Final Report Summary - MD-THIV (Migration and Differentiation of Th17 Cells in HIV/SIV Infection)
More than 25 years after the discovery of human immunodeficiency virus (HIV) as the causative agent of AIDS, the mechanisms governing pathogenesis and disease progression are still not fully understood. Indeed, a progressive impairment of the immune system, with alterations that affect both innate and adaptive immunity, characterises HIV-1 infection.
The loss of CD4+ CCR5+ T cells in the gut associated lymphoid tissue (GALT) has been well documented both in natural host and in pathogenic models of SIV infection. A decrease in the frequency of Th17 cells, a newly discovered subset of effector T cells involved in the immune response against extracellular bacteria, has been described, in the mucosa of SIV infected rhesus macaques and in HIV infected individuals. Th17 cells are primarily localised at mucosal sites and they express the homing marker for the skin and the gut CCR6. Recent data show that CCR6+ cells and therefore Th17 cells are more susceptible to HIV-1 infection compared to other subset of T-helper cells, thus suggesting a direct effect of the virus on Th17 cell depletion. Indeed the mechanisms that are responsible for Th17 cell loss during HIV infection have not been investigated in details so far.
Aim of this project was to investigate the mechanisms that mediate Th17 cells trafficking and activities at mucosal sites together with their decrease in frequency during HIV infection. We have also focused on understanding how changes in chemokines/cytokines expression during infection might influence the migratory capacity and differentiation of Th17 cells to mucosal sites.
In order to achieve the research objectives we set up collaborations with the German Primate Center (G?ttingen, Germany) for the analysis of the macaque model of SIV-infection and with the Ospedale Regionale di Lugano (Lugano, Switzerland) for the study of HIV infected patients. The following four objectives were achieved during the research project:
1) Characterise and quantify IL-17 producing cells in the GALT
We have analysed the phenotype of IL17+ cells both on healthy human gut tissues and on biopsies from healthy rhesus macaques. Our analysis demonstrates that a variety of cells, among which T-cells, neutrophils and mast-cells, express IL-17. The expression of this cytokine mainly localises to the lamina propria and not to Peyer's Patches. Surprisingly mast-cells and not T-cells represent the major source of IL-17.
Moreover we have quantified by immuhistochemistry CD3, CD4 and IL-17 expressing cells on serial sections of colon tissues collected longitudinally during the acute and chronic phase of SIV-infection from three rhesus macaques. While the number of CD4+ cells progressively declines, IL-17+ cells are significantly increased in the chronic phase of infection. These results suggest that other cells that are not lymphocytes might produce this cytokine during SIV-infection.
2) Determine whether HIV/SIV infection influences Th17 cells homing to the GALT
Chemokines are essential to drive lymphocyte trafficking/homing to tissues both in homeostatic and inflammatory/pathological conditions. We used the chemokines receptors CCR6 and CXCR3 as markers for the discrimination of Th17 and Th1 cells respectively, in order to determine whether SIV/HIV infection alters T-helper homing to tissues. A total of 12 healthy and 11 SIV-infected rhesus macaques have been enrolled in this study. The frequency and the Mean Fluorescence Intensity (MFI) of CD3, CD4, CCR6 and CXCR3 on PBMCs have been compared between uninfected and infected animals. As expected, the frequency of CD4+ T-cells is significantly reduced in infected animals. Moreover, the frequency of both CCR6+/CD4+ and CXCR3+/CD4+ T-cells declines very rapidly during the course of infection and remains significantly low during the chronic phase. The expression of CCR6 on CD4+ T-cells is significantly decreased in the chronic phase of SIV-infection, while the expression of CXCR3 is diminished only during the first phase of infection and subsequently recovers. Similar experiments have been performed on humans. A total of 18 healthy volunteers and 16 patients with HIV infection have been enrolled in the study. HIV+ individuals were under Highly Active Anti-Retroviral Therapy (HAART) at the time of bleeding. CD4+ T-lymphocytes are significantly decreased in HIV+ individuals, but no alterations in the relative frequency of Na?ve, Central Memory (Cm) or Effector Memory (Em) T-helper cells have been found. In contrast with what we observed in macaques, we did not detect changes in the frequency of total CCR6+/CD4+ T-cells or in the intensity of CCR6 expression on CD4+ T-cells. Nevertheless the percentage of CCR6+ cells in the Em compartment was significantly reduced. Moreover CXCR3+/CD4+ T-cells and CXCR3 expression on CD4 T-cells was significantly higher in HIV+ individuals compared to healthy volunteers.
Interestingly, we observed a significant reduction in the migratory response of CCR6+ cells to their natural-ligand CCL20 during the course of infection, both in macaques and humans. These data suggest a diminished ability of CCR6+ cells to home to mucosal tissues during the infection.
The differences we have highlighted between macaques and humans could be an outcome of Anti-Retroviral Therapy, suggesting that the therapy is efficient in preserving most of CCR6+ cells. To confirm this hypothesis the study will continue by comparing also HIV+ individuals that are not receiving any treatment.
