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. 2013 Jun 30;44(2):92-9.
eCollection 2013 Apr.

Expression and activation of intracellular receptors TLR7, TLR8 and TLR9 in peripheral blood monocytes from HIV-infected patients

Guillermo J Valencia Pacheco  1 , Francisc Pinzón Herrera  1 , Juan J Cruz López  1 , Ligia Del Carmen Vera Gamboa  1 , Norma Pavía Ruiz  1 , Adrián Santos Rivero  2 , Saulo Sánchez Lugo  2 , Fernando Puerto  1
Affiliations

Expression and activation of intracellular receptors TLR7, TLR8 and TLR9 in peripheral blood monocytes from HIV-infected patients

Guillermo J Valencia Pacheco et al. Colomb Med (Cali). .

Abstract
in English, Spanish

Introduction: TLR´s play a role in host defense in HIV infection recognizing the viral DNA or RNA. Their activation induces a signaling pathway that includes the proteins MyD88, IRAK4, TRAF6 and the transcription factor NF-kBp65.

Objective: To determine the expression of TLR7, TLR8 and TLR9, and activation of its signaling pathway in monocytes from patients infected with HIV. Methods. Expression of TLR7, TLR8 and TLR9 was determined in monocytes from HIV-infected patients (n= 13) and control subjects (n= 13), which were activated with specific ligands. The expression of MyD88 and NF-kBp65 were determined by flow cytometry; IRAK4 and TRAF6 were studied by immunoblotting.

Results: No statistical difference was found in the expression of TLR7, 8 and 9 in monocytes from patients compared to controls, but we observed the non-significant increased expression of TLR9 in patients. The activation showed no significant difference in the expression of MyD88 and NF-kBp65 in patients when compared to controls, but were decreased in stimulated cells over non-stimulated cells. IRAK4 and TRAF6 were not detected.

Conclusions: No statistical difference was observed in the expression of intracellular TLRs, MyD88 and NFkBp65 in monocytes from patients compared to controls. This was probably due to effective antiretroviral therapy being received at the time of study entry. Additional studies are needed under controlled conditions that include infected patients with and without ARVT, responders and non-responders, and work with different cell populations.

Introducción: En la infección por VIH los TLR juega un papel en la defensa del huésped al reconocer el ADN o ARN viral. Su activación induce la vía de señalización que incluye las proteínas MyD88, IRAK4, TRAF6 y el factor de transcripción NF-kBp65.

Objetivo: Determinar la expresión del TLR7, TLR8 y TLR9, y activación de su vía de señalización en monocitos de pacientes infectados con VIH.

Métodos: Se determinó la expresión de TLR7, TLR8 y TLR9 en monocitos de pacientes infectados con VIH (n= 13) y sujetos control (n= 13), se activaron con ligandos específicos y se determinó la expresión de MyD88 y NF-kBp65 por citometría de flujo. IRAK4 y TRAF6 fueron estudiadas por inmunoelectrotransferencia.

Resultados: No se observó diferencia estadística en la expresión de TLR7, 8 y 9 en los monocitos de pacientes con respecto a los controles, pero observamos aumento no significante del TLR9 en los pacientes. La activación no mostró diferencia significativa en la expresión de MyD88 y NF-kBp65 en pacientes con respecto a los controles, pero se encontraron disminuidas en células estimuladas con respecto a las no estimuladas. IRAK4 y TRAF6 no se detectaron.

Conclusiones: No se observó diferencia en la expresión de los TLR, ni en la expresión de MyD88 y NFkBp65, en monocitos de pacientes con respecto a los controles probablemente debido a la terapia antirretroviral recibida al momento del estudio. Se sugieren estudios con pacientes con y sin TARV, respondedores y no respondedores, y trabajar con diferentes poblaciones celulares.

Keywords: HIV; Innate immunity; monocytes; toll-like receptors.

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Conflict of interest statement

Conflict of interest : The authors declare that there is no real or potential conflict of interest regarding the possible publication of this work.

Figures

Figure 1
Figure 1. Expression of monocytes The PBMC of patients (n= 13) and control subjects (n= 13) were immunostained for DC14 and analyzed by flow cytometry as described in the methodology section. Data are presented as the mean and standard deviation of the percentage of positive DC14 cells. No significant differences between patient and control group (p > 0.05) were found. Statistical tests used: bilateral paired t and Mann-Whitney.
Figure 2.
Figure 2.. Expression of intracellular TLR in peripheral blood monocytes The PBMC (n= 13) of patients and control subjects (n= 13) were immune-stained for DC14, TLR7, TLR8, TLR9, and analyzed by flow cytometry, as described in material and methods section. Data are the mean and standard deviation of the percentage of DC14 positive cells expressing TLR7, TLR8, and TLR9 (A) and the mean fluorescent intensity (MFI) of TLRs (B). No significant difference between patients and control groups were found (p > 0.05). Statistical tests used; bilateral paired t and Mann-Whitney.
Figure 3
Figure 3. Expression of MyD88 (A) and NF-kBp65 (B) in peripheral blood monocytes. The PBMC (n = 12) of patients and control subjects (n = 12) were immune-stained for DC14, MyD88 and NF-kBp65 and analyzed by flow cytometry, as described in material and methods section. Data presented are the mean and standard deviation of the percentage of DC14 positive cells expressing MyD88 (A), and NF-kBp65 (B). No significant difference between patients and control groups were found (p> 0.05). Statistical tests used: bilateral paired t and Mann-Whitney.
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