CHAPTER 5: In vivo and in vitro effects of injectable hormonal contraceptives on
6.5 Discussion
Previous studies have suggested that injectable HC use might facilitate contact between cervico-vaginal mucosal target cells and HIV-1 by reducing the integrity and/or thickness of vaginal epithelium and density of tight junction proteins (Chandra et al., 2012; Ildgruben et al., 2003; Miller et al., 2000; Wieser et al., 2001; Wira et al., 2011a). In this study comparing vaginal epithelial thickness in acutely HIV-1 infected women using DMPA, Net-EN or non-HCs, no difference was observed between those using injectable HCs and non-HC users in vaginal epithelial thickness. Injectable HC use may also alter the inflammatory and/or chemotactic environment of the genital mucosa (Ildgruben et al., 2003; Miller et al., 2000; Wieser et al., 2001; Wira et al., 2011b; Wira and Veronese, 2011) and this may indirectly increase the recruitment of HIV-1 susceptible immune cells to the mucosa. Vaginal biopsies from acutely HIV-1 infected women using injectable HCs had increased frequencies of genital CD4+T cells compared to non-injectable HC users. Unlike HIV-1 negative women discussed in Chapter 3, injectable HC use in acutely HIV-1 infected women was not associated with lower concentrations of cytokines or growth factors in genital secretions.
However, several chemokines, hematopoietic and adaptive cytokines in CVLs during acute infection were associated with increased frequencies of vaginal tissue CD68+
macrophages, but not CD4+cells. Those women with the highest plasma viral loads during acute infection had the lowest numbers of CD68+ cells in vaginal tissue.
Studies have suggested that the thickness of the stratified squamous epithelium is altered by DMPA use (Smith et al., 2000; Smith et al., 2004; Trunova et al., 2006) and hormonal fluctuations during the menstrual cycle (King, 1983; Owen, 1975;
Poonia et al., 2006; Veazey et al., 2012). In this study, epithelial thickness was similar in women using injectable HCs compare to non-injectable HC users. This study did find a difference in epithelial thickness measurement depending on the method used to calculate thickness, with the automated method being adopted for all subsequent analysis because of its ability to make thousands of measurements in an unbiased way. In agreement with this observation about measurement bias, Chandra and colleagues observed up to a 34% inter-observer variability in measurements using different methods of measurement (Chandra et al., 2013). They have suggested that the conflicting results reported in the literature to date on epithelial thickness following HC use may reflect differences in the method of measurement, observer bias, timing of tissue sampling, pharmacokinetics, and inter-individual variability in the biological response to DMPA. Through automated measurements, the whole stratified squamous epithelium including sinuous patterning of the epithelial rete pegs that extend into the dermal papillae was included in all measurements. A recent study suggest that a non-viable stratum corneum from the viable stratum malpighii should be measured instead, to exclude the variance that may occur from the epithelial rete pegs (Tjernlund et al., 2015).
Studies have suggested that DMPA may influence the frequency of immune cells in the vaginal mucosa (Chandra et al. 2012; Ildgruben et al. 2003; Miller et al. 2000). In this Chapter, women using injectable HCs had increased numbers of CD4+T cells in their vaginal stratified epithelium compared to non-HC users. In HIV negative women, previous studies found no difference in numbers of CD4+ T cells in the stratified epithelium post DMPA treatment (Chandra et al., 2013; Mitchell et al.,
2014). The increased numbers of CD4+T cells observed in the current study may be due to the fact that these women were already HIV-1 infected at the time of biopsy collection. CD68+ cells in women who use injectable HCs were not significantly different from non-injectable HC users. Density of vaginal immune cells did not predict faster disease progression rates.
Previous studies suggested that blood HIV-1 RNA concentrations positively predicted risk for heterosexual transmision of HIV-1 (Mayaux et al., 1997; Mock et al., 1999;
Quinn et al., 2000). Baeten et al. (2007) suggested that DMPA use influenced how infectious an HIV-1 infected women was to her sexual partner as well as the rate at which she progressed during her clinical course of HIV-1 infection (Baeten et al., 2007b). In this study, women using DMPA did not have higher viral load set-points or more severe CD4 decline over 12 months than women using non-HCs, suggesting that the rate of disease progression was similar.
