Plasma and Vaginal-associated Immune Responses in Women with Symptomatic Vulvo-Vaginal Candidiasis
CHAPTER 5 Discussion and Conclusions
Although symptomatic vulvo-vaginal candidiasis (VVC) is not classified as an AIDS-defining condition, women with HIV-associated immunosuppression, particularly those who are ART- naïve, are more likely to develop symptomatic VVC. However, findings from this study showed that more than one pathogenic mechanism can explain the occurrence of symptomatic VVC in HIV infected women:
(i) HIV-induced immunosuppression:
Level of HIV-induced immunosuppression, as measured by CD4+ T lymphocytes category, was significantly related to increased odds of symptomatic vulvo-vaginal candidiasis (VVC) only among women with severe immunosuppression (CD4 levels <200/mm3). When compared with HIV negative women, odds of VVC increased by >7-fold for women whose CD4 levels were
<200/mm3 and there was no increased risk for other immunosuppressed women whose CD4 levels were between 200 – 500/mm3. However, when considering only HIV positive women and comparing risk of developing VVC among the 3 WHO stages of HIV-induced immunosuppression (WHO, 2005), odds of VVC increased by 9-fold for women with advanced immunosuppression (CD4 levels 200–349/mm3) and by 60-fold for women with severe immunosuppression (CD4 levels <200/mm3) with HIV loads not included in the multivariate model. There was no increased risk observed for women with mild immunosuppression (CD4 levels 350–499/mm3). Furthermore, among HIV-seropositive women, odds of VVC increased 4- fold and 8-fold respectively for women whose genital and plasma HIV load was >1000 copies/mL when CD4 levels were not included in the multivariate model; but odds highly increased by almost 100-fold for HIV positive women with plasma HIV load ≥10 000
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copies/mL. Use of HAART was significantly associated with reduced odds of VVC, regardless of whether HIV load or CD4 count was included in the multivariate model.
There were limitations in this study that might prevent generalization of the above findings: (1) a cross-sectional study design with a small sample size; (2) although multivariate regression analyses were used to control numerous confounding factors, stratification of continuous variables (i.e. CD4+ T cells and HIV loads), particularly when the sample size is small, may lead to residual confounders that might require further adjustment-based methods that were not considered in this study; (3) to prevent the effect of multicollinearity among variables that showed strong biological correlations (CD4+ T cells, HIV loads and use of HAART), CD4+ T cell levels and plasma/genital HIV viral loads were not included in the same multivariate models regardless of whether the use of HAART was included in these models.
Taken together, as compared with HIV negative women, the hypothesis that VVC is due to HIV- induced immunosuppression might be relevant to only a group of patients, particularly those with severe immunosuppression. This hypothesis is further supported in this study by the fact that the cohort of HIV infected women with severe immunosuppression had higher levels of plasma MIP-1β when co-infected with VVC as compared to women without VVC. It is well established that HIV induces lymphoid activation despite CD4+ T cell depletion in patients with severe immunosuppression, reflected by increased production of MIP-1β, and other markers of T cell activation such as CD38 and HLA-DR (Trumpfheller et al., 1998, Paiardini and Muller-Trutwin, 2013). This chronic immune activation is usually associated with HIV viral persistence and increased cases of opportunistic infections such as mucosal candidiasis. However, controversies exist in the literature regarding the role played by HIV-induced immunosuppression in the occurrence of VVC (Ohmit et al., 2003). During a longitudinal study by Ohmit et al (2003) with
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a large cohort of HIV seropositive women, findings did not demonstrate a significant correlation between CD4+ lymphocyte count and the occurrence of VVC (Ohmit et al., 2003). The authors did not also found that the use of HAART prevented the occurrence of VVC; hence suggested that VVC may be less likely related to cellular immunodeficiency (Ohmit et al., 2003). However, in agreement with our findings, the Ohmit et al showed that HIV load independently influenced the odds of VVC because higher HIV loads were found to be significantly associated with increased odds of VVC (Ohmit et al., 2003). Prior to this report, Sobel et al. (2000) found out that higher HIV loads rather than lower CD4+ T-lymphocyte counts were associated with statistically significant increased odds for both persistent candidal vaginal colonization and symptomatic VVC (Sobel et al., 2000).
We can therefore conclude that if severe cellular immunodeciency induced by HIV does not directly influence the occurrence of VVC, the corresponding persistent and higher HIV load does. In the present study, we found a significant linear correlation between plasma HIV viral load and genital HIV shedding. I can be hypothesized that during HIV-induced severe immunosuppression with subsequently observed higher HIV loads, HIV particles might change the vaginal environment, hence promoting virulence of Candida species by switching from its nonpathogenic form into a filamentous form that causes symptomatic VVC. A further study is required to ascertain this hypothesis.
