Figures
Abstract
Objective
Sexual transmission of HIV occurs across a mucosal surface, which contains many soluble immune factors important for HIV immunity. Although the composition of mucosal fluids in the vaginal and oral compartments has been studied extensively, the knowledge of the expression of these factors in the rectal mucosa has been understudied and is very limited. This has particular relevance given that the highest rates of HIV acquisition occur via the rectal tract. To further our understanding of rectal mucosa, this study uses a proteomics approach to characterize immune factor components of rectal fluid, using saliva as a comparison, and evaluates its antiviral activity against HIV.
Methods
Paired salivary fluid (n = 10) and rectal lavage fluid (n = 10) samples were collected from healthy, HIV seronegative individuals. Samples were analyzed by label-free tandem mass spectrometry to comprehensively identify and quantify mucosal immune protein abundance differences between saliva and rectal fluids. The HIV inhibitory capacity of these fluids was further assessed using a TZM-bl reporter cell line.
Results
Of the 315 proteins identified in rectal lavage fluid, 72 had known immune functions, many of which have described anti-HIV activity, including cathelicidin, serpins, cystatins and antileukoproteinase. The majority of immune factors were similarly expressed between fluids, with only 21 differentially abundant (p<0.05, multiple comparison corrected). Notably, rectal mucosa had a high abundance of mucosal immunoglobulins and antiproteases relative to saliva, Rectal lavage limited HIV infection by 40–50% in vitro (p<0.05), which is lower than the potent anti-HIV effect of oral mucosal fluid (70–80% inhibition, p<0.005).
Citation: Romas LM, Hasselrot K, Aboud LG, Birse KD, Ball TB, Broliden K, et al. (2014) A Comparative Proteomic Analysis of the Soluble Immune Factor Environment of Rectal and Oral Mucosa. PLoS ONE 9(6): e100820. https://doi.org/10.1371/journal.pone.0100820
Editor: Donald L. Sodora, Seattle Biomedical Research Institute, United States of America
Received: February 6, 2014; Accepted: May 30, 2014; Published: June 30, 2014
Copyright: © 2014 Romas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is funded through the Canadian Institute for Health Research (www.cihr-irsc.gc.ca; CIHR HOP123802), the Public Health Agency of Canada (www.phac-aspc.gc.ca), the Swedish Research Council (www.vr.se), and the Swedish Physicians against AIDS foundation (www.aidsfond.se). 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.
Introduction
Men who have sex with men (MSM) are one of the highest risk groups for HIV acquisition worldwide and are severely impacted by HIV/AIDS pandemic [1]. HIV acquisition is highest in the receptive MSM partner, who can be exposed at the oral or rectal mucosae during unprotected receptive oral intercourse (UROI) and unprotected receptive anal intercourse (URAI). Infection rate per sexual exposure through UROI is estimated to be 0.00–0.04% while URAI is highest at 1.4% [2]. This heterogeneity in transmission at receptive exposure sites is well observed in the literature [3]–[6], but little is known on the biological factors that influence this phenomenon.
HIV first contacts the mucosal epithelium during sexual exposure, which serves as an immune barrier to infection. The oral cavity consists of multi-layered squamous epithelia, while the rectal mucosa consists only of a single layer of columnar epithelia. The reduced thickness of the rectum leads to a higher risk of trauma to the rectal compartment during intercourse. This can cause abrasions in the epithelia for HIV to enter underlying target cells and is thought to be a major contributor to the relatively high risk of HIV acquisition over the oral cavity. The mucosal fluid that overlies the epithelia also contributes to HIV susceptibility, as it is replete with immune proteins to limit pathogen invasion. Soluble protein factors found in oral mucosa such as immunoglobulins, high CC-chemokine levels and HIV binding inhibitors (RANTES and SLPI) have been found to be important in impeding HIV infection at this exposure site [7]–[10]. Certain factors have also been shown to correlate with reduced susceptibility in HIV-exposed uninfected individuals, and are therefore associated with reduced risk of HIV acquisition. These include elevated CC-chemokines and bPRP2 within the saliva of HESN MSM [11], [12]. As well, HIV-neutralizing salivary IgA has been correlated with HIV protection in resistant individuals, and the potent antiviral activity of IgA has made it an attractive vaccine target [13]–[15]. Mucosal immune proteins within the rectum, including potential anti-HIV factors, have yet to be comprehensively described as they have been at other sites of HIV exposure (Table 1). This represents a significant gap in our knowledge of HIV pathogenesis and is a major barrier to understanding HIV transmission through URAI.
