Tonsil epithelial factors may influence oropharyngeal human immunodeficiency virus transmission

Abstract

Tonsil epithelium has been implicated in human immunodeficiency virus (HIV) pathogenesis, but its role in oral transmission remains controversial. To study characteristics of this tissue, which may influence susceptibility or resistance to HIV, we performed microarray analysis of the tonsil epithelium. Our data revealed that genes related to immune functions such as antibody production and antigen processing were increasingly expressed in tonsil compared with the epithelium of another oropharyngeal site, the gingival epithelium. Importantly, tonsil epithelium highly expressed genes associated with HIV entrapment and/or transmission, including the HIV co-receptor CXCR4 and the potential HIV-binding molecules FcRgammaIII, complement receptor 2, and various complement components. Immunohistochemical staining confirmed the increased presence of CXCR4 in the tonsil epithelium compared with multiple oral epithelial sites, particularly in basal and parabasal layers. This increased expression of molecules involved in viral recognition, binding, and entry may favor virus-epithelium interactions in an environment with reduced innate antiviral mechanisms. Specifically, secretory leukocyte protease inhibitor, an innate molecule with anti-HIV activity, was minimal in the tonsil epithelium, in contrast to oral mucosa. Collectively, our data suggest that increased expression of molecules associated with HIV binding and entry coupled with decreased innate antiviral factors may render the tonsil a potential site for oral transmission.

Figure 1

LCM and differential and shared gene expression between tonsil and gingival epithelium. A: The epithelium area was outlined for both the tonsil surface epithelium and gingival epithelium and subsequently isolated by LCM. Sections are shown before, during, and after epithelium removal. B: Top 10 expressed genes (absolute intensity) in both tonsil and gingival epithelium.

Figure 2

GO scan analysis of differential gene expression between tonsil and gingival epithelium. Affymetrix probe set IDs were linked to gene identifiers and functional Gene Ontology annotations using the Netaffx website at http://www.affymetrix.com/analysis/index.affx. Statistically differentially expressed genes were organized into functional categories using the Gene Ontology (GO) database (http://www.geneontology.org) and the GO-Significant Collections of Annotations (GOSCAN) program (http://abs.cit.nih.gov/goscan/). Ratios of tonsil to gingival epithelium gene expression is reported as tonsil/gingiva and colored areas indicate the functional category(ies) that each gene belongs to.

Figure 3

Differential expression of HIV-related genes. A: Parallel plot showing gene expression intensity for B-cell markers (CD19, CD79), antibody-related genes (Ig J polypeptide), and T-cell marker (CD3) in gingival (n = 7) and tonsil (n = 5) samples. B: Parallel plot showing expression levels/intensity for genes associated with HIV entrapment/transmission in the tonsil (n = 5) and gingival (n = 7) epithelium. C: Parallel plot showing gene expression intensity for the HIV receptor CD4 and co-receptors (GalCer, CXCR4, and CCR5) in the tonsil and gingival epithelium, *FDR < 10% and P < 0.05 of differential gene expression between sample groups. Immunohistochemical staining for CD19 in the tonsil (D) and CD3 (E). Immunohistochemical staining for ICAM-3 (F), for CD4 (H), and GalCer (I) in the tonsil epithelium. Immunofluorescence FcR-CD32 (red), pan-cytokeratin (green) and 4,6-diamidino-2-phenylindole (G). Arrows indicate positive staining. Original magnifications: ×20 (D–F, H, and I); ×63 (G).

Figure 4

CXCR4 and CCR5 protein expression in the tonsil and oral epithelia. A and B: Immunohistochemical staining for CXCR4 in the tonsil epithelium. Staining of high intensity (grade 3) is seen in the basal and parabasal cell layers (A) and in the spinous layer of select samples (B, arrows indicate staining of high intensity in A and B). Weak to moderate CXCR4 staining is also seen in the oral tissues keratinized (C) and nonkeratinized (D, arrows indicate staining of moderate intensity in C and D). E: Immunohistochemical staining for CCR5 in the tonsil epithelium (E and F, negative control) and in the parakeratinized (G) and nonkeratinized (H) oral epithelium. Staining is moderate (shown by arrows) and weak in most areas. Original magnifications: ×20 (A–D, and F); ×10 (E, G, and H).

Figure 5

SLPI expression in the tonsil and oral epithelia. A: Mean fold change of gene expression between tonsil and gingiva for the innate immune factors lysozyme, defensin-β1, defensin-β4, SLPI, and thrombospondin-1. *P ≤ 0.05. B: Quantitation of percent positive SLPI, defensin-β1, and defensin-β4 cells in the tonsil (n = 5) and oral epithelium (n = 5). *P ≤ 0.05. C: SLPI staining in the tonsil is weak (grade 1) with isolated cells staining moderately (grade 2, arrow showing higher intensity staining). D: Negative control. In the oral mucosa, SLPI staining is abundant and of high intensity (grade 2 to 3) for both keratinized (E) and nonkeratinized (F) oral epithelia (arrow shows high-intensity staining). Original magnifications: ×20 (D–F).