(ii) Th17 cells rather than Th1/Th2 paradigm were key players in the defense and development of effector responses in HIV infected women with symptomatic VVC:
Using discriminant analysis, it was found that mean levels of circulatory TGF-β3 were significantly higher in women with VVC regardless of their HIV serostatus. In addition, after
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adjusting for multiple comparisons using post hoc Bonferroni pairwise tests, mean concentrations of genital IL-6, IL-8, IL-10, IL-17 and GM-CSF were significantly higher in HIV infected women co-infected with VVC. Collectively, it has been demonstrated that in the presence TGF-β3 and IL-6, naïve CD+ T cells differentiate to Th17 cells (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013). Th17 cells produce IL-6, IL-8 and IL-17 as effector cytokines (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013).
Although initial studies in human subjects pointed out that TGF-β was not a key factor for Th17 differentiation, more recent findings indicate that the presence of TGF-β is essential for the generation of functional Th17 cells (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez- Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013). Other key cytokines involved in Th17 cell differentiation and expansion are IL-21 and IL-23 but these cytokines were not measured in this present study (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013).
The prototype cytokine for Th17 cell response, IL-17, coordinates tissue inflammation through the induction of proinflammatory cytokines and chemokines, such as IL-6 and IL-8 (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013). IL-6 is a multifunctional pleiotropic cytokine involved in the regulation of immune responses, acute-phase responses, hematopoiesis, and inflammation (Kishimoto, 2010). IL-8 is a neutrophil-specific chemotactic factor classified as a member of the CXC chemokine family (Lin et al., 2004). The major effector function of IL-8 is the activation and recruitment of neutrophils, NK cells, T cells, basophils, and GM-CSF to the site of infection
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(Lin et al., 2004). It has been demonstrated that the generation of classical Th17 cells in the presence of TGF-β favors the production of higher levels of IL-10 as also shown in our study. It has been demonstrated that in the presence of TGF-β and IL-10, CD4+ T cells will differentiate to regulatory T cells (Treg cells) resulting in the production of more IL-10 (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013). Therefore, IL-10 produced by Treg cells or directly by the more regulatory classical Th17 cells blocks Th17-mediated inflammation, suggesting the existence of regulatory mechanisms to avoid excessive tissue damage (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013).
In this study, although some Th1 cytokines (IL-2 and IL-12) were significantly higher among HIV infected women with VVC, IFN-γ, “a prototype Th1 cytokine” was shown to be very low.
In general, infection of myeloid cells typically elicits production of IL-12, which induces differentiation of IFN-γ-producing Th1 cells (Liu et al., 2005). In 2007, Steinman reported on a central paradox that the signature Th1 effector cytokine, IFNγ, was significantly less important in various disease settings than was IL-12, the key Th1 inductive cytokine (Steinman, 2007, Hernandez-Santos and Gaffen, 2012). This significant overhaul in the prevailing paradigm of CD4-mediated immunity became evident with the discovery of Th17 cells, which reconciled these discrepancies (Hernandez-Santos and Gaffen, 2012). Of particular importance is the finding that the p40 subunit of IL-12 is shared with IL-23 and the IL-12Rβ1 is a subunit of the IL-23R (Ghilardi and Ouyang, 2007). Thus, mice lacking either IL-12p40 or IL-12Rβ1 are deficient not only in IL-12 (hence, lacking Th1 cells), but also IL-23 (hence, lacking Th17 cells) (Ghilardi and Ouyang, 2007).
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In conclusion, there is Th1/Th2 paradigm shift in the pathogenesis of VVC among HIV infected women (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013). Of major importance was the observed significant level of TGF-β3, a member of TGF-β family of cytokines. It is well established that the immunoregulatory properties of these cytokines can explain the normal absence of cell- medicated immunity against candida in the vagina (LeBlanc et al., 2006, Kosonen et al., 2006, Romani, 2000). In addition, Th17 cells rather than Th1/Th2 paradigm were demonstrated to be key players in the defense and development of effector responses in HIV infected women with symptomatic VVC (Yano et al., 2012, Littman and Rudensky, 2010, Hernandez-Santos and Gaffen, 2012, Cypowyj et al., 2012, Bixler and Mattapallil, 2013). Studies have shown that during the course of HIV infection, there are progressive decline of Th17 cells as well as a decrease of Th17/Treg ratio (Bixler and Mattapallil, 2013). This unbalance seems to be more important in advanced HIV disease (Bixler and Mattapallil, 2013). Most of these studies however have been assessing the loss of Th17 cells in the gastro-intestinal mucosa of HIV infected patients (Bixler and Mattapallil, 2013). The role of Th17 cells during Candida infection in different other anatomical sites remains largely unknown. If there is no loss of Th17 cells (at the mucosal surface of the vagina) in HIV infected women with VVC, then an inflammatory but non-protective response induced by Th17 cells during Candida infection following a strong recruitment of PMN cells can explain the presence of symptoms in patients with VVC. Based on data from a human life challenge model, Fidel Jr. et al. hypothesized that following the interaction of Candida with vaginal epithelial cells, symptomatic VVC was associated with signals that promoted a non-protective inflammatory leukocyte response and concomitant