Potential mechanisms for increased HIV-1 transmission across the endocervical epithelium during C. trachomatis infection.

Among the now pandemic sexually transmitted infections (STIs), Chlamydia trachomatis (C. trachomatis) is the predominant bacterial pathogen and human immunodeficiency virus type 1 (HIV-1) is the most lethal of the viral pathogens. The female genital tract is the primary site for heterosexual transmission of both C. trachomatis and HIV-1. Infection with C. trachomatis, and with a variety of other STIs, increases the risk for transmission of HIV-1, although the mechanisms for this finding remain unclear. We have used in vitro modeling to assess the mechanisms by which infection with genital C. trachomatis serovars might increase the transmission of HIV-1 across the female genital tract. C. trachomatis infection of an immortalized endocervical epithelial cell line (A2EN) increases the cell surface expression of the HIV-1 alternative primary receptor, galactosyl ceramide (GalCer), and of the HIV-1 co-receptors, CXCR4 and CCR5. C. trachomatis infection also increases the binding of HIV-1 to A2EN cells, and, subsequently, increases levels of virus in co-cultures of HIV-exposed A2EN and susceptible MT4-R5 T cells. Finally, in vivo endocervical cell sampling reveals a dramatic increase in the number of CD4+, CXCR4 and/or CCR5 positive T cell targets in the endocervix of C. trachomatis positive women when compared to those who are C. trachomatis negative. This combination of in vitro and in vivo results suggests several mechanisms for increased transmission of HIV-1 across the endocervices of C. trachomatis-infected women.

A distinct cellular profile is seen in the human endocervix during Chlamydia trachomatis infection.

PROBLEM: The endocervix is a major target of Chlamydia trachomatis infection, but little is known about the immune repertoire in this tissue, or its response to these common bacteria. METHOD OF STUDY: Using a cytobrush, we isolated cells from the endocervix of 20 women during C. trachomatis infection, and post-antibiotic treatment. Endocervical swabs and blood were taken in parallel. Endocervical cells were enumerated, and endocervical and blood T cells immunophenotyped. Chlamydia trachomatis was genotyped by sequence analysis of the OmpA gene, and quantified by culture. RESULTS: Chlamydia trachomatis genotypes were D, E, F and Ia, and infectious burden varied considerably. Endocervical T cell and neutrophil numbers were highly elevated during infection, with both CD4 and CD8 T-cell subsets accumulating. Regardless of the presence or absence of infection, the endocervical cell infiltrate was dominated by effector memory T cells, and the numbers of CCR5 and CD103 expressing T cells was significantly higher than in the blood. Human leukocyte antigen (HLA-DR) expression by endocervical T cells was significantly increased during infection. CONCLUSION: The human endocervix exhibits a distinct cellular response to C. trachomatis infection that can be longitudinally evaluated by cytobrush sampling. Infecting organisms can be sampled and analyzed in parallel.

Quantification and comparison of toll-like receptor expression and responsiveness in primary and immortalized human female lower genital tract epithelia.

PROBLEM: To better understand innate immune responses to sexually-transmitted infection (STI) and the appropriateness of epithelial cell (EC) models of the vaginal and cervical mucosa, quantified toll-like receptor (TLR) expression from a population of women is needed. METHOD OF STUDY: TLR gene expression was quantified in primary and immortalized endocervical, ectocervical, and vaginal EC from multiple donors. TLR bioactivity was evaluated by cytokine elaboration. RESULTS: TLR1-3 and 5-9 were expressed in each EC type with TLR2, 3, 5, 6 and CD14 expressed most abundantly. TLR4 was expressed by endocervical and vaginal EC. Agonist stimulation of TLR2, 3, 5 and 6 elicited cytokines. TLR4 and 7-9 were minimally expressed and were not consistently bioactive. Immortalized EC generally modeled primary cultures but elaborated significantly reduced cytokine levels. CONCLUSION: TLR expression patterns were remarkably conserved across the study population and evaluated tissues indicating a predictable responsiveness to STI. The results support cautious use of immortalized cells for genital tract modeling.

CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity.

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.

An intravaginal live Candida challenge in humans leads to new hypotheses for the immunopathogenesis of vulvovaginal candidiasis.

Acute and recurrent vulvovaginal candidiasis (VVC) remains a significant problem in women of childbearing age. While clinical studies of women with recurrent VVC (RVVC) and animal models have provided important data about a limited protective role of adaptive immunity, there remains a paucity of information on the protective mechanisms or factors associated with susceptibility to infection. In the present study, an intravaginal live Candida challenge in healthy adult women showed a differential susceptibility to symptomatic VVC, where 3 (15%) of 19 women with no history of VVC acquired a symptomatic infection compared to 6 (55%) of 11 women with an infrequent history of VVC. Furthermore, these studies revealed that protection against infection is noninflammatory while symptomatic infection correlates with a vaginal infiltration of polymorphonuclear neutrophils (PMNs) and a high vaginal fungal burden. Thus, the presence of symptomatic infection appears more dependent on host factors than on properties of the organism. Finally, vaginal lavage fluid from women with a symptomatic infection, but not those asymptomatically colonized, promoted the chemotaxis of PMNs. These results suggest that rather than RVVC/VVC being caused by an aberrant adaptive immune response, symptoms that define infection appear to be due to an aggressive innate response by PMNs.