Protective and Pathologic T Cell Response in Cutaneous and Mucosal Leishmaniasis

Protective and Pathologic T Cell Response in Cutaneous and Mucosal Leishmaniasis EDGAR M. CARVALHO Serviço de Imunologia Hospital Universitário Prof. Edgard Santos Universidade Federal da Bahia Salvador-Bahia-Brazil

Endemic Areas of Leishmaniasis

Clinical Forms of L.brasiliensis Infection Subclinical DTH (+) Cutaneous Leishmaniasis Disseminated Leishmaniasis Mucosal Leishmaniasis

Protective and Pathological T cell Response in Cutaneous and Mucosal Leishmaniasis To compare sistemic and lesion site immune response in cutaneous and mucosal leishmaniasis. To correlate immunological response with pathology in cutaneous and mucosal leishmaniasis. To evaluate the mechanisms involved in the exagerated immunologic response in cutaneous and mucosal leishmaniasis.

Cytokine Profile in Patients with Cutaneous Leishmaniasis 4000 300 IFN-γ and TNF-α (pg/ml) 3000 2000 1000 200 100 IL-10 and IL-5 (pg/ml) 0 IFN TNF IL-10 IL-5 0

Cytokine Profile in Patients with Mucosal Leishmaniasis IFN-γ and TNF- α (pg/ml) 10000 7500 5000 2500 0 IFN-γ TNF-α IL-10 IL-5 750 500 250 0 IL-10 and IL-5 (pg/ml)

Macrophages are the Main Source of TNF-α in Cutaneous and Mucosal Lesions Clinical form Total TNF-alpha+ cells CD68+ CD68- % contribution of CD68+ cells CL 1144+/- 691 909+/- 547 253+/- 233 78+/-13 ML 1354+/- 993 1148+/- 937 206+/- 139 80+/-13

High Numbers of Cells Expressing IL-10 in Both Cutaneous and Mucosal Lesions Clinical form Total CD68+ IL-10+ cells % contribution of CD68+ cells Commitment of CD68+ cells CL 2025+/- 785 1267 +/- 598 62 +/- 13 79 +/- 16 ML 2468 +/- 1597 1148 +/- 1158 53 +/- 17 91 +/- 4 * *ML > CL, p<0,05

Protective and Pathological T cell Response in Cutaneous and Mucosal Leishmaniasis To compare sistemic and lesion site immune response in cutaneous and mucosal leishmaniasis. To correlate immunological response with pathology in cutaneous and mucosal leishmaniasis. To evaluate the mechanisms involved in the exagerated immunologic response in cutaneous and mucosal leishmaniasis.

Evidence that Tissue Damage in Cutaneous and Mucosal Leishmaniasis is Due to Inflammatory Response Tissue damage is associated to lymphocyte and macrophage infiltrate and few parasites in tissue. Bitencourt 1991 Exagerated type 1 immune response is more evidenciated in mucosal than in cutaneous leishmaniasis patients. Bacellar,2002 Decreasing in IFN-γ and TNF-α levels follows successful therapy with antimony in mucosal leishmaniasis. Lessa,2002 Antimony therapy in the pre-ulcerative phase of the disease do not prevent ulcer development in cutaneous leishmaniasis. Machado 2003 Molecules that down modulate immune response associated with antimony cure refractory cutaneous and mucosal leishmaniasis. Almeida,1999; Lessa 2002

Antimony Therapy Previous to the Ulcerative Phase do not Prevent Ulcer Development in Cutaneous Leishmaniasis

Granulomatous Vasculitis in Cutaneous Leishmaniasis

Correlation between Immunological Response and Lesion Size in Cutaneous Leishmaniasis

inos Expression: Correlation with Tissue Inflammation in Cutaneous but not Mucosal Leishmaniasis Total Infiltrate 1800 1700 1600 1500 1400 1300 1200 Cutaneous R 2 = 0,847 p<0,02 1000 1100 1200 1300 1400 1500 1600 inos + Total Infiltrate 2200 2000 1800 1600 1400 R 2 = 0,100 p<0,6 Mucosal 800 1000 1200 1400 1600 inos + 3000 2500 2000 1500 1000 500 Total IFN-gamma+ cells/field 3500 0 1000 1100 1200 1300 1400 1500 1600 inos + Linear Fit R 2 = 0.75 P=0,05 4000 3000 2000 1000 800 1000 1200 1400 1600 inos + Total IFN-gamma+ cells/field5000 Linear Fit R 2 = 0.035 P=0,75

