Porphyromonas gingivalisSelectively Up-Regulates the HIV-1 Coreceptor CCR5 in Oral Keratinocytes

Abstract

Primary infection of oral epithelial cells by HIV-1, if it occurs, could promote systemic infection. Most primary systemic infections are associated with R5-type HIV-1 targeting the R5-specific coreceptor CCR5, which is not usually expressed on oral keratinocytes. Because coinfection with other microbes has been suggested to modulate cellular infection by HIV-1, we hypothesized that oral keratinocytes may up-regulate CCR5 in response to the oral endogenous pathogen Porphyromonas gingivalis by cysteine-protease (gingipains) activation of the protease-activated receptors (PARs) or LPS signaling through the TLRs. The OKF6/TERT-2-immortalized normal human oral keratinocyte line expressed CXCR4, whereas CCR5 was not detectable. When exposed to P. gingivalis ATCC 33277, TERT-2 cells induced greater time-dependent expression of CCR5-specific mRNA and surface coreceptors than CXCR4. By comparing arg- (Rgp) and lys-gingipain (Kgp) mutants, a mutant deficient in both proteases, and the action of trypsin, P. gingivalis Rgp was strongly suggested to cleave PAR-1 and PAR-2 to up-regulate CCR5. CCR5 was also slightly up-regulated by an isogenic gingipain-deficient mutant, suggesting the presence of a nongingipain-mediated mechanism. Purified P. gingivalis LPS also up-regulated CCR5. Blocking TLR2 and TLR4 receptors with Abs attenuated induction of CCR5, suggesting LPS signaling through TLRs. P. gingivalis, therefore, selectively up-regulated CCR5 by two independent signaling pathways, Rgp acting on PAR-1 and PAR-2, and LPS on TLR2 and TLR4. By inducing CCR5 expression, P. gingivalis coinfection could promote selective R5-type HIV-1 infection of oral keratinocytes.

Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains

“…As we have reported (Giacaman et al, 2007), P. gingivalis Rgp upregulates CCR5 through PAR-1 and -2 signalling. Hence, the PARs enhance the responses of keratinocytes to cytokines.…”

“…For example, P. gingivalis Rgp upregulates IL-8 expression in gingival fibroblasts (OidoMori et al, 2001), but not in oral epithelial cells as we show here. Furthermore, P. gingivalis gingipains are generally copresented with LPS, which can synergize or simulate PARmediated transcriptional responses in oral keratinocytes (Giacaman et al, 2007). Since all of the P. gingivalis mutants used in our study express LPS, differential regulation of pro-inflammatory cytokines argues in favour of gingipain-associated cytokine induction signalled by selective PAR cleavage, rather than from synergistic LPSmediated activation of TLRs.…”

“…Since the TLRs are intact and functional on the oral keratinocytes (Giacaman et al, 2007), differences in keratinocyte responses to gingipain mutants are unlikely to be attributable to differential signalling through the TLRs. In our system, P. gingivalis cleaves PAR-1 (Fig.…”

“…OKF6/TERT-2 (TERT-2), an immortalized oral epithelial cell line provided by J. Rheinwald (Harvard Medical School, Cambridge, MA) (Dickson et al, 2000), was used to model primary gingival epithelial cells. TERT-2 cells were grown in 5 % CO 2 at 37 uC in keratinocyte serum-free and calcium-free medium (Invitrogen Life Technologies) supplemented with 0.4 mM CaCl 2 , 25 mg ml 21 bovine pituitary extract and 0.2 ng ml 21 epidermal growth factor, as previously reported (Giacaman et al, 2007). Culture medium was changed every 3 days and cells were subcultured when they reached approximately 60 % confluence.…”

“…Since incubation with P. gingivalis for longer than 6 h causes cells to detach from the flasks (Nisapakultorn et al, 2001), keratinocytes were incubated in TERT medium with P. gingivalis for 3 h, as previously described (Giacaman et al, 2007), washed five times with DPBS and then incubation continued for the times indicated. At times up to 48 h after initial exposure to P. gingivalis, TERT-2 cells were harvested.…”