XVII International AIDS Conference Effects of HIV-1 Tat Protein on Trypanosomatid Replication in HIV-1-Infected Macrophages Dumith Chequer Bou-Habib Oswaldo Cruz Institute – Fiocruz Rio de Janeiro, RJ Brazil XVII International AIDS Conference Mexico City, August 2008

35 WHO, 2003

1911 Leishmania/HIV co-infection cases reported in south-western Europe by early 2001 (Desjeux & Alvar, 2003) Brazil: 326 cases of HIV/Leishmania co-infection cases from 2000 to 2006, ~ 2% of visceral leishmaniasis reported in the country (SINAN)

Some clinical and laboratory findings of HIV-1/Leishmania co-infected patients Diffuse cutaneous leishmaniasis due to parasitic dissemination (ulcers containing parasites) Chronic progress and relapses of leishmaniasis Increased replication of HIV-1 and Leishmania High levels of serum cytokines (IL-4, IL-6, IL-10) Decreased IFN- levels

At the cellular level…

HIV-1/Leishmania co-infected macrophages “Something happens” Increases Leishmania replication

Tat (Trans-activator protein) HIV-1 Genome Tat (Trans-activator protein) TGF-b Adapted from Wahl et al. (2003). J Leukoc Biol 74:726-735

Evaluation of Leishmania Infection Model: HIV-1-infected Macrophages + Leishmania Evaluation of Leishmania Infection Endocytic Index = % of Leishmania-infected Macrophages X Mean of Parasites per Cell

Replication of both pathogens is augmented in HIV-1/Leishmania co-infected macrophages * p<0.0001 ** p<0.04 Barreto-de-Souza et al. (2006)

HIV-1-mediated enhancement of Leishmania growth is inhibited by anti-Tat antibody * p<0.025 * p<0.025 Barreto-de-Souza et al. 2006.

Purified HIV-1 Tat augments Leishmania replication in macrophages Barreto-de-Souza et al. 2006.

HIV-1 Tat inhibits leishmanicidal effect induced by IFN- * p<0.035

TGF-1 is partially responsible for the enhancement of Leishmania growth mediated by Tat Recombinant Tat induced TGF-b1 secretion by uninfected macrophages (p<0.002 )

Tat induces the expression of COX-2 enzyme and PGE2 synthesis in human macrophages PGE2 Med Tat COX-2 b-actin 0.716±0.03 1.387±0.23 (ng/mL) Recombinant Tat was LPS free (< 0.15 EU/mg)

Inhibition of the enzyme COX-2 reverts Tat effect on Leishmania growth * p<0.02

Inhibition of TGF-1 abolishes PGE2 effect on Leishmania growth * p<0.03

Conclusions Tat exacerbates Leishmania growth in macrophages co-infected with HIV-1, or infected only with Leishmania Tat effects on Leishmania replication are mediated by: - Induction of COX-2 and subsequent PGE2 production - Induction of TGF-b1 secretion (probably via PGE2) - Inhibition of leishmanicidal effect induced by IFN-

Induces the expression of COX-2 HIV-1 Genome Induces the expression of COX-2 TGF-b Adapted from Wahl et al., 2003.

At the cellular level… Work in progress

Next question: Could HIV-1 infection deactivate the natural microbicidal activity of human macrophages? (Leading to an establishment/growth of a non-pathogenic protozoan?)

Reports of AIDS patients presenting Leishmania-like infection caused by otherwise non-pathogenic trypanosomatids AIDS patient with a visceral lesions caused by Leptomonas pulexsimulantis (Pacheco RS et al., 1998). AIDS patients infected by non-pathogenic trypanosomatids have been reported since the 80’s (Chicharro C & Alvar J, 2003).

Blastocrithidia culicis as a model for HIV-co-pathogen studies Monoxenic trypanosomatid (entire life cycle limited to one host- hematophagous insects) Endosymbiont-bearing trypanosomatid, containing a single bacterium/protozoan cell. Normal human macrophages usually kill Blastocrithidia in few days (Barreto-de-Souza et al., 2008) Some monoxenic protozoa were detected in AIDS patients (mainly Herpetomonas and Crithidia)

Replication of both agents is augmented in Blastocrithidia/HIV-1 co-infected macrophages Barreto-de-Souza et al., 2008

HIV-1 infected Macrophage harboring B.culicis cells

Dividing forms of the protozoan in Blastocrithidia/HIV-1 co-infected macrophages Barreto-de-Souza et al., 2008

Neutralization of Tat reduces B Neutralization of Tat reduces B. culicis growth in HIV-1 co-infected macrophages Barreto-de-Souza et al. 2008

rTat promotes Blastocrithidia growth in macrophages Barreto-de-Souza et al. 2008

The increment of B.culicis growth is mediated by Tat-induced TGF-1 * p<0.05

Acknowledgments Oswaldo Cruz Institute/Fiocruz Federal University of Dept. Immunology Victor Barreto de Souza Thalyta Medeiros Dept. Physiology, Pharmacodinamics Adriana R. Silva Patrícia T. Bozza Hugo C. Castro Faria-Neto Federal University of Rio de Janeiro Dept. Immunology, IMPPG Elvira Saraiva Graziela J. Pacheco Biophysics Institute, IBCCF Cristina Motta Reagents: NIH AIDS Reagent Program, Hemotherapy Service (HUCCF - UFRJ) Financial Support: Papes/Fiocruz, CNPq, Faperj