An important first step in understanding rectal HIV susceptibility due to mucosal fluid is to describe the soluble immune components of rectal mucosa as well as assess the capacity of this fluid to inhibit HIV infection. Characterization of immune factors in these secretions is imperative for our understanding of the frontline role of mucosae at the portals of entry for HIV, and must be considered in the design of preventative strategies or therapeutics that would limit HIV transmission at these sites. Using a proteomic analysis of mucosal secretions, this study is the first comprehensive proteomic analysis of the mucosal proteins within the rectal compartment, using oral mucosa as a reference, in order to address this gap in knowledge.
Methods
Ethics
The ethical committee at Karolinska Institutet has approved this study and all participants gave written, informed consent.
Sample collection and pooling
Mucosal samples were collected from healthy male participants recruited by an advertisement at a blood donor clinic through the Gay Men's Health Clinic in Stockholm, Sweden (n = 10 salivary fluid; n = 10 rectal lavage). Whole, un-stimulated saliva was collected in 50 ml vials, aliquoted and frozen at −80°C; participants were instructed not to eat or drink two hours preceding. During the same visit, rectal lavage was collected after installing 5 ml of sterile PBS and then aspirating the fluid, which was subsequently filtered to remove debris and immediately frozen at −70°C. Low risk individuals were classified as men who have had 0–1 sexual partners and tested negative for HIV (regular plasma screen), chlamydia (throat, urine and rectum) and gonorrhea (throat, urethra and rectum) [14], [16]. The protein concentration of each sample was determined by BCA assay (Novagen). Equal amounts of protein/samples (10 µg) from each individual were combined to create pooled samples (100 µg) for both saliva and rectal lavage for subsequent assays.
HIV infection assays
HIV infection assays were performed under previously established conditions [17]. Briefly, the TZM-bl reporter cell line was cultured in DMEM media completed with 10% Fetal Bovine Serum (Hyclone Media) and 5% Penicillin-Streptomycin (Fisher Scientific) and incubated at 37°C and 5% CO2 for three days [18]–[22]. The TZM-bl reporter cell line was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TSM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. Two days prior to infection, 96 well plates were seeded at 10,000 TZM-bl cells per well at the same culture conditions. Just prior to infection, culture media was removed and cells were incubated with 300 µl whole, sterile filtered (0.2 µM) lavage fluid. Salivary fluid was diluted 1∶2 in PBS and rectal lavage fluid was diluted 1∶1.5 in PBS to retain similar concentrations of protein (39.12–0.31 µg/ml salivary fluid and 40.73–0.32 µg protein/ml rectal lavage). Immediately following, an R5-tropic HIV-1 virus (BaL) was added at an M.O.I. of 0.2 (3.92 µl/well), and incubated with cells for 3 hours. Our assays used an R5-tropic strain of HIV that utilize the CD4+ and CCR5+ co-receptors as these are the major infectious strains found to establish a founder population in mucosal tissues, [23] and more specifically, within mucosal T lymphocytes [24]. Negative control wells, containing only virus, cells and PBS, and a positive control containing 10 µM azidothymidine (AZT), virus, cells and PBS were included. Virus and mucosal fluid were then removed and cells were incubated in complete DMEM. TZM-bl cell cytotoxicity in the presence of mucosal fluids was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to manufacturer's instructions. Cell viability was measured by a luciferase reaction that produced a luminescent signal proportional to the amount of ATP produced in culture, which was directly proportional to the number of viable cells in each culture. Experimentally treated TZM-bl cells were screened for viral infection after 72 hours incubation (37°C, 5% CO2) according to a β-Gal Screen System (Life Technologies) in relative light units (RLUs). Percent infectivity of experimental wells was calculated relative to the negative control and conditions were compared using a two-tailed t-test (α = 0.05).
Mass spectrometry analysis
Protein isolation, digestion into peptides, and label-free mass spectrometry analysis was performed as described [12], [25] to identify and quantify host proteins (Methods S1). All proteins identified were annotated by function using the peer-reviewed UniProtKB database (www.uniprot.com), and proteins with a known function in immunity were selected for further analysis. Average mucosal immune protein expression was compared across anatomical sites. The average abundance of each protein was calculated within mucosal pools by mass spectrometry and was converted to fold-difference values relative to the mean expression of that protein across mucosal fluids. Data was normalized to a Gaussian distribution using log2 transformation, and normalization was confirmed using normal quantile plots. Log2(fold-difference) values were further normalized to average protein content so that all positive values correspond to an heightened relative protein abundance, while all negative values correspond to a lower relative abundance of protein. Differentially abundant proteins were determined with two-tailed, independent t-tests (α = 0.05 corrected for multiple comparisons using the Benjimani-Hochberg method) using GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla, CA) and restricted to those with 2.4 or greater fold-difference (effect size = 1.4) between groups to retain an experimental power of 0.8. A full proteins list is available in the supplemental methods (Table S1).
