs. eight ML patients expressing activation markers (CD62_low, CD28−, CD69) were compared. Conditions were tested in triplicate. Statistical comparisons were done using the Mann–Whitney test.