Caracterización fenotípica y funcional del monocito como célula implicada en el estado de inflamación sistémica persistente en ancianos y en pacientes infectados por VIH-1 como modelo de envejecimiento prematuro

tttt 2 UNIVERSIDAD DE SEVILLA Facultad de Medicina Departamento de Medicina Doctorado en Biología Molecular y Biomedicina Caracterización fenotípica y funcional del monocito como célula implicada en el estado de inflamación sistémica persistente en ancianos y en pacientes infectados por VIH-1 como modelo de envejecimiento prematuro Tesis Doctoral REBECA DE PABLO BERNAL Directores D. EZEQUIEL RUIZ-MATEOS CARMONA D. MANUEL LEAL NOVAL Tutor D. JOSÉ VILLAR ORTIZ Sevilla, Octubre 2015 3 UNIVERSIDAD DE SEVILLA Facultad de Medicina Departamento de Medicina Doctorado en Biología Molecular y Biomedicina D. MANUEL LEAL NOVAL, Profesor Asociado del Departamento de Medicina de la Universidad de Sevilla e Investigador Responsable del Laboratorio de Inmunovirología del Instituto de Biomedicina de Sevilla (IBIS). D. EZEQUIEL RUIZ-MATEOS CARMONA, Investigador del Sistema Nacional de Salud por el instituto de Salud Carlos III, "Miguel Servet" y Co-Investigador Responsable del Laboratorio de Inmunovirología del Instituto de Biomedicina de Sevilla (IBIS). D. JOSÉ VILLAR ORTIZ, Catedrático del Departamento de Medicina, de la Universidad de Sevilla. CERTIFICAN: Que el trabajo titulado “Caracterización fenotípica y funcional del monocito como célula implicada en el estado de inflamación sistémica en ancianos y en pacientes infectados por VIH-1 como modelo de envejecimiento prematuro", presentado por la Licenciada en Veterinaria Dña. REBECA DE PABLO BERNAL, ha sido realizado bajo nuestra dirección y asesoramiento en el Laboratorio de Inmunovirología del Instituto de Biomedicina de Sevilla (IBIS). Concluido el trabajo experimental y bibliográfico, autorizamos la presentación y la defensa de esta Tesis Doctoral, para que sea juzgada por el tribunal correspondiente. Sevilla, a 14 de Octubre de 2016. Fdo. Dr. Manuel Leal Noval Fdo. Dr Ezequiel Ruiz Mateos Carmona Fdo. Dr José Villar Ortiz 4 Introducción El desarrollo y evolución en la terapia antirretroviral combinada (cART), por suprimir la replicación viral hasta niveles sanguíneos de Carga Viral (CV) indetectables, ha supuesto un hito en la historia de la infección por el Virus de la Inmunodeficiencia Humana tipo 1 (VIH-1), el acceso al cART en los países desarrollados ha favorecido la reducción de la morbilidad y mortalidad asociada a SIDA (1,2). Sin embargo, esta situación más favorable ha hecho emerger otra realidad clínica, caracterizada por una mayor prevalencia de enfermedades no SIDA (ENOS) (3). En este sentido, ha sido descrito por ciertos autores una mayor prevalencia y precocidad en el desarrollo de enfermedades como cáncer, cardiovascular, ósea, renal, diabetes y transtornos neurocognitivos (4,5) (también denominadas como enfermedades asociadas a la edad) en pacientes infectados por VIH bajo cART que en la población general de similar edad. Esto es debido, a que el cART podría no restaurar completamente el daño inmunológico que se produce en la infección por el VIH (6). En este sentido, incluso algunos autores han señalado que ciertos fármacos antirretrovirales podrían inducir efectos proinflamatorios y aumento de la activación celular, estas observaciones han sido descritas a partir de cultivos de células humanas in-vitro y modelos murinos in-vivo (7, 8), sugiriendo un papel activo y deletéreo sobre el sistema inmune de los pacientes infectados por VIH bajo ciertos cART. Por analogía con el envejecimiento natural, definido como la disminución progresiva de la respuesta inmunitaria que afecta a todos los componentes del sistema inmunológico tanto innato como adaptativo (9), lo que contribuye de manera importante a la morbilidad y mortalidad de los ancianos, ya que favorece una mayor incidencia y reactivación de enfermedades infecciosas (10), así como el desarrollo de patologías autoinmunes y cáncer (11-17). Ha sido sugerido por varios autores que el paciente infectado por VIH posee un proceso de envejecimiento prematuro y acentuado del proceso natural de senescencia (4, 18). Esto es debido a que el paciente infectado por VIH naïve y los ancianos no infectados por el virus a menudo presentan similares rasgos de desgaste inmunológico como reactivaciones de infecciones silentes como citomegalovirus (CMV), reducción de la función del timo, disminución del número de células naïve y expansión del número de células T CD57+, entre otros (19-28) (resumidos en Tabla 1), que además se asocian a la aparición de enfermedad en ambos escenarios (29). Tabla 1. Posibles similitudes entre los cambios inmunológicos asociados a la edad y a la infección por VIH. Parámetro VIH- ≥65años VIH+ naïve VIH+ cART Aumento activación céls.T Controvertido SÍ Posible Expansión céls.T CD57+ SÍ SÍ Sí Expansión clones específicos CMV SÍ SÍ SÍ Aumento títulos IgG anti-CMV SÍ SÍ SÍ Disminución ratio céls. naïve/memoria SÍ SÍ Posible Bajo potencial proliferativo céls. T SÍ SÍ Posible Reducida respuesta a vacunas SÍ SÍ Posible Reducción función del timo SÍ SÍ Desconocido Aumento niveles plasmáticos IL6 SÍ SÍ Desconocido 5 Sin embargo, si el tratamiento antirretroviral a largo plazo restaura esas alteraciones está siendo estudiado, siendo los defectos en el compartimento celular T, los mejores descritos hasta la actualidad. Si el tratamiento a largo plazo normaliza o reduce la alteración en marcadores inflamatorios solubles de pacientes infectados por VIH y en qué medida con respecto a poblaciones no infectadas de diferentes edades, es todavía incierto. Es por este motivo, que el primer objetivo de la presente tesis doctoral, fue comparar los niveles de mediadores de inflamación solubles en pacientes infectados por VIH naïves al tratamiento antirretroviral y bajo cART a largo plazo con dos poblaciones de referencia no infectadas por VIH de diferentes edades: 1) jóvenes, apareados por edad a la población infectada por VIH y 2) ancianos. Este objetivo fue analizado en el trabajo: “TNF-α levels in HIV-infected patients after long-term suppressive cART persist as high as in elderly, HIV-uninfected subjects.” (J Antimicrob Chemother. 