Acutely HIV-1 infected women in this study who had the higher concentrations of pro-inflammatory cytokines RANTES, MCP-1, IP-10, and IL-17; and adaptive/hematopoetic cytokines IL-9 and IL-7 in CVLs also had the highest numbers of CD68+ macrophages in the vaginal stratified epithelium. RANTES is part of the β- chemokine family, upregulated during inflammation, and responsible for the recruitment of lymphocytes (including basophils, eosinophils, natural killer cells and monocytes) to the site of infection (Baggiolini et al., 1997). RANTES, like MCP-1, has variable affinities for its receptors CCR1, CCR2, CCR3, CCR4 and CCR5, which are expressed on the surface of mature macrophages (Baggiolini et al., 1997; Decrion
et al., 2005; Kaufmann et al., 2001). In addition, MCP-1 promotes immune cell activation and recruitment (Decrion et al., 2005). IP-10 is a biomarker for antiviral immune responses (Luster et al., 1985), while IL-17 plays a vital role in pathogen clearance and mediates pro-inflammatory responses by increasing the production of several other cytokines (Freel et al., 2010). This suggests that these cytokines have a potential to increase the risk of HIV-1 acquisition, promoting activation and recruitment of HIV-1 target cells to the genital mucosa.
Although CD68+ macrophage density did not change with HC use, this study also found that CD68+ macrophage density in vaginal biopsies decreased by 5 cell/mm2 of tissue with every 1-Log increase in plasma viral load. Like CD4+ T cell and DCs, macrophages are the first immune cells to fight the virus at mucosal surface and may also be infected. Macrophages produce cytokines that recruit CD4+ T cell to the site of infection and support viral pathogenesis by increasing the number of primary target cells available for HIV-1 replication during acute HIV-1 infection (Cicala et al., 2011;
Koppensteiner et al., 2012). In this study, the density of CD4+ target cells in vaginal tissue did not correlate with the density of CD68+ macrophages. In tissues, macrophages are long-lived reservoirs of HIV-1 and contibute to the inability to achieve complete HIV-1 clearance during acute infection (Galiwango et al., 2012;
Wu, 2011). These findings support evidence that vaginal macrophages are available to become productively infected during early HIV-1 infection.
Genital tract cytokine concentrations did not predict vaginal CD4+ T cell densities.
Previous studies have shown that pro-inflammatory cytokines in the genital tract
during acute HIV-1 infection were associated with decreased CD4+ T cell counts in blood and increased viral loads during both acute infection (Bebell et al., 2009) and at 12 months post-infection (Roberts et al., 2012). In this study, CD4+T cells in the stratified squamous epithelium of the vagina from acute infection tended to be negatively associated with pro-inflammatory cytokine concentrations (including IL- 12p70, IL-1β, IL-6, TNF-α, IL-12p40, IL-18, IL-1α, MIF, TNF- β and TRAIL).
Plasma viral load was also not associated with vaginal CD4+T cell depletion.
A limitation of this study is that HIV-1 target cells and epithelial thickness were measured in tissue available from women after they seroconverted only, who were predominantly injectable HC users, and no control groups including HIV-1 infected women from other low risk communities or women who remained uninfected from the same CAPRISA 004 communities were available for this study. This study is also relatively small.
In conclusion, injectable HC users had increased frequencies of CD4+T cells in their vaginal stratified epithelium than in women not using injectables. However, injectable HC use was not associated with thinning of the vaginal epithelial barrier or faster HIV-1 disease progression. Although there was no injectable use effect, the density of CD68+ macrophages in cervicovaginal tissue correlated with a broad panel of mucosal cytokines and inversely correlated with plasma viral loads during acute HIV- 1 infection. This study provides valuable insight into possible underlying mechanisms by which genital inflammation may increase HIV-1 risk and subsequent clinical phenotypes during HIV-1 disease course, such as viral set point.