Protective and Pathological T cell Response in Cutaneous and Mucosal Leishmaniasis To compare sistemic and lesion site immune response in cutaneous and mucosal leishmaniasis. To correlate immunological response with pathology in cutaneous and mucosal leishmaniasis. To evaluate the mechanisms involved in the exaggerated immunologic response in cutaneous and mucosal leishmaniasis.

Possible Mechanisms Associated with Exaggerated T Cell Response in Mucosal Leismaniasis Increasing expression of co-stimulatory molecules. Decreased apoptosis. Lack of function of regulatory cytokines or regulatory T cells. Increased frequency of memory/effectors T cells. Increased production of IL-17.

Diseases Associated with IL-17 Rheumatoid Arthritis Crohn s Diseasis Ulcerative retocolitis Psoriasis Kidney allograft rejection

IL-17 Levels in Supernates of Lymphocyte Cultures Stimulated with Leishmania Antigen or PPD 350 300 IL-17 (pg/ml) 250 200 150 100 50 0 24 hs 48 hs 96 hs TIME leishmania antigen PPD

Frequency of Cells Expressing Co-Stimulatory Molecules in CL and ML Patients 150 Cutaneous (n=8) Mucosal (n=8) 100 50 Frequency (%) MHCII/CD14 CD80/CD14 CD86/CD14 CD40/CD14 CD40L/CD4 CTLA4/CD4 0

Decreased Suppression of SLA-induced IFN-γ in PBMC by Addition of CTLA-4 in CL and ML Patients SLA + CTLA-4 Cutaneous (n=9) Mucosal Controls (n=6) (n=6) SLA + CTLA-4 PPD + CTLA-4 0 20 40 60 80 100 IFN-γ supression (%)

Inability of IL-10 in Down-Regulation IFN-γ Production in Mucosal Leishmaniasis Patients 3000 Leish Ag IFN- γ (pg/ml) 2000 1000 * 19% * 48% * 86% Leish Ag+IL-10 5ng Leish Ag Leish Ag +IL-10 5ng PPD PPD+ IL-10 5ng 0 ML patients CL patients Healthy controls * % suppression

Expression of IL-10 Receptor in ML and CL Lesions 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 % cells expressing IL-10 receptor 300 250 200 150 100 50 0 A B 0.0 1 2 1 2 CL ML CL ML * Number of IL-10 receptor+ cells 3500.0 3000.0 2500.0 2000.0 1500.0 1000.0 500.0 0.0 * C Intensity of IL-10 receptor Intensity expression of IL-10 receptor 1 2 CL ML

Cell Activation Markers in Mucosal and Cutaneous Leishmaniasis Frequency (%) 50 40 30 20 Cutaneous Mucosal 10 0 CD4+/CD62L- CD4+/CD28- CD+/CD69+

Conclusions 1. Immunological response at lesion site in cutaneous and mucosal leishmaniasis is charactherized by over expression of IFN-γ,TNF-α, inos, IL-10 and increased frequency of cells expressing granzyme. 2. Exacerbated type 1 immune response in cutaneous and mucosal leishmaniasis is associated with pathology. 3. There is a decreased ability of T cells from cutaneous and mucosal leishmaniasis patients to be modulated by IL-10 and CTLA-4.

Collaborators Lucas P. Carvalho Olívia Bacellar Sara Passos Hélio Lessa Argemiro D Oliveira Júnior Marcus Lessa Luís Henrique Guimarães Amélia Ribeiro de Jesus Albert Schriefer Roque Almeida Maria Elisa Rosa Paulo Machado Walderez Dutra Kenneth Gollob TMRC-NIH AI 30639