3) Determine whether HIV/SIV infection impairs Th17 differentiation
Th17 cells differentiation is driven by IL-1b, IL-6 and IL-23, that are produced by antigen presenting cells at mucosal sites. It has been described that the number of circulating DCs and their maturation status is altered during the course of HIV/SIV infection. In order to confirm these data mDC and pDCs from healthy donors and HIV+ individuals have been analysed. Our data indicate that in HIV+ individuals the frequency of circulating DCs is not altered, but both mDC and pDCs show a reduced expression of the maturation marker HLA-DR.
Moreover we quantified the production of cytokines that are important for Th17 cell differentiation by healthy donors'or HIV+ individuals'mo-DC stimulated with Candida Albicans in the hyphal form, or LPS. This analysis shows that there is no reduction in local cytokines levels accounting for the Th17 cells loss.
4) Determine whether differences in the levels of chemokines that are expressed at mucosal sites during HIV infection can affect Th17 cells recruitment
Our group has shown that natural non-ligand chemokines can act in synergism with agonists of CCR7, CCR4, CCR2, and CXCR4. HIV infection is characterised by an increased expression of CXCL10 at mucosal sites. In homeostatic conditions CCR6+ cells migrate in vitro toward its agonist, CCL20, only at high concentrations. Our data, obtained both using CCR6-transfected cells and CCR6+ cells isolated from buffy-coats, show that in the presence of CXCL10, migration of CCR6+ cells is triggered at lower CCL20 concentrations. Therefore, chemokines like CXCL10, might influence the ability of CCR6+ to migrate into the tissue.
In contrast, during HIV infection, the migration of CCR6+ cells toward low concentrations of CCL20 is not fully recovered in the presence of CXCL10.
CONCLUSIONS:
Previous reports have demonstrated that Th-17 cells are drastically reduced during the course of SIV/HIV infection. Here we show that mast-cells and not T-cells represent the major source of IL 17 in the gut and during SIV-infection. Importantly, the data obtained from our experiments clearly show a reduction in the number of circulating CCR6+ T-cells and suggest a diminished ability of these cells to home to mucosal tissues in macaques and humans. Moreover, we could not detect any difference in the ability of DCs to produce cytokines that are important for driving Th17 differentiation, despite their altered maturation status. We also show that CXCL10 can synergise with CCL20 and affect CCR6+ lymphocyte trafficking.
Based on the evidence obtained, Th17 cell depletion in the GALT may be due both to direct viral infection and to defects in cell trafficking. A therapeutic approach aimed at preserving the number and activity of Th17 cells in the gut could be beneficial in the treatment of HIV positive individuals. The present study, including both the analysis of human and rhesus macaques samples, could provide insight on novel and important mechanisms that can be explored for future vaccination strategies against HIV and even indicate a novel approach for the study of others infectious diseases
The loss of CD4+ CCR5+ T cells in the gut associated lymphoid tissue (GALT) has been well documented both in natural host and in pathogenic models of SIV infection. A decrease in the frequency of Th17 cells, a newly discovered subset of effector T cells involved in the immune response against extracellular bacteria, has been described, in the mucosa of SIV infected rhesus macaques and in HIV infected individuals. Th17 cells are primarily localised at mucosal sites and they express the homing marker for the skin and the gut CCR6. Recent data show that CCR6+ cells and therefore Th17 cells are more susceptible to HIV-1 infection compared to other subset of T-helper cells, thus suggesting a direct effect of the virus on Th17 cell depletion. Indeed the mechanisms that are responsible for Th17 cell loss during HIV infection have not been investigated in details so far.
Aim of this project was to investigate the mechanisms that mediate Th17 cells trafficking and activities at mucosal sites together with their decrease in frequency during HIV infection. We have also focused on understanding how changes in chemokines/cytokines expression during infection might influence the migratory capacity and differentiation of Th17 cells to mucosal sites.
In order to achieve the research objectives we set up collaborations with the German Primate Center (G?ttingen, Germany) for the analysis of the macaque model of SIV-infection and with the Ospedale Regionale di Lugano (Lugano, Switzerland) for the study of HIV infected patients. The following four objectives were achieved during the research project:
1) Characterise and quantify IL-17 producing cells in the GALT
We have analysed the phenotype of IL17+ cells both on healthy human gut tissues and on biopsies from healthy rhesus macaques. Our analysis demonstrates that a variety of cells, among which T-cells, neutrophils and mast-cells, express IL-17. The expression of this cytokine mainly localises to the lamina propria and not to Peyer's Patches. Surprisingly mast-cells and not T-cells represent the major source of IL-17.
Moreover we have quantified by immuhistochemistry CD3, CD4 and IL-17 expressing cells on serial sections of colon tissues collected longitudinally during the acute and chronic phase of SIV-infection from three rhesus macaques. While the number of CD4+ cells progressively declines, IL-17+ cells are significantly increased in the chronic phase of infection. These results suggest that other cells that are not lymphocytes might produce this cytokine during SIV-infection.
