Human macrophage model for selective evaluation of CD8⁺ and γδ⁺ cytotoxic T cell function in tuberculosis

The human macrophage cell line U937 was investigated as an in vitro model for human macrophage function in mycobacterial infections. This involved evaluating the ability of differentiated U937 cells to phagocytose Mycobacterium tuberculosis, control intracellular mycobacterial growth, and present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs). Differentiation of U937 cells usmg IFN-γ, 1,25-(OH)₂ vitamin D₃, or PMA significantly enhanced their ability to phagocytose M. tuberculosis but failed to induce a subsequent respiratory burst response. Following infection, U937 cells were found to be permissive to the intracellular growth of both the virulent H37Rv strain of M. tuberculosis and the attenuated vaccine strain of M. bovis BCG. U937 cells have been shown to constitutively express high levels of cell surface HLA class I while expressing undetectable levels of HLA class II both at the mRN A level and at the cell surface. HLA class II expression was neither up-regulated following infection with M. tuberculosis nor inducible using IFN-γ, 1,25-(OH)₂ vitamin D₃, PMA, GM-CSF or a combination of these agents. In contrast, chronic infection of U937 cells with virulent H37Rv M. tuberculosis (but not with BCG) resulted in the cell surface expression of HLA class I being significantly up-regulated. Taken together, these characteristics made U937 cells a very attractive model for further investigations into their ability to present mycobacterial antigens to human HLA class I-restricted CTLs. Differentiation of U937 cells was found to completely abrogate their sensitivity to non-antigen specific cytolysis mediated by NK or LAK cells. Following infection with M. tuberculosis, U937 target cells were lysed by M. tuberculosis-primed CTLs from HLA class I-matched donors in an antigen-specific manner and with a similar efficiency to autologous macrophage targets. This cytolytic activity was restricted to live organisms since only U937 cells infected with virulent H37Rv M. tuberculosis and BCG but not those pulsed with soluble PPD were lysed by the HLA class I-matched effector cells. On the other hand, M. tuberculosisstimulated but ALA-mismatched CTLs failed to lyse infected U937 cells in an antigen-specific manner. T cell subset fractionation of the HLA class I-matched M. tuberculosis-primed CTL population and limiting dilution cloning demonstrated that the cytolytic activity was mediated by CD8⁺ cytolytic T cells and confirmed that CD4⁺ T cells showed no significant ability to lyse infected U937 target cells. Furthermore, this study found that M. tuberculosis-infected U937 target cells were lysed by CD8⁺ CTLs more rapidly and strongly than similarly infected autologous macrophage targets demonstrating the sensitivity of this in vitro model as an indicator for CD8⁺ cytolytic function in mycobacterial infections. M. tuberculosis-infected U937 cells were found to be highly sensitive to mycobacterial antigenspecific cytolysis mediated by γδ⁺ CTL. Mycobacterial antigen-specific γδ⁺ CTLs consistently showed stronger cytolytic activity against infected U937 target cells than γδ⁺ CTL but were not restricted to classical HLA class I or class II molecules. A panel of cytolytic human M. tuberculosis-reactive γδ⁺ CTL clones was established to investigate more thoroughly the role of γδ⁺ CTL lytic activity in human mycobacterial infections. This study examined the mechanism of cellular cytotoxicity used by these mycobacterial-specific γδ⁺ CTL clones against infected U93 7 targets and further investigated the effect of γδ⁺ T cell-mediated cytolysis on intracellular mycobacterial survival. Cytolysis mediated by the γδ⁺ T cell clones was found to be dependent on cell-to-cell contact. Furthermore, the ability of the γδ⁺ CTL clones to lyse infected targets was found to be strongly Ca²⁺-dependent, sensitive to cyclosporine A (a specific inhibitor of granule exocytosis ), and completely abrogated following Sr²⁺-induced de-granulation of the γδ⁺ T cell effectors, indicating that cytoxicity was mediated predominantly by the granule exocytosis/ perforin pathway. Despite being strongly cytolytic against infected U937 cells, however, the γδ⁺ CTL clones did not have any impact on the survival of intracellular M. tuberculosis. The major conclusions of this study are that U937 cells not only provide a useful in vitro human macrophage model allowing for selective evaluation of HLA class I-restricted CD8⁺ CTL function in mycobacterial infections but also provided a highly sensitive indicator for γδ⁺ CTL cytolytic activity

