Regulatory T cells control HIV replication in activated T cells through a cAMP-dependent mechanism.

We hypothesized that regulatory T cells (Tregs) could play a beneficial role during HIV infection by controlling HIV replication in conventional T cells (Tcons). Purified Tregs and Tcons from healthy donors were activated separately. Tcons were infected with the X4 or R5 HIV strains and cultured with or without autologous Tregs. Coculture of Tcons and Tregs resulted in a dose-dependent inhibition of Tcon infection, which was significant when a 1:1 Treg:Tcon ratio was used. Treg suppression of HIV infection was largely mediated by contact-dependent mechanisms. Blockage of cytotoxic T-lymphocyte-associated antigen-4 did not significantly reduce Treg function. In contrast, Tregs acted through cAMP-dependent mechanisms, because the decrease of cAMP levels in Tregs, the blockade of gap junction formation between Tregs and Tcons, the blockage of CD39 activity, and the blockage of protein kinase A in Tcons all abolished Treg-mediated suppression of HIV replication. Our data suggest a complex role for Tregs during HIV infection. Although Tregs inhibit specific immune responses, their inhibition of HIV replication in Tcons may play a beneficial role, particularly during early HIV infection, when the effector immune cells are not yet activated. Such a protective role of Tregs could have a profound impact on infection outcome.

HIV-induced T-cell activation/exhaustion in rectal mucosa is controlled only partially by antiretroviral treatment.

Peripheral blood T-cells from untreated HIV-1-infected patients exhibit reduced immune responses, usually associated with a hyperactivated/exhausted phenotype compared to HAART treated patients. However, it is not clear whether HAART ameliorates this altered phenotype of T-cells in the gastrointestinal-associated lymphoid tissue (GALT), the main site for viral replication. Here, we compared T-cells from peripheral blood and GALT of two groups of chronically HIV-1-infected patients: untreated patients with active viral replication, and patients on suppressive HAART. We characterized the T-cell phenotype by measuring PD-1, CTLA-4, HLA-DR, CD25, Foxp3 and granzyme A expression by flow cytometry; mRNA expression of T-bet, GATA-3, ROR-γt and Foxp3, and was also evaluated in peripheral blood mononuclear cells and rectal lymphoid cells. In HIV-1+ patients, the frequency of PD-1(+) and CTLA-4(+) T-cells (both CD4+ and CD8+ T cells) was higher in the GALT than in the blood. The expression of PD-1 by T-cells from GALT was higher in HIV-1-infected subjects with active viral replication compared to controls. Moreover, the expression per cell of PD-1 and CTLA-4 in CD4(+) T-cells from blood and GALT was positively correlated with viral load. HAART treatment decreased the expression of CTLA-4 in CD8(+) T cells from blood and GALT to levels similar as those observed in controls. Frequency of Granzyme A(+) CD8(+) T-cells in both tissues was low in the untreated group, compared to controls and HAART-treated patients. Finally, a switch towards Treg polarization was found in untreated patients, in both tissues. Together, these findings suggest that chronic HIV-1 infection results in an activated/exhausted T-cell phenotype, despite T-cell polarization towards a regulatory profile; these alterations are more pronounced in the GALT compared to peripheral blood, and are only partiality modulated by HAART.

cAMP during HIV infection: friend or foe?

Intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP) are important regulators of immune cells, partially determining the balance between activation and suppression. In this review, we discuss the mechanisms by which HIV infection increases cAMP levels in T cells, as well as the effect of cAMP on HIV-specific responses and its effect on HIV replication and infection. Results suggest that increased cAMP levels during HIV infection may have a dual and opposite roles. On the one hand, they could have a protective effect by limiting viral replication in infected cells and decreasing viral entry. On the other hand, they could have a detrimental role by reducing HIV-specific antiviral immune responses, thus reducing the clearance of the virus and contributing to T cell dysfunction. Future studies are thus needed to further define the beneficial versus detrimental roles of cAMP, as they could help establish new therapeutic targets to combat HIV replication and/or identify novel ways to boost antiviral immune responses.

In vitro susceptibility of Trypanosoma cruzi strains from Santander, Colombia, to hexadecylphosphocholine (miltefosine), nifurtimox and benznidazole.

