A comparative proteomic analysis of the soluble immune factor environment of rectal and oral mucosa

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).

Conclusions: This study reveals that rectal mucosa contains many innate immune factors important for host immunity to HIV and can limit viral replication in vitro. This indicates an important role for this fluid as the first line of defense against HIV.

Figure 1. Rectal lavage shows mild inhibitory activity against R5-tropic HIV in vitro.

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).

Figure 2. Biological functional categories of immune factors identified in saliva and rectal lavage fluid according to their gene ontology.

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.