Vaginal microbiomes associated with aerobic vaginitis and bacterial vaginosis

A healthy vaginal microbiota is considered to be significant for maintaining vaginal health and preventing infections. However, certain vaginal bacterial commensal species serve an important first line of defense of the body. Any disruption of this microbial barrier might result in a number of urogenital conditions including aerobic vaginitis (AV) and bacterial vaginosis (BV). The health of the vagina is closely associated with inhabitant microbiota. Furthermore, these microbes maintain a low vaginal pH, prevent the acquisition of pathogens, stimulate or moderate the local innate immune system, and further protect against complications during pregnancies. Therefore, this review will focus on vaginal microbial “health” in the lower reproductive tract of women and on the physiological characteristics that determine the well-being of reproductive health. In addition, we explore the distinct versus shared characteristics of BV and AV, which are commonly associated with increased risk for preterm delivery

Semen IgM, IgG1, and IgG3 Differentially Associate With Pro-Inflammatory Cytokines in HIV-Infected Men

Genital inflammation significantly increases the risk for HIV infection. The seminal environment is enriched in pro-inflammatory cytokines and chemokines. Here, we investigated the interplay between semen cytokines and humoral immunity to understand whether the characteristics of semen antibodies are associated with genital inflammation. In 36 HIV-infected and 40 HIV-uninfected mens' semen, HIV-specific antibodies (gp120, gp41, p66, and p24), immunoglobulin (Ig) subclasses, isotypes and cytokines, using multiplex assays, were measured. Semen IgG1, IgG3, and IgM were significantly higher in HIV-infected compared to HIV-uninfected men (p < 0.05). In HIV-uninfected men, pro-inflammatory cytokines IL-6, IL-8, and MCP-1 significantly correlated with IgG1 and total IgG (IgG1+IgG2+IgG3+IgG4) (both r≥0.55; p≤0.001). Total IgG in HIV-infected men correlated to HIV-specific antibodies in the semen irrespective of antiretroviral (ARV) use. In HIV-infected, ARV-treated men, p66 and gp41-specific antibodies were inversely correlated with IL-6 and MIP-1α (both r≥−0.65, p≤0.03). In HIV-infected, ARV-naïve men, p24 and gp120-specific antibodies correlated significantly with pro-inflammatory TNF-α (r≥0.44, p≤0.03), while p24 antibodies correlated significantly with chemokine MIP-1β (r = 0.45; p = 0.02). Local cytokines/chemokines were associated with the mucosal-specific Ig subclasses which likely effect specific antibody functions. Together, these data inform on mucosal-specific immunity that may be elicited in the male genital tract (MGT) in future vaccines and/or combination HIV prevention strategies

Papanicolaou smears and cervical inflammatory cytokine responses

In a case-control study among 2064 South African women to investigate the risk of clinically invasive cancer of the cervix, we found a marked reduction in the risk of cervical cancer among women who gave a history of ever having undergone even a single Pap smear, and a statistically significant decline in the HPV positivity rate correlated with the lifetime number of Pap smears received. HPV infections and their associated low-grade lesions commonly regress, indicating that most often there is an effective host immune response against HPV infection. We hypothesized that act of performing a Pap smear is associated with inflammatory responses at the site of trauma, the cervix, and that this inflammatory signalling may be an immunological factor initiating these productive anti-HPV responses. In the present study, a randomized controlled trial, we enrolled 80 healthy young women to investigate the impact of performing a Pap smear on cervical inflammation. Forty one women, in the intervention group, received a Pap smear at enrollment and cervicovaginal lavages (CVLs) were collected at baseline and 2 weeks later. Thirty nine women received no intervention at enrollment (control group) but CVLs were collected at enrolment and 2 weeks later. We assessed various markers of inflammation including IL-12 p70, TNF-α, IL-8, IL-6, IL-10, and IL-1β in CVL specimens. While CVL levels of IL-8, IL-1β and IL-6 remained unchanged following a Pap smear, markers of cell mediated immunity (IL-12 p70 and TNF-α) and T cell regulation (IL-10) were significantly elevated

Relationship between levels of inflammatory cytokines in the genital tract and CD4+ cell counts in women with acute HIV-1 infection.