Results and Discussion
The capacity of rectal mucosal fluid to directly limit HIV replication in vitro has, to our knowledge, not been assessed in the literature. HIV inhibition assays were performed by incubating an R5-tropic HIV lab strain (BaL) with TZM-bl reporter cells in the presence of diluted salivary or rectal mucosal fluid. Rectal mucosa demonstrated the ability to inhibit HIV infection by significantly limiting HIV production by approximately 40% at mucosal protein concentrations of 2 µg/µl to a maximum of 61.5% at 64 µg/µl (p = 0.05; Figure 1a). The inhibitory capacity of rectal lavage fluid demonstrated in our assay is relatively mild compared to saliva, which has previously been shown to have a potent effect on HIV infection [26]–[28]. In agreement with the literature, our assays demonstrated that salivary fluid possessed higher antiviral activity, limiting HIV infectivity by 80% at 2 µg/µl (p value = 0.005, Figure 1b). This demonstrates that mucosal fluid can inhibit HIV at physiologically relevant concentrations (6 µg/ml to 68 µg/ml), albeit with lower capacity than saliva. This may have relevance to what is observed in vivo, as demonstrated by a much higher incidence of infection upon rectal exposure than through oral exposure [4]. Previously, the low occurrence of HIV orally has been, in part, attributed to high levels of soluble immune factors such as CC-chemokines and the antimicrobial peptides SLPI, LL-37 and defensins [31]. In an attempt to understand the role of these, and other soluble factors in HIV infection at different sites of exposure, we used mass spectrometry to comprehensively define proteins contained within oral and rectal mucosal fluid, and define natural differences within these fluids that may be responsible for the observed discrepancy in HIV inhibition.
Inhibition assays of HIV BaL replication within the CCR5+/CXCR4+ TZM-bl reporter cells in the presence of varying concentrations of rectal lavage and salivary fluid protein were performed. Rectal lavage exhibited a significant, mild inhibitory effect on HIV infection in TZM-bl cells (∼40% inhibition) beginning at 2 µg/ml of protein relative to a negative control (p = 0.05, triplicate assays) (A). Parallel assays demonstrated that salivary fluid had a stronger anti-HIV capacity (∼70–80% inhibition) at as low as 0.3 µg/ml relative to the negative control (p<0.005, triplicate assays) (B). Mucosal fluids were determined to have a negligent effect on cell death based on a luciferase assay that indirectly measured the number of viable cells in each culture via their ATP production (data not shown).
Our proteomic analysis identified 315 human proteins expressed in both rectal and salivary mucosal fluid (Table S1). Both fluids contained numerous immune factors with 72 common proteins found to play a role in host defence and immunity (Figure 2). A small portion of proteins were unique to either fluid (one protein was unique to saliva [0.3%] and four proteins were unique to rectal mucosa [1.3%], but none had known roles in immunity (Table S1). The majority of immune proteins identified in both mucosal fluids have known roles in role in inflammation (9.6% of all 315 proteins identified) and/or antimicrobial defence (8.8%; Figure 2). Several other categories of immune proteins were identified within our data set, which included the following: antiproteases (4.0%), immunoglobulins (4.0%), wound healing (3.1%), acute phase response (2.1%), platelet activation (1.2%) and MHC Immunity (1.2%). Within our dataset we identified many proteins without immune function (Table S1); however, our downstream analysis was focused on proteins with known immune function to best determine immunological differences between compartments.
Proteins identified in both rectal and salivary mucosal fluid pools were annotated by function using the UniprotKB database. Functional analysis found 72 of the 315 identified proteins had functions in immunity. The proportion of the total number of proteins known to possess each given function is displayed (proteins may be found under multiple categories if they have displayed more than one function). Differential expression analysis identified 49 proteins commonly expressed between fluids (grey), 15 overabundant proteins in rectal lavage (red) and 8 proteins underabundant in rectal lavage (green), relative to saliva (p<0.05, corrected for multiple comparisons). Differentially expressed proteins were found in multiple functional categories. The complete list is shown in in Tables 2a and 2b. Functions of proteins that have no known role in immunity are included in Table S1.