INTRODUCTION: The current chemotherapy for Chagas disease is unsatisfactory with only two drugs available for treatment. Research to discover new drugs for Chagas disease is urgent. Hexadecyl-phosphocholine (HPC, miltefosine) has been demonstrated to have in vitro activity against Trypanosoma cruzi parasites, but its activity on different Colombian T. cruzi strains is not known. OBJECTIVE: To evaluate the in vitro susceptibility of T. cruzi strains isolated from humans and vectors in Santander, Colombia. to miltefosine, nifurtimox and benznidazole. MATERIALS AND METHODS: Eight T. cruzi Colombian strains and three reference strains (Esmeraldo, SilvioX10 and Y) were studied. Drug activities against extracellular epimastigotes and intracellular amastigotes were determined by microscopic counting. The results were expressed as the concentrations that inhibited 50% and 90% growth (IC50 and IC90). RESULTS: For miltefosine a similar range of drug activity was observed against all the Colombian strains, all parasites being more susceptible to miltefosine than to the reference drugs. The intracellular amastigotes were more susceptible to miltefosine (IC50 0.08 to 0.63 microM and IC90 0.21 to 2.21 microM) than extracellular forms (IC50 <0.92 to 2.29 microM and IC90 1.38 to 4.76 microM). For reference drugs, parasites were more susceptible to nifurtimox than to benznidazole and some differences in activity of benznidazole between T. cruzi strains was observed. CONCLUSIONS: The results showed the significant in vitro activity of miltefosine against T. cruzi stages, and the expected results for the reference drugs. Further in vivo studies with miltefosine are planned.

AMPc: una molécula clave en los eventos de regulación inmune y en el control de la replicación del VIH / cAMP: A keymolecule in events of immune regulation and in the control of HIV replication

El monofosfato de adenosina cíclico (AMPc) induce la activación de la proteína cinasa A, la cual regula negativamente la activación, la proliferación celular y la producción de IL-2, en células T. En células infectadas con el virus de inmunodeficiencia humana, el monofosfato de adenosina cíclico suprime la actividad de transcripción del promotor del virus y el paso del ADN viral del citoplasma al núcleo. El incremento del monofosfato de adenosina cíclico mediado por células T reguladoras CD4+, empleando la inyección de esta molécula en células blanco a través de las uniones comunicantes o empleando el eje CD39-CD73 para generar adenosina es utilizado para suprimir otras poblaciones celulares. En esta revisión se propone que la modulación del monofosfato de adenosina cíclico por las células T reguladoras CD4+ podría tener un papel dual durante la evolución de la infección por el virus de inmunodeficiencia humana. Su papel benéfico se centraría principalmente en el control de la replicación viral y factores de transcripción, o evitando la infección de nuevas células blanco por disminución en la expresión de los receptores virales. Paradójicamente, la segunda posibilidad es que el aumento del monofosfato de adenosina cíclico podría tener un papel perjudicial, debido al efecto negativo sobre la proliferación, activación, respuesta citotóxica y en la producción de citocinas que se observa durante la infección viral.

Cyclic adenosine monophosphate induces the activation of protein kinase A, which negatively regulates activation, proliferation and IL-2 production in T cells. In cells infected with human immunodeficiency virus, cyclic adenosine monophosphate suppresses the transcriptional activity of long terminal repeats and the amount of viral DNA from the cytoplasm to the nucleus. The increase in cyclic adenosine monophosphate mediated by CD4+ regulatory T cells, using either the influx of this molecule in target cells through the GAP junctions or by CD39-CD73 to generate adenosine, is used by CD4+ regulatory T cells to suppress other cell populations. In this review, we suggest that modulation of cyclic adenosine monophosphate by CD4+ regulatory T cells may have a dual role during the evolution of human immunodeficiency virus infection. The beneficial role would be mainly focused on the control of viral replication and transcription factors to replicate the virus, and/or preventing the infection of new target cells, decreasing the expression of the viral co-receptors. Paradoxically to this beneficial role, the second possibility is that increased cyclic adenosine monophosphate could have a detrimental role, due to the negative effect on proliferation, activation, cytotoxic response and cytokine production, which occurs during viral infection.

Regulación inmune durante la coinfección por el virus de la inmunodeficiencia humana y el Mycobacterium tuberculosis / Immune regulation during Human Immunodeficiency Virus and mycobacterium tuberculosis coinfection

Durante las infecciones crónicas la regulación inmune constituye un mecanismo esencial para controlar los procesos inflamatorios; sin embargo, la excesiva regulación impide el desarrollo de una respuesta efectora adecuada. Las células T reguladoras, las células dendríticas y algunas moléculas inhibitorias, como CTLA-4, PD-1, IL-10, TGF-ß dioxigenasa, participan en la modulación de la respuesta inmune contra el virus de la inmunodeficiencia humana (VIH) y Mycobacterium tuberculosis. La mayoría de los hallazgos sustentan un efecto negativo de la regulación durante ambas infecciones, debido a que permiten la replicación de los patógenos. La acumulación de células T reguladoras funcionales y la expresión de estas moléculas se han asociado a un mecanismo compensador, en respuesta a la hiperactivación celular y a una inducción directa por parte de los microorganismos. En la coinfección, el VIH favorece la reactivación de M. tuberculosis y el desarrollo de formas extrapulmonares de la enfermedad. La infección por M. tuberculosis facilita la entrada del virus a la célula blanco y su replicación. Asimismo, se evidencia un aumento del estado de hiperactivación inmune, junto a una menor respuesta efectora. Aunque la inmunopatogénesis durante la coinfección ha sido poco estudiada, es probable que el estado proinflamatorio y de hiperactivación, característico de ambas infecciones, facilita el desarrollo de mecanismos de regulación que alteren aún más el equilibrio de la respuesta protectora durante la coinfección y facilitan la gravedad de la enfermedad.