2014). Los mecanismos implicados en el estado inflamatorio crónico de bajo grado observado tanto en pacientes como ancianos son desconocidos. Nosotros pensamos que una de las causas que podría asociarse a los altos niveles de marcadores proinflamatorios observados en ambos escenarios, podría ser la elevada activación de monocitos circulantes. Los monocitos, son esencialmente células efectoras y mediadoras de la respuesta inflamatoria (30,31). Los monocitos son fagocitos que derivan de la médula ósea, poseen vida corta (1-3 días en circulación sanguínea) y están implicados en la respuesta inmune innata ante patógenos (32-35). En humanos, recientemente se han descrito tres subpoblaciones de monocitos dependiendo de la expresión de dos de sus receptores de superficie el CD14 (receptor del complejo de lipopolisacárido y de la proteína de unión al lipopolisacárido o LBP) y CD16. (FcγRIII). Así se identifica una subpoblación de monocitos clásicos (CD14++CD16−), los intermedios (CD14++CD16+) y los no clásicos (CD14dimCD16+) (36). Uno de los rasgos a destacar entre las tres subpoblaciones es que presentan heterogeneidad: en tamaño, complejidad, receptores (de membrana e intracelulares). De forma interesante, en cuanto a la funcionalidad celular también se han descrito diferencias (37-39). La identificación de las tres subpoblaciones es relativamente reciente (2010) y actualmente su caracterización está siendo objeto de estudios básicos. A continuación se resumen las principales características de las tres subpoblaciones: A. Monocitos clásicos (CD14++CD16−): subpoblación de mayor tamaño y complejidad de núcleo, mayor capacidad de fagocitosis y productora de ROS in-vitro. Mayor respuesta frente a agonistas de TLR-2 y 4. B. Monocitos intermedios (CD14++CD16+): características intermedias entre las tres subpoblaciones en cuanto a tamaño y complejidad, mayor expresión en membrana de MHCII. C. Monocitos no clásicos (CD14dimCD16+): subpoblación más sencilla y pequeña. Respuesta ante agonistas endosómicos TLR 7 y 8. Implicada en el rodamiento y adhesión al endotelio vascular 0102103104105 <Qdot 655-A>: cd14 0 103 104 105 <PE-CF594-A>: cd16 24.7 10.8 64.4 A BC CD14 CD16 6 Algunos autores, han descrito alteraciones en marcadores de activación, así cómo en el número y distribución de las diferentes subpoblaciones de monocitos en el envejecimiento natural. Existen asociaciones entre el número de monocitos no clásicos, la disminución de la expresión de la selectina CD62L (40) y la disminución de la capacidad fagocítica con la edad avanzada (41). A pesar, de la relevancia de estas células como potencial mecanismo implicado en el estado de inflamación persistente que ha sido descrito en ancianos, los cambios fenotípicos, y funcionales y su relación con parámetros inflamatorios solubles y otros factores que podrían estar asociados como son, la infección latente por CMV y la enfermedad neurocognitiva en ancianos son desconocidos. El siguiente objetivo de la presente tesis doctoral, fue analizar los cambios fenotípicos y funcionales en las tres subpoblaciones de monocitos con la edad. Así como analizar su asociación con el estado de inflamación persistente observado en ancianos, y su relación con la infección latente por CMV y con el deterioro neurocognitivo. Este objetivo fue abordado en el trabajo: “Monocyte phenotype and polyfunctionality are associated with elevated soluble inflammatory markers, cytomegalovirus infection, and functional and cognitive decline in elderly adults.” (J Gerontol A Biol Sci Med Sci 2015, en prensa). Recientemente en el escenario de la infección por VIH han aparecido trabajos que describen la elevada activación de monocitos (42,43) y marcadores solubles asociados a estas células (44-46). Estas alteraciones, que se ha visto que incluso persisten en pacientes infectados por VIH bajo cART a largo plazo, además se han asociado con el desarrollo de ENOS en estos pacientes (47-49). En este sentido, la cuestión que se deriva de estas observaciones es si los mecanismos asociados a la alteración inflamatoria en pacientes bajo cART y en el envejecimiento natural son similares y en qué medida comparten rasgos. Recientemente, algunos autores han sugerido que los monocitos de pacientes presentan alteraciones compatibles con un envejecimiento prematuro de estas células (50, 51). Sin embargo, todavía la funcionalidad (en cuanto a la capacidad de producción de citoquinas ex-vivo e in-vitro) simple y compuesta por más de una citoquina de forma simultánea (polifuncionalidad) de los monocitos de pacientes bajo cART es desconocida. Por esto, el siguiente objetivo de esta tesis doctoral fue, caracterizar de forma detallada los cambios fenotípicos y funcionales de los monocitos de pacientes infectados por VIH bajo cART, incluyendo las tres subpoblaciones de monocitos, tanto ex-vivo como in-vitro y compararlo con los de una población no infectada por VIH envejecida. Este objetivo fue analizado en la publicación “Phenotype and polyfunctional deregulation involving IL-6- and IL-10- producing monocytes of HIV-infected patients on cART differ from those of aging” (The Journal of Infectious Diseases 2015, en prensa). En la Figura 1, se resumen los efectos de la infección por VIH-1 y su tratamiento en inflamación e inmunosenescencia. 