The large majority of proteins identified by mass spectrometry were commonly expressed between the two fluids, with only 29% differentially abundant between saliva and rectal lavage (p<0.05; Tables 2 and 3). However, certain immune factors were found to be higher in abundance in rectal lavage fluid; notably, mucosal immunoglobulins IgA and IgM, known to be important for the binding and clearing of pathogens, increased phagocytosis of microbes and complement activation (Table 2) [29], [30], This may suggest a stronger reliance on immunoglobulin-associated mechanisms of defence in the gut, and supports recent findings on the importance of secreted immunoglobulins in maintaining gut homeostasis [30]. As well, several antiproteases (serpins, inter-alpha trypsin inhibitor, and alpha-2 macroglobulin), known to be important in control of inflammation and tissue remodeling, wound healing proteins (fibronectin) were found overabundant in rectal lavage (Table 2); this may reflect an increased need for the rectal mucosa to control inflammation and repair the thin, damage-prone epithelial layer. Though salivary and rectal mucosal fluid have a similar abundance of most immune proteins, several differentially expressed factors may suggest slightly different immune mechanisms at each mucosal surface that appears to reflect their unique immune requirements. Apart from this trend, the high similarity between these fluids is intuitive when considering that both compartments are a part of the gastrointestinal system.
Mass spectrometry analysis found that saliva and rectal mucosa expressed the majority of detected immune factors at similar levels. The oral and rectal mucosae are constantly exposed to food antigens and commensal bacteria, as well as harmful pathogens as a part of the digestive tract; therefore, the oral and rectal mucosal defence systems must be similarly equipped to maintain a defensive barrier against pathogens while avoiding severe immunopathology from constant stimulation [31]–[33]. Both fluids contained defensive pro-inflammatory proteins (complement components and S100 proteins) that promote the activation and recruitment of immune cells, and regulatory anti-inflammatory factors (apolipoproteins) that act to counter inflammation through attenuation of inflammation-signaling pathways (Tables 4 and 5). Mass spectrometry was also able to characterize many proteins with antimicrobial functions outside of inflammatory mechanisms such as pathogen binding/clearing (mucins, deleted in malignant brain tumors 1, peptidoglycan recognition protein) or direct microbicidal activity (lysozyme c, lactoperoxidase and myeloperoxidase); some of these have been found to have specific anti-HIV mechanisms (antileukoproteinase and cathelicidin) and are listed in Table 6. Furthermore, we found most antiproteases (serpins and cystatins) to be commonly expressed between fluids (Table 7). Antiproteases have an emerging role in immune defense at the mucosal surface as they have been found to regulate inflammation, and have been found to be overexpressed in an HIV-exposed yet seronegative (HESN) population of commercial sex workers, implicating a potential role in susceptibility to HIV infection (Table 1). Overall, mass spectrometry was able to provide a novel comprehensive characterization of immune proteins that likely play a role in transmission of HIV across the rectal mucosa.
As this study was an examination of the mucosal proteome of Caucasian men from Sweden it is possible that these findings are restricted to this gender and/or certain populations. Variation between the male and female rectal compartment, as well as variation between populations have been previously described and may influence immune protein expression at each site. For example, current research in women suggests that mucosal factors fluctuate with hormone levels during the menstrual cycle [34], [35]. As the rectal compartment contains hormone receptors, such as luteinizing hormone (LH) receptor, that have been found to fluctuate throughout the menstrual cycle in mammalian rectal tissue [36], it is plausible that sex hormone differences between genders may impact mucosal immunity in the rectal compartment; however, further studies are needed to fully elucidate the role of LH and other sex hormones, such as estrogen, progesterone and testosterone, in mucosal immunity in both men and women to fully determine this impact. As well, the ethnic profile of our population may not apply to other populations for several reasons, including underlying genetic factors or environment. The effect of diet and microflora composition on secreted mucosal immune factors is not fully understood; however, both are known to influence the intestinal immune system [37]. These factors may cause variability in secreted immune factors and result in variation between populations and would be an important consideration for the interpretation of future proteomic data from different cohorts.
Our characterization of rectal mucosa is an important early step in understanding the natural soluble components of this immune barrier. Our HIV infectivity assays demonstrated the capacity of rectal mucosa to inhibit HIV in vitro, establishing it as a determinant of HIV infection that warrants future investigation to understand its role in HIV susceptibility. Our findings suggest that, at the level of the proteome, rectal mucosa is equipped with many immune factors known to be important in HIV acquisition through the oral compartment; however, the relatively high abundance of mucosal immunoglobulins and antiproteases in rectal mucosa suggests that its mucosal defense may rely on immunoglobulin-mediated and/or anti-inflammatory immune mechanisms; this could be exploited for HIV vaccine development. The lower inhibition capacity of rectal fluid compared to saliva may contribute to relative susceptibility between compartments to HIV. This difference in inhibitory capacity between fluids may also be due to other factors below the detection threshold of our proteomic analysis. This includes altered levels of CC-chemokines/cytokines which are known to be in high abundance in salivary fluid [7], and other short antimicrobial peptides [38] that are critical factors in HIV infection, and have previously been characterized within this cohort in the context of saliva [11]. Further studies into the HIV inhibitory capacity and soluble immune composition of rectal mucosa from individuals may be warranted to better understand natural variations in HIV susceptibility within populations. Furthermore, investigation into small immune proteins/peptides, as well as secreted factors from commensal bacteria will be necessary to fully understand the soluble immune response within rectal mucosa.