2) Determine whether HIV/SIV infection influences Th17 cells homing to the GALT
Chemokines are essential to drive lymphocyte trafficking/homing to tissues both in homeostatic and inflammatory/pathological conditions. We used the chemokines receptors CCR6 and CXCR3 as markers for the discrimination of Th17 and Th1 cells respectively, in order to determine whether SIV/HIV infection alters T-helper homing to tissues. A total of 12 healthy and 11 SIV-infected rhesus macaques have been enrolled in this study. The frequency and the Mean Fluorescence Intensity (MFI) of CD3, CD4, CCR6 and CXCR3 on PBMCs have been compared between uninfected and infected animals. As expected, the frequency of CD4+ T-cells is significantly reduced in infected animals. Moreover, the frequency of both CCR6+/CD4+ and CXCR3+/CD4+ T-cells declines very rapidly during the course of infection and remains significantly low during the chronic phase. The expression of CCR6 on CD4+ T-cells is significantly decreased in the chronic phase of SIV-infection, while the expression of CXCR3 is diminished only during the first phase of infection and subsequently recovers. Similar experiments have been performed on humans. A total of 18 healthy volunteers and 16 patients with HIV infection have been enrolled in the study. HIV+ individuals were under Highly Active Anti-Retroviral Therapy (HAART) at the time of bleeding. CD4+ T-lymphocytes are significantly decreased in HIV+ individuals, but no alterations in the relative frequency of Na?ve, Central Memory (Cm) or Effector Memory (Em) T-helper cells have been found. In contrast with what we observed in macaques, we did not detect changes in the frequency of total CCR6+/CD4+ T-cells or in the intensity of CCR6 expression on CD4+ T-cells. Nevertheless the percentage of CCR6+ cells in the Em compartment was significantly reduced. Moreover CXCR3+/CD4+ T-cells and CXCR3 expression on CD4 T-cells was significantly higher in HIV+ individuals compared to healthy volunteers.
Interestingly, we observed a significant reduction in the migratory response of CCR6+ cells to their natural-ligand CCL20 during the course of infection, both in macaques and humans. These data suggest a diminished ability of CCR6+ cells to home to mucosal tissues during the infection.
The differences we have highlighted between macaques and humans could be an outcome of Anti-Retroviral Therapy, suggesting that the therapy is efficient in preserving most of CCR6+ cells. To confirm this hypothesis the study will continue by comparing also HIV+ individuals that are not receiving any treatment.
3) Determine whether HIV/SIV infection impairs Th17 differentiation
Th17 cells differentiation is driven by IL-1b, IL-6 and IL-23, that are produced by antigen presenting cells at mucosal sites. It has been described that the number of circulating DCs and their maturation status is altered during the course of HIV/SIV infection. In order to confirm these data mDC and pDCs from healthy donors and HIV+ individuals have been analysed. Our data indicate that in HIV+ individuals the frequency of circulating DCs is not altered, but both mDC and pDCs show a reduced expression of the maturation marker HLA-DR.
Moreover we quantified the production of cytokines that are important for Th17 cell differentiation by healthy donors'or HIV+ individuals'mo-DC stimulated with Candida Albicans in the hyphal form, or LPS. This analysis shows that there is no reduction in local cytokines levels accounting for the Th17 cells loss.
4) Determine whether differences in the levels of chemokines that are expressed at mucosal sites during HIV infection can affect Th17 cells recruitment
Our group has shown that natural non-ligand chemokines can act in synergism with agonists of CCR7, CCR4, CCR2, and CXCR4. HIV infection is characterised by an increased expression of CXCL10 at mucosal sites. In homeostatic conditions CCR6+ cells migrate in vitro toward its agonist, CCL20, only at high concentrations. Our data, obtained both using CCR6-transfected cells and CCR6+ cells isolated from buffy-coats, show that in the presence of CXCL10, migration of CCR6+ cells is triggered at lower CCL20 concentrations. Therefore, chemokines like CXCL10, might influence the ability of CCR6+ to migrate into the tissue.
In contrast, during HIV infection, the migration of CCR6+ cells toward low concentrations of CCL20 is not fully recovered in the presence of CXCL10.
CONCLUSIONS:
Previous reports have demonstrated that Th-17 cells are drastically reduced during the course of SIV/HIV infection. Here we show that mast-cells and not T-cells represent the major source of IL 17 in the gut and during SIV-infection. Importantly, the data obtained from our experiments clearly show a reduction in the number of circulating CCR6+ T-cells and suggest a diminished ability of these cells to home to mucosal tissues in macaques and humans. Moreover, we could not detect any difference in the ability of DCs to produce cytokines that are important for driving Th17 differentiation, despite their altered maturation status. We also show that CXCL10 can synergise with CCL20 and affect CCR6+ lymphocyte trafficking.
Based on the evidence obtained, Th17 cell depletion in the GALT may be due both to direct viral infection and to defects in cell trafficking. A therapeutic approach aimed at preserving the number and activity of Th17 cells in the gut could be beneficial in the treatment of HIV positive individuals. The present study, including both the analysis of human and rhesus macaques samples, could provide insight on novel and important mechanisms that can be explored for future vaccination strategies against HIV and even indicate a novel approach for the study of others infectious diseases