7 Hipótesis y Objetivos Las hipótesis y objetivos de la presente tesis doctoral fueron: Hipótesis 1: el estado inflamatorio crónico de pacientes infectados por VIH-1 comparte alteraciones con el observado en envejecimiento natural. Presentando, sin embargo, rasgos propios que hacen que no sea compatible con el proceso natural de envejecimiento prematuro de su sistema inflamatorio. Objetivo 1: Comparar los niveles de mediadores de inflamación solubles en pacientes infectados por VIH naïves al tratamiento y bajo cART a largo plazo con dos poblaciones de referencia no infectadas por VIH de diferentes edades: jóvenes y ancianos. Este objetivo fue analizado en el trabajo: “TNF-α levels in HIV-infected patients after long-term suppressive cART persist as high as in elderly, HIV- uninfected subjects.” (J Antimicrob Chemother. 2014). Hipótesis 2: la activación y alteración en la función de monocitos con avanzada edad se asocia con el estado de inflamación crónica persistente observado en ancianos y con otros factores como la infección latente por CMV y deterioro neurocognitivo. Objetivo 2: analizar los cambios fenotípicos y funcionales en las tres subpoblaciones de monocitos con la edad. Así como analizar su asociación con el estado de inflamación persistente observado en ancianos, y su relación con la infección latente por CMV y con el deterioro neurocognitivo. Este objetivo fue abordado en el trabajo: “Monocyte phenotype and polyfunctionality are associated with elevated soluble inflammatory markers, cytomegalovirus infection, and functional and cognitive decline in elderly adults.” (J Gerontol A Biol Sci Med Sci 2015, en prensa). Hipótesis 3: los monocitos de pacientes infectados por VIH bajo cART presentan alteraciones fenotípicas y funcionales diferentes a las observadas en el envejecimiento natural. Objetivo 3: caracterizar de forma detallada los cambios fenotípicos y funcionales de los monocitos de pacientes infectados por VIH bajo cART, incluyendo las tres subpoblaciones de monocitos, tanto ex-vivo como in-vitro y compararlo con los de una población envejecida. Este objetivo fue analizado en la publicación “Phenotype and polyfunctional deregulation involving IL-6- and IL-10-producing monocytes of HIV-infected patients on cART differ from those of aging” (The Journal of Infectious Diseases 2015, ,en prensa). 8 Material, Métodos y Resultados TNF-alpha levels in HIV- infected patients after long-term suppressive cART persist as high as in elderly, HIV-uninfected subjects. De Pablo-Bernal RS, Ruiz-Mateos E, Leal M, et al. J Antimicrob Chemother 2014; 69:3041–6 9 16 17 18 19 20 21 22 23 24 Phenotype and polyfunctional deregulation involving IL- 6 and IL-10-producing monocytes of HIV- infected patients on cART differ from those of aging De Pablo-Bernal RS, Ruiz-Mateos E, Leal M, et al. The Journal of Infectious Diseases, 2015 (en prensa) 25 Title: Phenotype and polyfunctional deregulation involving IL-6- and IL-10- producing monocytes of HIV-infected patients on cART differ from those of aging De Pablo-Bernal RS1, Ramos R2, Genebat M1, Cañizares J2, Benhnia MR1,3, Muñoz- Fernández MA4, Pacheco YM1, Galvá MI2, Leal M1#, Ruiz-Mateos E1#* #these authors contributed equally to this work 1 Laboratory of Immunovirology, Clinic Unit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine of Seville, IBiS, Virgen del Rocío University Hospital/CSIC/University of Seville, Seville, Spain 2 Heliopolis nursing home, Seville, Spain 3Department of Biochemistry and Molecular Biology & Immunology, Medical School, University of Seville, Seville, Spain 4 Laboratory of Molecular Immuno-Biology Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBERBBN), Madrid, Spain. Running head: Monocyte aging and chronic HIV Text word count: 3418 Abstract word count: 196 *Correspondence: Ezequiel Ruiz-Mateos, (PhD), Immunovirology Laboratory (Lab 211), Institute of Biomedicine of Seville, Virgen del Rocío University Hospital, Avda Manuel Siurot s/n CP 41013, Seville, Spain ([email protected]) +34955923107. 32 RESULTS Characteristics of the study subjects The demographic and immunovirological characteristics of all study participants are shown in Table 1. The HIV and Young groups were predominantly men, while only 45% of the elderly subjects were male. The median age was 83 [67-90] years for this group. The absolute total monocyte counts in the HIV group were similar to the Elderly counts but higher than the Young counts. We did not find differences in the frequency of monocyte subsets between the groups (data not shown).Of the 60 participants, 100% were CMV-seropositive. Similar higher anti-CMV-IgG titers were found in the HIV and Elderly when compared to the Young group. The HIV group showed a median nadir CD4 T cell count of 330 [266-375] cells/mm3, the median time since HIV+ diagnosis was 6 [3-9] years and the median time since HIV-RNA was undetectable in plasma was 6 [3-9] years. None of HIV-infected patients had histories of prior Category C clinical stage. Neither patients nor controls had prior HCV exposure (anti-HCV negative) and all of them were HBsAg negative. HIV-infected patients showed a more activated phenotype of monocytes than the elderly