Conclusions
This comprehensive proteomic analysis of rectal mucosa provides critical information on the immune factor composition of this fluid that may be important for HIV acquisition. Our study includes important information on the rectal proteome in the context of HIV, but highlights several gaps in our knowledge of the subject. High susceptibility in the rectum combined with a paucity of knowledge on HIV transmission dynamics at the rectal mucosa emphasizes the critical need to further investigate this front-line barrier. This research may help in the development of preventative technologies against the rectal transmission of HIV.
Supporting Information
Table S1.
A comparative proteomic analysis of the soluble immune factor environment of rectal and oral mucosa: Proteins Identified by Label Free MS/MS in Rectal and Salivary Mucosal Fluid.
https://doi.org/10.1371/journal.pone.0100820.s002
(XLSX)
Acknowledgments
We would like to thank all study subjects, as well as the staff at the Gay Men's Health Clinic in Stockholm, Sweden, especially L. Persson, P. Säberg, G. Bratt and E. Sandström. As well, we would like to thank S. McCorister and G. Westmacott from the Mass Spectrometry Core at the Public Health Agency of Canada for providing technical support.
Author Contributions
Conceived and designed the experiments: KH KB TBB AB. Performed the experiments: LR LA KDB. Analyzed the data: LR LA AB. Contributed reagents/materials/analysis tools: KB KH TBB AB. Wrote the paper: LR AB.
References
- 1.
UNAIDS (2009) Report on the global AIDS epidemic.
- 2.
UNAIDS (2011) Criminalisation of HIV Non-Disclosure, Exposure and Transmission: Scientific, Medical, Legal and Human Rights Issues. Geneva, Switzerland.
- 3. Campo J, Perea MA, del Romero J, Cano J, Hernando V, et al. (2006) Oral transmission of HIV, reality or fiction? An update. Oral Dis 12: 219–228.
- 4. Edwards S, Carne C (1998) Oral sex and the transmission of viral STIs. Sex Transm Infect 74: 6–10.
- 5. Baggaley RF, White RG, Boily MC (2010) HIV transmission risk through anal intercourse: systematic review, meta-analysis and implications for HIV prevention. Int J Epidemiol 39: 1048–1063.
- 6. Baggaley RF, White RG, Boily MC (2008) Systematic review of orogenital HIV-1 transmission probabilities. Int J Epidemiol 37: 1255–1265.
- 7. Shugars DC, Alexander AL, Fu K, Freel SA (1999) Endogenous salivary inhibitors of human immunodeficiency virus. Arch Oral Biol 44: 445–453.
- 8. Shugars DC (1999) Endogenous mucosal antiviral factors of the oral cavity. J Infect Dis 179 Suppl 3: S431–435.
- 9. Robinovitch MR, Ashley RL, Iversen JM, Vigoren EM, Oppenheim FG, et al. (2001) Parotid salivary basic proline-rich proteins inhibit HIV-I infectivity. Oral Dis 7: 86–93.
- 10. McNeely TB, Dealy M, Dripps DJ, Orenstein JM, Eisenberg SP, et al. (1995) Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro. J Clin Invest 96: 456–464.
- 11. Hasselrot K, Bratt G, Duvefelt K, Hirbod T, Sandstrom E, et al. (2010) HIV-1 exposed uninfected men who have sex with men have increased levels of salivary CC-chemokines associated with sexual behavior. AIDS 24: 1569–1575.
- 12. Burgener A, Mogk K, Westmacott G, Plummer F, Ball B, et al. (2012) Salivary basic proline-rich proteins are elevated in HIV-exposed seronegative men who have sex with men. AIDS 26: 1857–1867.
- 13. Hirbod T, Kaul R, Reichard C, Kimani J, Ngugi E, et al. (2008) HIV-neutralizing immunoglobulin A and HIV-specific proliferation are independently associated with reduced HIV acquisition in Kenyan sex workers. AIDS 22: 727–735.