Inflammatory responses at mucosal surfaces after human immunodeficiency virus type 1 (HIV-1) transmission may influence disease outcome. We evaluated levels of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, IL-8, IL-10, and IL-12 in genital tract and plasma specimens from 44 women with acute HIV infection and 29 HIV-negative control women (13 of whom were women in the acute HIV infection cohort who had preinfection samples available for analysis). Women with acute HIV infection had significantly elevated levels of IL-6, IL-10, and IL-12 in genital tract specimens and elevated levels of IL-1beta, IL-8, and IL-10 in plasma specimens, compared with HIV-negative control women. Levels of IL-1beta, IL-6, and IL-8 in cervicovaginal specimens from women with acute HIV infection showed a significant inverse correlation with systemic CD4(+) cell counts, suggesting that mucosal inflammation is associated with low CD4(+) cell counts during acute HIV infection

Genital inflammation, immune activation and risk of sexual HIV acquisition.

CAPRISA, 2016.Abstract available in pdf

Inflammatory Cytokine Profiles of Semen Influence Cytokine Responses of Cervicovaginal Epithelial Cells

Genital inflammatory cytokine responses increase HIV risk. Since male partner semen is a complex mixture of immune-modulatory prostaglandins and cytokines, we hypothesized that exposure to semen may influence genital inflammation in women. Here, we investigated cytokine response kinetics of cervical cells following stimulation with seminal plasma from HIV-negative and HIV-positive men characterized as having low or high concentrations of inflammatory cytokines. Irrespective of the HIV status or semen cytokine profile, in vitro stimulation of cervical cells with seminal plasma resulted in significantly elevated concentrations of secreted IL-6, IL-8, TNF-β, MCP-1, GM-CSF, and VEGF within 8 h of stimulation, which tended to decline by 24 h, although this was only significant for TNF-β. Consistent with this, cervical cells responded to seminal plasma with increases in IL-8 and IL-1β mRNA expression of 10-fold. These findings suggest that the impact of semen on local female genital cytokines is likely transient. Although these findings suggest that the impact of semen on local female genital cytokines may not be sustained long-term, this heightened genital inflammation may have implications for HIV risk in women

Effect of female genital schistosomiasis and anti-schistosomal treatment on monocytes, CD4+ T-cells and CCR5 expression in the female genital tract

BACKGROUND: Schistosoma haematobium is a waterborne parasite that may cause female genital schistosomiasis (FGS), characterized by genital mucosal lesions. There is clinical and epidemiological evidence for a relationship between FGS and HIV. We investigated the impact of FGS on HIV target cell density and expression of the HIV co-receptor CCR5 in blood and cervical cytobrush samples. Furthermore we evaluated the effect of anti-schistosomal treatment on these cell populations. Design The study followed a case-control design with post treatment follow-up, nested in an on-going field study on FGS. METHODS: Blood and cervical cytobrush samples were collected from FGS negative and positive women for flow cytometry analyses. Urine samples were investigated for schistosome ova by microscopy and polymerase chain reaction (PCR). RESULTS: FGS was associated with a higher frequency of CD14 + cells (monocytes) in blood (11.5% in FGS+ vs. 2.2% in FGS-, p = 0.042). Frequencies of CD4 + cells expressing CCR5 were higher in blood samples from FGS+ than from FGS- women (4.7% vs. 1.5%, p = 0.018). The CD14 + cell population decreased significantly in both compartments after anti-schistosomal treatment (p = 0.043). Although the frequency of CD4+ cells did not change after treatment, frequencies of CCR5 expression by CD4+ cells decreased significantly in both compartments (from 3.4% to 0.5% in blood, p = 0.036; and from 42.4% to 5.6% in genital samples, p = 0.025). CONCLUSIONS: The results support the hypothesis that FGS may increase the risk of HIV acquisition, not only through damage of the mucosal epithelial barrier, but also by affecting HIV target cell populations, and that anti-schistosomal treatment can modify this

Modulation of female genital tract-derived dendritic cell migration and activation in response to inflammatory cytokines and toll-like receptor agonists.

CAPRISA, 2016.Abstract available in pdf