Unbiased Proteomics Analysis Demonstrates Significant Variability in Mucosal Immune Factor Expression Depending on the Site and Method of Collection

et al. (2013) Unbiased Proteomics Analysis Demonstrates Significant Variability in Mucosal Immune Factor Expression Depending on the Site and Method of Collection. PLoS ONE 8(11): e79505. doi:10.1371/journal.pone.0079505 Unbiased Proteomics Analysis Demonstrates Significant Variability in Mucosal Immune Factor Expression Depending on the Site and Method of Collection Kenzie M. Birse 0 Adam Burgener 0 Garrett R. Westmacott 0 Stuart McCorrister 0 Richard M. Novak 0 T. Blake Ball 0 Clive M. Gray, University of Cape Town, South Africa 0 1 Department of Medical Microbiology, University of Manitoba , Winnipeg, Manitoba , Canada , 2 Proteomics Group, National Lab for HIV Immunology, Public Health Agency of Canada , Winnipeg, Manitoba , Canada , 3 Mass Spectrometry Core Facility , National Microbiology Laboratory, Public Health Agency of Canada , Winnipeg, Manitoba , Canada , 4 Section of Infectious Diseases, University of Illinois , Chicago , Illinois, United States of America, 5 Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya, 6 National Lab for HIV Immunology, Public Health Agency of Canada , Winnipeg, Manitoba , Canada Female genital tract secretions are commonly sampled by lavage of the ectocervix and vaginal vault or via a sponge inserted into the endocervix for evaluating inflammation status and immune factors critical for HIV microbicide and vaccine studies. This study uses a proteomics approach to comprehensively compare the efficacy of these methods, which sample from different compartments of the female genital tract, for the collection of immune factors. Matching sponge and lavage samples were collected from 10 healthy women and were analyzed by tandem mass spectrometry. Data was analyzed by a combination of differential protein expression analysis, hierarchical clustering and pathway analysis. Of the 385 proteins identified, endocervical sponge samples collected nearly twice as many unique proteins as cervicovaginal lavage (111 vs. 61) with 55% of proteins common to both (213). Each method/site identified 73 unique proteins that have roles in host immunity according to their gene ontology. Sponge samples enriched for specific inflammation pathways including acute phase response proteins (p = 3.37610224) and LXR/RXR immune activation pathways (p = 8.82610222) while the role IL-17A in psoriasis pathway (p = 5.9861024) and the complement system pathway (p = 3.9161023) were enriched in lavage samples. Many host defense factors were differentially enriched (p,0.05) between sites including known/potential antimicrobial factors (n = 21), S100 proteins (n = 9), and immune regulatory factors such as serpins (n = 7). Immunoglobulins (n = 6) were collected at comparable levels in abundance in each site although 25% of those identified were unique to sponge samples. This study demonstrates significant differences in types and quantities of immune factors and inflammation pathways collected by each sampling technique. Therefore, clinical studies that measure mucosal immune activation or factors assessing HIV transmission should utilize both collection methods to obtain the greatest representation of immune factors secreted into the female genital tract. - Funding: This work was funded through the National Institutes of Health (http://www.nih.gov/), HIV Vaccine Trials Network (http://www.hvtn.org/), and the Public Health Agency of Canada (http://www.phac-aspc.gc.ca/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Mucosal secretions provide a barrier against invading pathogens and microorganisms. In the case of HIV-1, heterosexual intercourse is the main route of new infections [1], making the mucosa of the female genital tract (FGT) the first site of contact for male to female HIV-1 transmission. This mucosal surface is complex, and contains an abundance of soluble innate immune factors that are important for HIV-1 acquisition. Such factors include RANTES [2] MIPa, MIPb, SLPI [3], Elafin [4,5], LL-37 [6], a/b-defensins [7,8], Lysozyme, Lactoferrin, Calprotectin, Histone H2A [9], Cystatins, Serpins [10] as well as many other anti-proteases [11]. The composition and balance of these factors may influence susceptibility to HIV-1, as shown in studies of HIVexposed seronegative (HESN) individuals and individuals who succumb to infection [4,1116]. These factors may have an impact on local viral replication, establishing the viral load set point and the rate of disease progression [17]. Also, follow-up studies to determine the correlates of protection in HIV vaccines that have shown promise such as the Thai RV144 trial [18] have emphasized the importance of mucosal immune responses in reduced acquisition [19]. Therefore, it is critical that these factors are properly measured to understand early events in HIV pathogenesis and transmission. Recent clinical trials have indicated that increased immune activation in the FGT has been attributed to increased risk of HIV-infection. The importance of mucosal inflammation was exemplified by the failure of the detergent microbicide, nonoxynol-9, which increased HIV-infection risk and was associated with an increase in inflammation status in the FGT [20]. Furthermore, the lack of efficacy in the Centre for AIDS Programme of Research in South Africa (CAPRISA-004) microbicide trial may also have been attributed to increased baseline immune activation and pro-inflammatory cytokine production [16,21]. However, as the biological determinants of FGT inflammation and the immune pathways important for HIVsusceptibility have not yet been defined, defining techniques and protocols to efficiently and accurately monitor a broad range of factors involved with inflammation and immune activation in the mucosal compartment will be essential for future clinical trials and the development of future intervention technologies. The most commonly used techniques to sample the FGT mucosa involve the use of cervicovaginal lavages and/or Weck-Cel cervical sponges, which are largely standardised, inexpensive and minimally invasive [2224]. Each method collects secretions from different compartments of the FGT. Cervicovaginal lavages are designed to collect secretions primarily from the lower FGT, which includes the ectocervix and the vaginal vault, and Weck-Cel cervical sponges are designed to collect secretions primarily from the upper FGT, which includes the endocervix and the endometrium. However, it is unknown which technique and/or site sampled is most informative, and most relevant with respect to inflammation and the collection of immune factors. Although previous studies examining sampling techniques have noted some differences between the methods [23], these have been limited to a small list of pre-defined immune facto (...truncated)