- 14. Hasselrot K, Saberg P, Hirbod T, Soderlund J, Ehnlund M, et al. (2009) Oral HIV-exposure elicits mucosal HIV-neutralizing antibodies in uninfected men who have sex with men. AIDS 23: 329–333.
- 15. Hur EM, Patel SN, Shimizu S, Rao DS, Gnanapragasam PN, et al. (2012) Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice. Blood 120: 4571–4582.
- 16. Hasselrot K, Bratt G, Hirbod T, Saberg P, Ehnlund M, et al. (2010) Orally exposed uninfected individuals have systemic anti-HIV responses associating with partners' viral load. AIDS 24: 35–43.
- 17. Aboud L, Ball TB, Tjernlund A, Burgener A (2014) The role of serpin and cystatin antiproteases in mucosal innate immunity and their defense against HIV. Am J Reprod Immunol 71: 12–23.
- 18. Platt EJ, Bilska M, Kozak SL, Kabat D, Montefiori DC (2009) Evidence that ecotropic murine leukemia virus contamination in TZM-bl cells does not affect the outcome of neutralizing antibody assays with human immunodeficiency virus type 1. J Virol 83: 8289–8292.
- 19. Takeuchi Y, McClure MO, Pizzato M (2008) Identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research. J Virol 82: 12585–12588.
- 20. Wei X, Decker JM, Liu H, Zhang Z, Arani RB, et al. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46: 1896–1905.
- 21. Derdeyn CA, Decker JM, Sfakianos JN, Wu X, O′Brien WA, et al. (2000) Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J Virol 74: 8358–8367.
- 22. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D (1998) Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol 72: 2855–2864.
- 23. Shattock RJ, Moore JP (2003) Inhibiting sexual transmission of HIV-1 infection. Nat Rev Microbiol 1: 25–34.
- 24. Salazar-Gonzalez JF, Salazar MG, Keele BF, Learn GH, Giorgi EE, et al. (2009) Genetic identity, biological phenotype, and evolutionary pathways of transmitted/founder viruses in acute and early HIV-1 infection. J Exp Med 206: 1273–1289.
- 25.
Burgener A, Tjernlund A, Kaldensjo T, Abou M, McCorrister S, et al. (2013) A systems biology examination of the human female genital tract shows compartmentalization of immune factor expression. J Virol.
- 26. Bolscher JG, Nazmi K, Ran LJ, van Engelenburg FA, Schuitemaker H, et al. (2002) Inhibition of HIV-1 IIIB and clinical isolates by human parotid, submandibular, sublingual and palatine saliva. Eur J Oral Sci 110: 149–156.
- 27. Malamud D, Nagashunmugam T, Davis C, Kennedy S, Abrams WR, et al. (1997) Inhibition of HIV infectivity by human saliva. Oral Dis 3 Suppl 1: S58–63.
- 28. Archibald DW, Cole GA (1990) In vitro inhibition of HIV-1 infectivity by human salivas. AIDS Res Hum Retroviruses 6: 1425–1432.
- 29. Woof JM, Kerr MA (2006) The function of immunoglobulin A in immunity. J Pathol 208: 270–282.
- 30. Mantis NJ, Rol N, Corthesy B (2011) Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut. Mucosal Immunol 4: 603–611.
- 31.
Janeway CA, Jr., Travers P, Walport M, Shlomchik MJ (2001) Immunobiology: The Immune System in Health and Disase. New York: Garland Science.
- 32. Berbee JF, Havekes LM, Rensen PC (2005) Apolipoproteins modulate the inflammatory response to lipopolysaccharide. J Endotoxin Res 11: 97–103.
- 33. Kang SS, Jeon JH, Woo SJ, Yang JS, Kim KW, et al. (2012) IFN-gamma renders human intestinal epithelial cells responsive to lipopolysaccharide of Vibrio cholerae by down-regulation of DMBT1. Comp Immunol Microbiol Infect Dis 35: 345–354.
- 34. Rodriguez-Garcia M, Patel MV, Wira CR (2013) Innate and adaptive anti-HIV immune responses in the female reproductive tract. J Reprod Immunol 97: 74–84.
- 35. Rahman S, Rabbani R, Wachihi C, Kimani J, Plummer FA, et al. (2013) Mucosal Serpin A1 and A3 Levels in HIV Highly Exposed Sero-Negative Women are Affected by the Menstrual Cycle and Hormonal Contraceptives but are Independent of Epidemiological Confounders. Am J Reprod Immunol 69: 64–72.
- 36. Wang LH, Zhang W, Gao QX, Wang F (2012) Expression of the luteinizing hormone receptor (LHR) gene in ovine non-gonadal tissues during estrous cycle. Genet Mol Res 11: 3766–3780.
