Recombinant IL-8 was added to either saline (unfavorable control) or genital fluids (diluted 1:4) from 10 HIV-seropositive subjects and incubated at either 4C or 37C for 20 h. matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. Conclusions These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract. Introduction The chemokine Interleukin-8 (IL-8, CXCL8) is usually a member of the CXC chemokine family that plays a number of important functions in immunity including activation and attraction of neutrophils [1,2]. In vitro, IL-8 is usually EDC3 produced by neutrophils, macrophages, monocytes and epithelial cells when exposed to either microbial products derived from commensal bacteria or organisms that cause sexually transmitted infections (STI) [3C5]. IL-8 levels in lower genital tract Methotrexate (Abitrexate) secretions are increased in women with STIs [5C7] and also increased in response to non-STI alterations in lower genital tract microbiota [7C11]. IL-8 elevations in genital secretions and in cultures of epithelial cells have been used as a biomarker of inflammation in clinical trials of microbicides [12,13]. There are several proteases that have been reported to act on IL-8. A protease made by some strains of has been shown to cleave the C-terminal alpha helix of IL-8 resulting in IL-8 inactivation . In infected patients, disease severity correlated with the IL-8 Methotrexate (Abitrexate) protease activity expressed by the isolates . [19C21]. Strains of and are also present in the genital tract of some women. We hypothesized that some of these genital bacteria could express proteases that cleave IL-8. To explore this hypothesis, we used ELISA to assess a reduction of IL-8 detection after incubation with genital tract fluids collected from 200 different women. Since a reduction of reactivity in the ELISA does not necessarily show cleavage of IL-8, we used the terms cleavage/alteration and cleaving/altering throughout the paper to indicate reduction in ELISA activity. Material and Methods Subjects Genital samples were obtained from women in the Rwanda Womens Inter-association Study and Assessment (RWISA). RWISA is an observational Methotrexate (Abitrexate) prospective cohort study investigating the effectiveness and toxicity of antiretroviral therapy (ART) and comorbidities in HIV-infected Rwandan women. Written informed consent was obtained in accordance with protocols approved by the Rwanda National Ethics Committee and the Institutional Review Board of Montefiore Medical Center, Bronx NY. Genital samples were collected by cervicovaginal lavage (CVL) performed by irrigation of the cervix with 10 ml of nonbacteriostatic sterile saline, followed by aspiration from the posterior fornix. CVL were transported from the study site to the lab within two hours of collection, aliquoted and frozen. Measurement of IL-8 cleavage/alteration IL-8 cleavage/alteration was measured similarly to previously reports . CVL were clarified by centrifugation and diluted 1:4 with RPMI-1640 medium buffered with HEPES (Sigma, St. Louis, MO, added as a source of cations) and 0.1 ml of diluted fluid was added to 0.1 ml (2 ng/ml) of carrier-free recombinant human IL-8 (rhCXCL-8/IL-8, R&D Minneapolis, MN, USA). The mixtures were incubated for 20 h at 4C or 37C. Afterward, ELISA was used to determine the concentration of IL-8 (BD Bioscience, San Diego, CA USA). The cutoff for determining if samples were positive for IL-8 cleavage/alteration was set by calculating the standard deviation of unfavorable controls in multiple runs and multiplying by 3. In Methotrexate (Abitrexate) some experiments, protease inhibitors were added to the incubations; either General Protease Inhibitor (1:25 final concentration) (Sigma), EDTA (2 mmol final concentration), Marimastat (13 nM final concentration, Tocris, Bristol, UK) or CP471474 (16 nM final concentration, Tocris). A culture supernatant from Group A was used as a positive control in all experiments (a kind gift from Paul Sumby, Center for Molecular and Translational Human Infectious Diseases Research, Huston, Texas 77030). Pyrosequencing of the 16S rRNA gene and identification of bacteria Bacteria in Methotrexate (Abitrexate) CVL were pelleted by centrifugation and DNA was isolated using the Fast DNA Spin Kit for Ground (MP Biomedicals, Solon, OH USA). Multitag Pyrosequencing, as described previously [22,23], was performed using 12.