Figure 1 displays the percentage of surface expression of the activation and cell adhesion markers CD163, CD49d, CD62L, CD40, CD11b and CD38 in the three subsets of monocytes among the groups. The expression of the scavenger receptor CD163 in the HIV group was remarkably higher compared to both control groups in the three subsets of monocytes (p<0.001 for all comparisons). The expression of integrin CD49d, implicated in homing and stabilizing the adhesion of leukocytes to endothelial cells, was much higher in the HIV group compared to expression levels in both control groups in the classical and non-classical subsets (p<0.001 for all comparisons). Additionally, the HIV group expressed the highest levels of CD62L, implicated in homing and cell adhesion and migration, in the classical and intermediate monocytes (p=0.03 and p=0.001, respectively). The percentage of cells that expressed the costimulatory protein CD40 was significantly higher in the HIV group, compared to both control groups, only in the non-classical subset (p<0.001). The expression of CD11b, which mediates leukocyte adhesion and migration, was higher in the HIV group in the intermediate subset (p=0.001). No differences were found for CD38 expression among the groups. 33 HIV-infected patients showed higher levels of monocytes producing single and multiple cytokines under unstimulated conditions and in response to TLR agonists, when compared to elderly donors We addressed the characterization of the functionality, represented as IL-1α, IL- 1β, IL-6, IL-8, IL-10 and TNF-α intracellular production in the unstimulated condition, and the responsiveness to LM-MS (TLR2 agonist), LPS (TLR4 agonist) and ssRNA40 (TLR7 agonist) in vitro of the three monocyte subsets. Note, that the conditions utilized minimize the shedding of CD16 and allowed the identification of the non-classical subset (Supplementary Figure 1). The HIV group showed a significantly higher percentage of the three monocyte subsets that produced IL-6 and IL-10 (Table 2) under unstimulated conditions. Similar results were observed for the median intensity fluorescence (MFI) of positive cells (Supplementary Table 1). Figure 2A shows the percentage of classical monocytes producing simultaneous single and multiple cytokines per cell under unstimulated conditions. For the multiple combination of six cytokines, IL-1α+, IL-1β+, IL-6+, IL- 8+, IL-10+ and TNF-α+ are represented as, a+b+6+8+10+T+, single IL-6 production is represented as a-b-6+8-10-T- and IL-10 as a-b-6-8-10+T-. These functionalities were higher in the HIV group than in the controls (p<0.001, for all comparisons) (Figure 2A). The polyfunctional distribution of cytokines was different among groups (p<0.001, for all comparisons) in the unstimulated condition (Figure 2B). Remarkably prominent was the production of IL-10 in HIV-infected subjects (arcs in Figure 2B), particularly in the 1+ function. This high monofunctional distribution, likely due to the highly IL-10- producing monocytes (Figure 2B), overshadows the fact that monocytes from the HIV group are the highest producers of the a+b+6+8+10+T+ polyfunctionality (Figure 2A). In vitro, the HIV group showed a significantly higher percentage of classical monocytes that produced intracellular IL-10 in response to LM-MS and LPS (Figure 3 and Supplementary Table 2). Similar results were observed analyzing the data as MFI (Supplementary Table 3). Multiple cytokine production was higher in the HIV group after stimulation with LM-MS, LPS, and ssRNA40 in the classical and intermediate monocytes, (Figure 4A). It is notable that the simultaneous composition of six cytokines was only observed in the HIV group. A more in-depth analysis of the 63 possible multiple cytokine functionality profiles in response to TLR agonist in vitro revealed that the cells producing IL-10 (10+) in combination with other cytokines was significantly higher in the HIV group. For instance, Figure 4B displays the multiple combinations of 34 six and five simultaneous cytokines in response to LPS in the classical monocytes among groups. Note that the HIV group displays the highest levels in all the multiple combinations that include IL-10, but not in the absence of IL-10. Additional information about single cytokine representation among polyfunctional distribution is shown in arcs in Supplementary Figure 2. Association of soluble levels of inflammatory and coagulation biomarkers and the monocyte activation and functionality We determined the soluble concentrations of inflammatory and coagulation biomarkers. The IL-1β and IL-10 levels were below the detection limit of the assay. We found that the IL-6 levels were significantly higher in the HIV group compared to the young control group with a trend for hsCRP and β2M (Supplementary Table 4). Interestingly, we found that in the unstimulated condition, the single cytokine production was positively and strongly correlated with soluble inflammatory levels in the three monocyte subsets when analyzing all study subjects as a whole group (n=60). Remarkably, the β2M, D-dimer and hsCRP levels were strongly and positively associated with the production of almost all of the pro-inflammatory intracellular cytokines, especially in the classical and intermediate subsets (Supplementary Figure 3). However, most of these associations were lost after stratification into groups (young, HIV and elderly), probably due to the small number of studied cases. Interestingly, the analysis of the HIV group separately revealed strong associations only significant for this subgroup. There