- 37. Purchiaroni F, Tortora A, Gabrielli M, Bertucci F, Gigante G, et al. (2013) The role of intestinal microbiota and the immune system. Eur Rev Med Pharmacol Sci 17: 323–333.
- 38. Weinberg A, Krisanaprakornkit S, Dale BA (1998) Epithelial antimicrobial peptides: review and significance for oral applications. Crit Rev Oral Biol Med 9: 399–414.
- 39. Bergey EJ, Cho MI, Blumberg BM, Hammarskjold ML, Rekosh D, et al. (1994) Interaction of HIV-1 and human salivary mucins. J Acquir Immune Defic Syndr 7: 995–1002.
- 40.
Sheng YH, Triyana S, Wang R, Das I, Gerloff K, et al. (2012) MUC1 and MUC13 differentially regulate epithelial inflammation in response to inflammatory and infectious stimuli. Mucosal Immunol.
- 41. Steinstraesser L, Tippler B, Mertens J, Lamme E, Homann HH, et al. (2005) Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides. Retrovirology 2: 2.
- 42. Wong JH, Legowska A, Rolka K, Ng TB, Hui M, et al. (2011) Effects of cathelicidin and its fragments on three key enzymes of HIV-1. Peptides 32: 1117–1122.
- 43. Ogawa Y, Kawamura T, Matsuzawa T, Aoki R, Gee P, et al. (2013) Antimicrobial Peptide LL-37 Produced by HSV-2-Infected Keratinocytes Enhances HIV Infection of Langerhans Cells. Cell Host Microbe 13: 77–86.
- 44. Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75: 39–48.
- 45. Verani A, Lusso P (2002) Chemokines as natural HIV antagonists. Curr Mol Med 2: 691–702.
- 46. Ma G, Greenwell-Wild T, Lei K, Jin W, Swisher J, et al. (2004) Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection. J Exp Med 200: 1337–1346.
- 47. Bingle L, Tetley TD (1996) Secretory leukoprotease inhibitor: partnering alpha 1-proteinase inhibitor to combat pulmonary inflammation. Thorax 51: 1273–1274.
- 48. Wingens M, van Bergen BH, Hiemstra PS, Meis JF, van Vlijmen-Willems IM, et al. (1998) Induction of SLPI (ALP/HUSI-I) in epidermal keratinocytes. J Invest Dermatol 111: 996–1002.
- 49. Williams SE, Brown TI, Roghanian A, Sallenave JM (2006) SLPI and elafin: one glove, many fingers. Clin Sci (Lond) 110: 21–35.
- 50. Devito C, Hinkula J, Kaul R, Kimani J, Kiama P, et al. (2002) Cross-clade HIV-1-specific neutralizing IgA in mucosal and systemic compartments of HIV-1-exposed, persistently seronegative subjects. J Acquir Immune Defic Syndr 30: 413–420.
- 51. Devito C, Broliden K, Kaul R, Svensson L, Johansen K, et al. (2000) Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells. J Immunol 165: 5170–5176.
- 52. Lamm ME (1997) Interaction of antigens and antibodies at mucosal surfaces. Annu Rev Microbiol 51: 311–340.
- 53. Amado F, Lobo MJ, Domingues P, Duarte JA, Vitorino R (2010) Salivary peptidomics. Expert Rev Proteomics 7: 709–721.
- 54. Gu M, Haraszthy GG, Collins AR, Bergey EJ (1995) Identification of salivary proteins inhibiting herpes simplex virus 1 replication. Oral Microbiol Immunol 10: 54–59.
- 55. Newman F, Beeley JA, MacFarlane TW (1996) Adherence of oral microorganisms to human parotid salivary proteins. Electrophoresis 17: 266–270.
- 56. White SH, Wimley WC, Selsted ME (1995) Structure, function, and membrane integration of defensins. Curr Opin Struct Biol 5: 521–527.
- 57. Furci L, Sironi F, Tolazzi M, Vassena L, Lusso P (2007) Alpha-defensins block the early steps of HIV-1 infection: interference with the binding of gp120 to CD4. Blood 109: 2928–2935.
- 58. Furci L, Tolazzi M, Sironi F, Vassena L, Lusso P (2012) Inhibition of HIV-1 infection by human alpha-defensin-5, a natural antimicrobial peptide expressed in the genital and intestinal mucosae. PLoS One 7: e45208.
- 59. Levinson P, Kaul R, Kimani J, Ngugi E, Moses S, et al. (2009) Levels of innate immune factors in genital fluids: association of alpha defensins and LL-37 with genital infections and increased HIV acquisition. AIDS 23: 309–317.