During chronic infections, the immune regulation is an important mechanism to control inflammatory processes; however, the excessive regulation prevents the development of an appropriate effector immune response. The regulatory T cells (Treg), dendritic cells (DC) and some inhibitory molecules such as CTLA- 4, PD-1, IL-10, TGF-ß e IDO take part in the modulation of the immune response against the human immunodeficiency virus (HIV) and the Mycobacterium tuberculosis (M.tb). Most of the evidence supports a negative effect of the immune regulation during both infections, due to the fact that they allow the active replication of the pathogens. The accumulation of functional Treg cells and the expression of these molecules have been associated with a compensating mechanism, in response to a cellular hyper-activation and to these microorganisms direct induction. During the co-infection, the HIV favors the reactivation of M.tb and the development of extra-pulmonary TB forms. The M.tb infection promotes the entry of the virus into target cells and its replication. Likewise, an increase of the immune hyper-activation state has been reported along with low effector responses. Although the immune-pathogenesis during the co-infection has not been extensively studied, most likely the pro-inflammatory and immunological hyper-activation state, typical of both infections, promotes the development of immune regulatory mechanisms that further disturb the balance between the protective and pathogenic responses during co-infection, favoring the illness severity.

In vitro susceptibility of Trypanosoma cruzi strains from Santander, Colombia, to hexadecylphosphocholine (miltefosine), nifurtimox and benznidazole

Introduction. The current chemotherapy for Chagas disease is unsatisfactory with only two drugs available for treatment. Research to discover new drugs for Chagas disease is urgent. Hexadecyl-phosphocholine (HPC, miltefosine) has been demonstrated to have in vitro activity against Trypanosoma cruzi parasites, but its activity on different Colombian T. cruzi strains is not known. Objective. To evaluate the in vitro susceptibility of T. cruzi strains isolated from humans and vectors in Santander, Colombia, to miltefosine, nifurtimox and benznidazole. Materials and methods. Eight T. cruzi Colombian strains and three reference strains (Esmeraldo, SilvioX10 and Y) were studied. Drug activities against extracellular epimastigotes and intracellular amastigotes were determined by microscopic counting. The results were expressed as the concentrations that inhibited 50% and 90% growth (IC50 and IC90). Results. For miltefosine a similar range of drug activity was observed against all the Colombian strains, all parasites being more susceptible to miltefosine than to the reference drugs. The intracellular amastigotes were more susceptible to miltefosine (IC50 0.08 to 0.63 ìM and IC90 0.21 to 2.21 ìM) than extracellular forms (IC50 <0.92 to 2.29 ìM and IC90 1.38 to 4.76 ìM). For reference drugs, parasites were more susceptible to nifurtimox than to benznidazole and some differences in activity of benznidazole between T. cruzi strains was observed. Conclusions. The results showed the significant in vitro activity of miltefosine against T. cruzi stages, and the expected results for the reference drugs. Further in vivo studies with miltefosine are planned.

Introducción. Los tratamientos actuales para la enfermedad de Chagas son insatisfactorios y sólo existen dos medicamentos disponibles. La búsqueda de alternativas terapéuticas es prioritaria. La hexadecilfosfocolina (miltefosina) ha mostrado actividad in vitro contra Trypanosoma cruzi. Sin embargo, su actividad en aislamientos de T. cruzi obtenidos en Colombia aún no ha sido reportada. Objetivo. Evaluar la susceptibilidad in vitro a miltefosina, nifurtimox y benznidazole de cepas de T. cruzi aisladas de humanos y vectores en Santander, Colombia. Materiales y métodos. Se evaluó la susceptibilidad de los tres medicamentos en ocho cepas colombianas de T. cruzi y tres cepas de referencia: Esmeraldo, Silvio X10 y Y. La actividad de los compuestos fue determinada en epimastigotes extracelulares y amastigotes intracelulares, por conteo microscópico. Los resultados se expresaron en concentraciones inhibitorias 50 y 90 (CI50 y CI90). Resultados. Para la miltefosina, se observaron rangos similares en la actividad del medicamento entre las cepas colombianas; todos los parásitos fueron más susceptibles a la miltefosina que a los medicamentos de referencia. Los amastigotes intracelulares fueron más sensibles a la miltefosina (CI50, 0,08 a 0,63 µM y CI90, 0,21 a 2,21 µM) que las formas extracelulares (CI50, <0,92 a 2,29 µM y CI90, 1,38 a 4,76 µM). En los medicamentos de referencia, los parásitos fueron más susceptibles al nifurtimox que al benznidazole. Se observaron algunas diferencias en la actividad del benznidazole en las cepas estudiadas de T. cruzi. Conclusiones. Los resultados obtenidos de la actividad in vitro de miltefosina y de los medicamentos de referencia contra aislamientos de T. cruzi son satisfactorios y serán considerados en estudios posteriores in vivo.