was a strong inverse correlation between sCD14 levels and the IL- 10 production (p=0.009, rho= -0.594) in the intermediate subset without stimulation and between the sCD163 levels and the IL-8 production in intermediate and non-classical subsets of monocytes also, in the unstimulated condition (p=0.003, rho= -0.629; p=0.016, rho= -0.531, respectively). When this analysis was applied to multiple cytokine combinations, in the three subsets, the polyfunctionality in all participants was also positively correlated with the soluble inflammatory markers in the unstimulated condition (data not shown). Regarding the phenotype of activation markers, we found strong associations among activation cell surface markers on monocytes for the three groups of study. There was a strong positive correlation between CD11b and CD38 in the classical subset and a strong inverse association between CD49d and CD62L in the intermediate subset for each group (data not shown), strikingly the most significant and strongest associations between these markers were found for the HIV group (p<0.001, rho= 0.818; p<0.001, 35 rho= -0.68, respectively) Also, we have found associations between the activation phenotype and the soluble inflammatory concentrations. Of special interest were the associations of the scavenger receptor CD163 with the following in the elderly: sCD14 (p=0.045; rho=0.465), IL-6 (p=0.020; rho=0.542), hsCRP (p=0.029; rho=0.500) and β2M (p=0.004; rho=0.634). These associations were lost for all the plasma markers in the HIV group, with the exception of the sCD14 levels (p=0.028; rho=0.495). 36 DISCUSSION Our results demonstrate profound phenotypic and functional alterations in the three monocyte subsets of HIV-infected patients on cART. Moreover, we showed that those alterations described in patients are different from the age-related changes. The results presented herein are the first to comprehensively characterize higher single and multiple intracellular cytokine production by monocytes both under unstimulated conditions and in response to TLR agonist in vitro in HIV patients when compared to uninfected, agedmatched and elderly subjects. The results of the present study add novel evidence to the relevance of monocytes hyperactivation in the HIV infection scenario. Monocytes from immunologically wellconserved, young HIV-infected individuals on cART exhibited a higher expression of activation markers, which are implicated in activation, homing, migration and also stabilization of the adhesion of leukocytes to endothelial cells [9, 10, 14-16], even when compared to the elderly group (over 40 years older than the HIV group). These profound alterations may, therefore, promote the development of inflammatory diseases in HIV-infected patients by mechanisms different from those found in age-related illnesses. Regarding polyfunctionality, this work adds a new concept to innate immunosenescence. In the T-cell scenario, immunosenescence is related to the changing phenotypic composition of T cells with age and impaired T-cell polyfunctionality [17]. In the HIV field, enhanced polyfunctionality in HIV-1-specific CD8+ T cells has been associated with better control of the virus [18]. Our results suggest that monocyte functionality in HIV-infected patients and controls is not desirable, because the production of intracellular cytokines in the unstimulated condition is positively correlated with soluble inflammatory and coagulation biomarkers that are known to be associated with increased morbidity and mortality risk. Researchers have debated whether individuals with HIV infection experience accelerated aging (health risks associated with early aging), accentuated aging (health risks associated with aging are greater), or both. These speculations arise from the vast previous evidence from epidemiological studies [19, 20]. The critical question is whether HIV infection shares common mechanisms with the natural process of aging. Our results demonstrate that the concept of premature aging of monocytes and consequently the premature aging of the inflammatory systems of HIV-infected patients is a simplification of the mostly unknown and complex underlying alterations that HIV 37 infection produces in innate immune cells, which appear to be different from those of aging. Monocytes are the main producer of IL-10 [21]; this cytokine is an important regulator of myeloid cells and has an anti-inflammatory role by down-regulating the capacity of monocytes to secrete inflammatory mediators, including IL-1α and TNF-α [22], providing a negative-feedback control loop. Here, we have observed an immunosuppressive mechanism that failed to inhibit proinflammatory regulation and homeostasis balance in HIV-infected patients on cART and that differs consistently from inflammatory homeostasis in aged subjects. The high levels of IL-10-producing monocytes found in the HIV group might be due to an unsuccessful attempt to reduce the high IL-6 plasma concentrations and the intracellular IL-6 levels observed in monocytes from these patients. One possible explanation of the high percentage of IL- 10-producing monocytes that we have described as a specific alteration found in HIV patients, could be that the HIV trans-acting regulatory protein (Tat) directly induces IL- 10 production by monocytes [23], interestingly through a CD14 independent mechanism [24]. A recent report has also described that Tat up-regulates PD-L1 expression on monocyte-derived dendritic cells (MoDCs) through TNF-α- and TLR4- mediated mechanisms, modulating the PD-1/PD-L1 