- 60. Rapista A, Ding J, Benito B, Lo YT, Neiditch MB, et al. (2011) Human defensins 5 and 6 enhance HIV-1 infectivity through promoting HIV attachment. Retrovirology 8: 45.
- 61. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, et al. (1999) Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286: 525–528.
- 62. Quinones-Mateu ME, Lederman MM, Feng Z, Chakraborty B, Weber J, et al. (2003) Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication. AIDS 17: F39–48.
- 63. Ellison RT 3rd, LaForce FM, Giehl TJ, Boose DS, Dunn BE (1990) Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+. J Gen Microbiol 136: 1437–1446.
- 64. Harmsen MC, Swart PJ, de Bethune MP, Pauwels R, De Clercq E, et al. (1995) Antiviral effects of plasma and milk proteins: lactoferrin shows potent activity against both human immunodeficiency virus and human cytomegalovirus replication in vitro. J Infect Dis 172: 380–388.
- 65. Viani RM, Gutteberg TJ, Lathey JL, Spector SA (1999) Lactoferrin inhibits HIV-1 replication in vitro and exhibits synergy when combined with zidovudine. AIDS 13: 1273–1274.
- 66. Goldman AS, Smith CW (1973) Host resistance factors in human milk. J Pediatr 82: 1082–1090.
- 67.
Heo SM, Choi KS, Kazim LA, Reddy MS, Haase EM, et al. (2013) Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus. Infect Immun.
- 68. Simpson AJ, Maxwell AI, Govan JR, Haslett C, Sallenave JM (1999) Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens. FEBS Lett 452: 309–313.
- 69. Henriksen PA, Hitt M, Xing Z, Wang J, Haslett C, et al. (2004) Adenoviral gene delivery of elafin and secretory leukocyte protease inhibitor attenuates NF-kappa B-dependent inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. J Immunol 172: 4535–4544.
- 70. Butler MW, Robertson I, Greene CM, O′Neill SJ, Taggart CC, et al. (2006) Elafin prevents lipopolysaccharide-induced AP-1 and NF-kappaB activation via an effect on the ubiquitin-proteasome pathway. J Biol Chem 281: 34730–34735.
- 71. Gettins PG (2002) Serpin structure, mechanism, and function. Chem Rev 102: 4751–4804.
- 72. Fahey JV, Bodwell JE, Hickey DK, Ghosh M, Muia MN, et al. (2011) New approaches to making the microenvironment of the female reproductive tract hostile to HIV. Am J Reprod Immunol 65: 334–343.
- 73. Mangan MS, Kaiserman D, Bird PI (2008) The role of serpins in vertebrate immunity. Tissue Antigens 72: 1–10.
- 74. Heutinck KM, ten Berge IJ, Hack CE, Hamann J, Rowshani AT (2010) Serine proteases of the human immune system in health and disease. Mol Immunol 47: 1943–1955.
- 75. Congote LF (2006) The C-terminal 26-residue peptide of serpin A1 is an inhibitor of HIV-1. Biochem Biophys Res Commun 343: 617–622.
- 76. Shapiro L, Pott GB, Ralston AH (2001) Alpha-1-antitrypsin inhibits human immunodeficiency virus type 1. FASEB J 15: 115–122.
- 77. Zhou X, Shapiro L, Fellingham G, Willardson BM, Burton GF (2011) HIV replication in CD4+ T lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by alpha-1-antitrypsin through altered IkappaBalpha ubiquitination. J Immunol 186: 3148–3155.
- 78. Munch J, Standker L, Adermann K, Schulz A, Schindler M, et al. (2007) Discovery and optimization of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide. Cell 129: 263–275.
- 79. Moriuchi H, Moriuchi M, Fauci AS (2000) Cathepsin G, a neutrophil-derived serine protease, increases susceptibility of macrophages to acute human immunodeficiency virus type 1 infection. J Virol 74: 6849–6855.
- 80. Wittek N, Majewska E (2010) [Cystatin C—modulator of immune processes]. Przegl Lek 67: 484–487.
- 81. Kopitar-Jerala N (2006) The role of cystatins in cells of the immune system. FEBS Lett 580: 6295–6301.
- 82. Luciano-Montalvo C, Melendez LM (2009) Cystatin B associates with signal transducer and activator of transcription 1 in monocyte-derived and placental macrophages. Placenta 30: 464–467.
- 83. Rivera-Rivera L, Perez-Laspiur J, Colon K, Melendez LM (2012) Inhibition of interferon response by cystatin B: implication in HIV replication of macrophage reservoirs. J Neurovirol 18: 20–29.