Salivary mucin 19 glycoproteins: innate immune functions in Streptococcus mutans-induced caries in mice and evidence for expression in human saliva.

Saliva functions in innate immunity of the oral cavity, protecting against demineralization of teeth (i.e. dental caries), a highly prevalent infectious disease associated with Streptococcus mutans, a pathogen also linked to endocarditis and atheromatous plaques. Gel-forming mucins are a major constituent of saliva. Because Muc19 is the dominant salivary gel-forming mucin in mice, we studied Muc19(-/-) mice for changes in innate immune functions of saliva in interactions with S. mutans. When challenged with S. mutans and a cariogenic diet, total smooth and sulcal surface lesions are more than 2- and 1.6-fold higher in Muc19(-/-) mice compared with wild type, whereas the severity of lesions are up to 6- and 10-fold higher, respectively. Furthermore, the oral microbiota of Muc19(-/-) mice display higher levels of indigenous streptococci. Results emphasize the importance of a single salivary constituent in the innate immune functions of saliva. In vitro studies of S. mutans and Muc19 interactions (i.e. adherence, aggregation, and biofilm formation) demonstrate Muc19 poorly aggregates S. mutans. Nonetheless, aggregation is enhanced upon adding Muc19 to saliva from Muc19(-/-) mice, indicating Muc19 assists in bacterial clearance through formation of heterotypic complexes with salivary constituents that bind S. mutans, thus representing a novel innate immune function for salivary gel-forming mucins. In humans, expression of salivary MUC19 is unclear. We find MUC19 transcripts in salivary glands of seven subjects and demonstrate MUC19 glycoproteins in glandular mucous cells and saliva. Similarities and differences between mice and humans in the expression and functions of salivary gel-forming mucins are discussed.

Oral colonization by Streptococcus mutans and caries development is reduced upon deletion of carbonic anhydrase VI expression in saliva.

Carbonic anhydrase VI (CA VI), encoded by type A transcripts of the gene Car6, is a secretory product of salivary glands and is found in the enamel pellicle. Because higher caries prevalence is associated with lower salivary concentrations of CA VI in humans, we tested whether CA VI protects enamel surfaces from caries induced by Streptococcus mutans, using Car6(-/-) mice, in which salivary CA VI expression is absent. We detected aberrant Car6 type A transcripts in Car6(-/-) mice, likely targets for nonsense-mediated mRNA decay. Expression of the intracellular stress-induced isoform of CA VI encoded by type B transcripts was restricted to parotid and submandibular glands of wild type mice. The salivary function of Car6(-/-) mice was normal as assessed by the histology and protein/glycoprotein profiles of glands, salivary flow rates and protein/glycoprotein compositions of saliva. Surprisingly, total smooth surface caries and sulcal caries in Car6(-/-) mice were more than 6-fold and 2-fold lower than in wild type mice after infection with S. mutans strain UA159. Recoveries of S. mutans and total microbiota from molars were also lower in Car6(-/-) mice. To explore possible mechanisms for increased caries susceptibility, we found no differences in S. mutans adherence to salivary pellicles, in vitro. Interestingly, higher levels of Lactobacillus murinus and an unidentified Streptococcus species were cultivated from the oral microbiota of Car6(-/-) mice. Collective results suggest salivary CA VI may promote caries by modulating the oral microbiota to favor S. mutans colonization and/or by the enzymatic production of acid within plaque.

Murine Salivary Amylase Protects Against Streptococcus mutans-Induced Caries.

Saliva protects dental surfaces against cavities (i. e., dental caries), a highly prevalent infectious disease frequently associated with acidogenic Streptococcus mutans. Substantial in vitro evidence supports amylase, a major constituent of saliva, as either protective against caries or supporting caries. We therefore produced mice with targeted deletion of salivary amylase (Amy1) and determined the impact on caries in mice challenged with S. mutans and fed a diet rich in sucrose to promote caries. Total smooth surface and sulcal caries were 2.35-fold and 1.79-fold greater in knockout mice, respectively, plus caries severities were twofold or greater on sulcal and smooth surfaces. In in vitro experiments with samples of whole stimulated saliva, amylase expression did not affect the adherence of S. mutans to saliva-coated hydroxyapatite and slightly increased its aggregation in solution (i.e., oral clearance). Conversely, S. mutans in biofilms formed in saliva with 1% glucose displayed no differences when cultured on polystyrene, but on hydroxyapatite was 40% less with amylase expression, suggesting that recognition by S. mutans of amylase bound to hydroxyapatite suppresses growth. However, this effect was overshadowed in vivo, as the recoveries of S. mutans from dental plaque were similar between both groups of mice, suggesting that amylase expression helps decrease plaque acids from S. mutans that dissolve dental enamel. With amylase deletion, commensal streptococcal species increased from ~75 to 90% of the total oral microbiota, suggesting that amylase may promote higher plaque pH by supporting colonization by base-producing oral commensals. Importantly, collective results indicate that amylase may serve as a biomarker of caries risk.

Harmine enhances the activity of the HIV-1 latency-reversing agents ingenol A and SAHA.

Infection with human immunodeficiency virus 1 (HIV-1) remains incurable because long-lived, latently-infected cells persist during prolonged antiretroviral therapy. Attempts to pharmacologically reactivate and purge the latent reservoir with latency reactivating agents (LRAs) such as protein kinase C (PKC) agonists (e.g. ingenol A) or histone deacetylase (HDAC) inhibitors (e.g. SAHA) have shown promising but incomplete efficacy. Using the J-Lat T cell model of HIV latency, we found that the plant-derived compound harmine enhanced the efficacy of existing PKC agonist LRAs in reactivating latently-infected cells. Treatment with harmine increased not only the number of reactivated cells but also increased HIV transcription and protein expression on a per-cell basis. Importantly, we observed a synergistic effect when harmine was used in combination with ingenol A and the HDAC inhibitor SAHA. An investigation into the mechanism revealed that harmine, when used with LRAs, increased the activity of NFκB, MAPK p38, and ERK1/2. Harmine treatment also resulted in reduced expression of HEXIM1, a negative regulator of transcriptional elongation. Thus, harmine enhanced the effects of LRAs by increasing the availability of transcription factors needed for HIV reactivation and promoting transcriptional elongation. Combination therapies with harmine and LRAs could benefit patients by achieving deeper reactivation of the latent pool of HIV provirus.