T-cell coinhibitory pathway of antigen-presenting cells by hijacking the TLR4 pathway [25]. In the view of these results, the involvement of the Tat/TLR4/TNF-α pathway in the modulation of PD- 1/PD-L1 and IL-10 production in monocytes in HIV-infected patients deserves further research. Our study was limited by the small number of patients; however, the differences among the groups were clear. Other fact is that with the present study design we could not analyze the effect of cART on the activation of monocytes. It has been described in in vivo and in vitro studies of human cells and murine models that some nucleoside reverse-transcriptase inhibitors induced the activation of leukocytes [26, 27]. It will be interesting to analyze in further studies in what extent cART and the different types of antiretroviral treatments potentially affect the monocyte function. Finally, with this study design we could not analyze any cause and effect relationship to the monocyte activation; to achieve this objective longitudinal studies are now being conducted. In summary, the chronic phenotype and inflammatory alterations associated with monocytes in HIV infection are maintained at a different level from those of aging. Our data points toward a crucial role of endogenously produced IL-10 in monocytes from 38 HIV-infected patients and provide evidence for an inflammatory homeostasis deregulation mechanism unable to constrain IL-6 production. The modulation of activated monocytes may have important clinical implications for mitigating the development of non-AIDS events (the main cause of morbidity and mortality in HIV- infected patients) and also for the design of therapeutic strategies in other chronic inflammatory conditions. 39 FIGURE LEGEND Figure 1. HIV-infected patients exhibited a more activated phenotype of monocytes than the elderly. Percentage of surface expression of activation markers of monocyte subsets in HIV-infected patients on cART (HIV) and in two different HIV-uninfected controls: young (Y) and elderly (E) groups. The Mann–Whitney U test was used to compare the levels of activation markers between the HIV and different-age control groups. *p< .05 and ** p< .001. ns, not significant. Figure 2. A) Percentage of classical monocytes producing six simultaneous cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-10 and TNF-α) and single IL-6 and IL-10 production in the unstimulated condition between groups. * p< .05, ** p< .001 and ns, not significant. B) Polyfunctional cytokine distribution in the three subsets of monocytes under unstimulated conditions. Colors represent the simultaneous production of up to six cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-10 and TNF-α) by monocytes among the groups (young (Y) and elderly (E)). The IL-10 production in polyfunctional distribution is shown in arcs. Statistically significant differences between the groups are shown as * p< .05,** p< .001 and ns, not significant. Pestle version 1.6.2 and Spice version 5.2 (M. Roederer. NIH. Bethesda. MD) were used. Figure 3. The percentage of classical monocytes producing intracellular IL-10 in response to LM-MS and LPS between HIV-infected patients on cART (HIV) and different HIV-uninfected controls: young (Y), elderly (E) groups. The responsiveness of cells is represented as the difference between the stimulated and unstimulated values. The Mann–Whitney U test was used to compare the levels of activation markers between the HIV and different-age control groups. * p< .05, ** p< .001 and ns, not significant. Figure 4. A) Polyfunctional cytokine distribution of the responsiveness in vitro to LMMS, LPS and ssRNA40 in the three subsets of monocytes among the groups (young (Y), elderly (E) groups). Pie charts show classical, intermediate and non-classical monocytes with up to six functional responses. Statistically significant differences between the findings are shown. * p< .05, ** p< .001, and ns, not significant. p <0.1 is represented with Ω. B) The percentage of classical monocytes simultaneously producing multiple combinations of six and five cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-10 and TNF-α) between groups in response to in vitro stimulation with LPS. Pestle version 1.6.2 and Spice version 5.2 (M. Roederer. NIH. Bethesda. MD) were used. * p< .05, ** p< .001 and ns, not significant. Supplementary Figure 1. A) Schematic diagram of the gating strategy of a representative HIV-infected subject. Note that the conditions utilized minimize the shedding of CD16 and allowed the identification of the non-classical subset.B) Histogram representation of cytokine production on classical monocytes under unstimulated conditions (grey line) and after LPS stimulation (red line). Supplementary Figure 2. Pie charts show the polyfunctional distribution of monocytes with up to six cytokines in response to LPS stimulation. Single cytokine representation among polyfunctional distribution is shown in arcs. Statistically significant differences between the findings are shown as * p< .05, ** p< .001 and ns, not significant. Pestle 40 version 1.6.2 and Spice version 5.2 software (M. Roederer. NIH. Bethesda. MD) were used. Supplementary Figure 3. A) Associations between soluble inflammatory markers and the intracellular cytokine production in the three subsets of monocytes for all study subjects (n=60). Spearman’s ρ correlation coefficient test was used. The p value is represented in italics and the rho value in bold. Statistical significance <0.05 is colored in black cells, p values <0.1 but >0.05 are colored in grey cells. B) Representative correlations of intracellular cytokine production and plasmatic concentrations in the three monocyte subsets for all study subjects (n=60). FUNDING: This study was supported by Redes Tematicas de Investigacion en SIDA (ISCIII RETIC RD12/0017/0029 and RD06/0006/0035), Proyecto de Excelencia, Consejeria de Innovacion, Ciencia y Empresa (P11-CTS-06313) and Consejeria Andaluza de Salud (PI-0278-2010). ERM received grants from Instituto de Salud Carlos III (CPII014/00025). This work was supported in part by a Ramon y Cajal grant from Ministry of Economy and Competitiveness RYC-2010-07419 to Benhnia RM. ACKNOWLEDGMENTS: The authors want to express their most sincere thanks to all of the volunteers of the HELIOPOLIS nursing home and the young control volunteers. We are grateful to Magdalena Rodriguez, Marien Gutierrez Sancho, and Ana Maria Guijarro from the Day Care Hospital (Infectious Diseases Department) and to Antonio Ángel Lugo for their outstanding assistance. We also want to thank María del Mar Rodriguez for her technical support. CONFLICT OF INTEREST: The authors do not have any associations that might pose a conflict of interest. AUTHOR CONTRIBUTIONS: RPB, ERM and ML designed the study.RPB produced the experimental data in the laboratory. RPB analyzed the data and prepared the manuscript with critical review from all of the authors. *Correspondence: Ezequiel Ruiz-Mateos, (PhD), Immunovirology Laboratory (Lab 211), Institute of Biomedicine of Seville, Virgen del Rocío University Hospital, Avda Manuel Siurot s/n CP 41013, Seville, Spain ([email protected]) +34955923107 41 REFERENCES 1. Deeks SG. The end of AIDS: HIV infection as a chronic disease. Lancet 2013; 382:1525-33. 2. Kalayjian RC, Landay A, Pollard RB et al. Age-related immune dysfunction in health and in human immunodeficiency virus (HIV) disease: association of age and HIV infection with naive CD8+ cell depletion, reduced expression of CD28 on CD8+ cells, and reduced thymic volumes. J Infect Dis 2003; 187:1924–33. 3. Vestergaard LS, Sembuche S et al. Plasma cytokine levels in Tanzanian HIV-1- infected adults and the effect of antiretroviral treatment. J Acquir Immune Defic Syndr 2009; 52:493-7. 4. Franceschi C, Capri M, Monti Det al. Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans. Mech Ageing Dev 2007; 128:92-105. 5. Singh T, Newman AB. Inflammatory markers in population studies of aging. Ageing Res Rev 2011; 10:319-29. 6. Kuller LH, Tracy R, Belloso W et al. Inflammatory and coagulation biomarkers and mortality in patients with HIV infection. PlosMed 2008; 5: e203. 7. Cesari M, Penninx BW, Newman AB et al. Inflammatory markers and onset of cardiovascular events: results from the Health ABC study. Circulation 2003; 108:2317–22. 8. De Pablo-Bernal RS, Ruiz-Mateos E, Rosado I, et al. TNF-α levels in HIV-infected patients after long-term suppressive cART persist as high as in elderly, HIV- uninfected subjects. J Antimicrob Chemother 2014; 69:3041–6. 9. Hearps AC, Martin GE, Angelovich TA, et al. Aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function. Aging Cell 2012;11:867–75. 10. Hearps AC, Maisa A, Cheng W-J, et al. HIV infection induces age-related changes to monocytes and innate immune activation in young males which persist despite cART. AIDS 2012; 26: 843–53. 11. Cros J, Cagnard N, Woollard K, et al. Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity 2010;33:375–86. 12. De Pablo-Bernal RS, Cañizares J, Rosado I, et al. Monocyte phenotype and polyfunctionality are associated with elevated soluble inflammatory markers, 48 Supplementary Table 3. Median fluorescence intensity (MFI) of the intracellular cytokine production of monocytes in response to TLR agonists among groups Values are expressed as Median and IQR. The Mann Whitney –U test was used to compare values between HIV and control groups. Cells are colored when HIV is different from both control groups. Bold values indicate when HIV is significantly different from both control groups. In-vitro LMMS LPS ssRNA40 p Elderly (1) HIV (2) Young (3) Elderly (1) HIV (2) Young (3) Elderly (1) HIV (2) Young (3) LMMS LPS ssRNA40 1 vs 2 2 vs 3 1 vs 2 2 vs 3 1 vs 2 2 vs 3 Classical (CD14++CD16-) IL-1α 13 [4-53] 32 [8-60] 1 [0.0-27] 50 [8-85] 91[48-172] 10 [0-26] 10 [0.0-36] 176 [46-225] 87 [0-172] 0.4 0.02 0.04 <0.001 <0.001 0.1 IL-1β 647 [430-1069] 433 [58-775] 51 [0-611] 2263 [1106-2794] 2210 [749-4326] 691 [17-1393] 1194 [346- 3137] 2590 [1452- .5619] 2586 [1076-4574] 0.04 0.1 0.8 0.06 0.07 0.8 IL-6 302 [215-357] 383 [192-441] 210 [17-407] 1213 [646-1868] 1472 [401-2714] 560 [331-1260] 219 [32-726] 247 [25-1139] 804 [150-1174] 0.1 0.03 0.4 0.7 0.6 0.9 IL-8 1083 [773- 1645] 1096 [776-1612] 839 [255-1205] 3347 [2407-4977] 3134 [1541- 6010] 1381 [997-3521] 383 [56-546] 1112 [378-1724] 800 [216-1703] 0.9 0.03 0.9 0.02 0.012 0.6 IL-10 2 [0-19] 5 [3-19] 1 [0-4] 0 [0.0-36] 4 [0-10] 0 [0-3] 0.0 [0.0-13] 0.0 [0.0-6] 6 [0-22] 0.06 0.001 0.02 0.02 0.7 0.09 TNF-α 718 [245-1343] 363 [67-660] 336 [6-826] 1207 [505-2324] 1258 [518-3419] 419 [0-1493 1841 [504- 3918] 5597 [4290- 7231] 3037 [1384-5882] 0.06 0.9 0.6 0.9 <0.001 0.1 Intermediate (CD14++CD16+) IL-1α 20 [3-55] 19[0-38] 18 [0-118] 69 [40-148] 98 [12-170] 11 [0-74] 7 [0-63] 100 [28-175] 66 [5-273] 0.6 0.7 0.8 0.03 0.05 0.8 IL-1β 668 [491-1428] 561 [382-986] 280 [13-614] 3262 [1694-3728] 1623 [628-5136] 872 [203-1601] 420 [179-1182] 719 [191-2338] 1160 [55-5045] 0.1 0.03 0.6 0.04 0.3 0.4 IL-6 438 [280-667] 170 [83-357] 165 [0-558] 1652 [862-2239] 713 [287-3501] 542 [48-1726] 86 [29-514] 36 [0-107] 294 [27-2993] 0.01 0.5 0.3 0.2 0.08 0.07 IL-8 693 [430-1068] 608 [328-1176] 334 [21-820] 2356 [980-4269] 1238 [613-6035] 1190 [353-1763] 112 [33-275] 159 [79-373] 536 [123-1847] 0.9 0.03 0.6 0.2 0.4 0.05 IL-10 0 [0-15] 3 [0-7] 2 [0-6] 0 [0-16] 6 [0-15] 4 [0-13] 0 [0-18] 2 [0-9] 1 [0-15] 0.6 0.9 0.6 0.8 0.7 0.7 TNF-α 1306 [929- 2291] 493 [46-965] 38 [0-410] 2540 [1382-5176] 1396 [592-4485] 445 [0-2414] 1368 [369- 4388] 1679 [1208- 6650] 2697 [447-10856] 0.03 0.6 0.2 0.1 0.2 0.6 Non-classical (CD14+CD16++) IL-1α 8 [0-27] 16 [0-68] 0 [0-3] 0 [0-33] 0 [0-32] 12 [0-30] 7 [0-14] 0.0 [0.0-62] 19 [0-75] 0.3 0.1 0.9 0.4 0.9 0.3 IL-1β 144 [0-312] 92 [0-182] 0 [0-283] 147 [0-360] 83 [0-273] 260 [0-400] 5 [0-201] 3 [0-30] 0 [0-479] 0.6 0.6 0.6 0.2 0.3 0.4 IL-6 42 [0-102] 14 [0-271] 159 [0-650] 119 [23-219] 61 [0-415] 57 [0-196] 29 [0-208] 0 [0-22] 5 [0-220] 0.8 0.3 0.8 0.6 0.05 0.2 IL-8 57 [0-187] 26 [0-68] 107 [0-493] 145 [0-343] 44 [0-146] 160 [74-366] 25 [0-136] 0 [0-47] 52 [0-400] 0.3 0.1 0.1 0.01 0.3 0.1 IL-10 3 [0-20] 3 [0-23] 6 [0-21] 8 [2-12] 2 [0-4] 3 [0-10] 0 [0-4] 2 [0-4] 1 [0-1] 0.8 0.4 0.05 0.2 0.8 0.6 TNF-α 72 [0-356] 84 [9-272] 166 [0-448] 97 [0-376] 145 [10-324] 74 [0-195] 0 [0-62] 17 [0-67] 100 [0-206] 0.9 0.7 0.7 0.3 0.4 0.3 49 Supplementary table 4. Soluble levels of inflammatory and coagulation biomarkers. Biomarker Elderly (n=20) HIV (n=20) Young (n=20) p (HIV vs. Elderly) p (HIV vs. Young) IL-8 (pg/mL) 17.8 [10.1-36.4] 10.9 [7.1-15.2] 8.1 [6.9-18.5] 0.04 0.7 IL-6 (pg/mL) 3.3 [1.8-4.2] 1.7 [0.9-2.1] 0.7 [0.5-1.6] 0.01 0.03 TNF-α (pg/mL) 2.6 [1.9-3.3] 2.1 [1.1-2.9] 1.7 [1.1-2.4] 0.1 0.2 sCD14 (ng/mL) 8396.9 [7800.5-12089.6] 9014.8 [7157.0-11588.6] 7984.8 [6304.5- 10159.9] 0.7 0.4 hsCRP (mg/L) 2.6 [1.0-4.5] 1.1 [0.5-2.4] 0.6 [0.4-1.4] 0.1 0.1 β2-microglobulin (mcg/mL) 2.5 [2.1-3.3] 1.6 [1.5-1.8] 1.5 [1.4-1.7] <0.001 0.1 D-dimer (µg/L) 820.0 [392.5-1647.5] 170.0 [170.0-465.5] 180.0 [170.0-250.0] <0.001 1 sCD163 (ng/mL) 1423.2 [1123.1-1651.0] 931.4 [657.9-1124.2] 778.3 [561.9-1184.4] <0.001 0.4 Median and IQR values are presented. The Mann–Whitney U test was used to compare the levels of inflammatory markers between group 50 51 52 53 54 55 56 57 Discusión La presente tesis doctoral profundiza en el conocimiento del proceso de inmunosenescencia prematura en el contexto de la infección por VIH. Concretamente, aporta datos que sugieren que el paciente infectado por VIH-1 bajo cART no posee un envejecimiento prematuro ni de su sistema inflamatorio, ni de su sistema inmunitario innato, (centrándonos para ello en el estudio del monocito). Para ello, en primer lugar, describimos que los niveles de mediadores de inflamación solubles en pacientes infectados por VIH son diferentes a los observados en ancianos. En segundo lugar, caracterizamos la activación del monocito como posible mecanismo implicado en esa alteración del estado inflamatorio en ancianos y analizamos los posibles factores asociados en el envejecimiento natural; para con ello, en tercer lugar, poder compararlos con los observados en pacientes bajo cART y así concluir que los monocitos de este tipo de pacientes presentan una mayor polifuncionalidad y producción de IL-6 e IL-10 que no es compatible con un envejecimiento prematuro de estas células en el contexto de la infección por VIH. En nuestras latitudes, en las que el acceso a la terapia antirretroviral combinada supresora de la CV es ampliamente extendida. Uno de los puntos más relevantes en relación a la patogénesis de la infección por VIH-1 es el manejo clínico del paciente bajo cART. Este tipo de pacientes, posee una creciente esperanza de vida (52), lo que conlleva que cada vez haya más pacientes que envejecen con la infección (5, 52, 53). En la actualidad, la mayoría de causas de muerte y enfermedad entre este tipo de pacientes son de origen inflamatorio (54-56). Por esto, profundizar en la caracterización de la alteración inflamatoria y en el estudio de los mecanismos asociados es una necesidad y un objetivo primordial por parte de la comunidad científica, que permitan desarrollar terapias de intervención en estos pacientes En la presente tesis doctoral describimos que los niveles de marcadores inflamatorios en pacientes infectados por VIH eran diferentes a los observados no sólo cuando comparábamos una población control de similar edad, sino incluso al compararlos con una población envejecida. Así mismo, comunicamos que los niveles de TNF-α persistían elevados en esos pacientes a similares niveles a los observados en ancianos. Además, describimos que aquellos pacientes que poseían un mayor tiempo desde el diagnóstico de la infección, presentaban mayores niveles de IL-6, añadiendo más evidencia a la necesidad de diagnosticar y tratar a los pacientes en el menor tiempo posible (57-59). A la luz de esos resultados, discutimos en ese artículo que una de las causas que podrían estar asociadas a los altos niveles de marcadores proinflamatorios observados tanto en ancianos como en pacientes infectados por VIH, podría ser la elevada activación de monocitos circulantes, siendo éstos las células inmunitarias productoras de citoquinas proinflamatorias entre ellas, por excelencia, el TNF-α. Los monocitos, son células heterogéneas. La caracterización de la funcionalidad de las tres subpoblaciones de monocitos en humanos es objetivo de estudios a nivel básico. Nosotros en la presente tesis doctoral, detallamos nuevos aspectos funcionales de las tres subpoblaciones con el 64 30. Auffray C, Sieweke MH, Geissmann F. 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