No abstract

Background-Accumulating evidence suggests a regulatory role of Wnt proteins in innate immune responses. However, the effects of Wnt3a signaling on TLR4-mediated inflammatory responses are controversial and the signaling crosstalk between TLR4 and Wnt3a remains uncertain.Methods-Gainand Loss-of function approaches were utilized to determine the function of Wnt3a signaling in TLR4-mediated inflammatory responses. Cytokine production at protein and mRNA levels and phosphorylation of signaling molecules were measured by ELISA, qRT-PCR, and Western Blot, respectively. Endotoxemia mouse model was employed to assess the effect of Wnt3a on systemic inflammatory cytokine levels and neutrophil infiltration.Results-LPS stimulation leads to an increase of Wnt3a expression and its downstream molecule, Dvl3, in primary monocytes. Inhibition or silence of Wnt3a or Dvl3 significantly increases the production of pro-inflammatory cytokines (IL-12, IL-6, TNFα), robustly reduces βcatenin accumulation, and enhances the phosphorylation of NF-κB P65 and its DNA binding

Porphyromonas gingivalis, a keystone pathogen in periodontitis (Hajishengallis et al., 2012; Lamont et al., 2018), is an endogenous inhabitant of the oral cavity, in particular the subgingival space. This region is a complex and dynamic microhabitat containing a multispecies microbial community with a fluid phase of gingival crevicular fluid (Lamont et al., 2018). Bacterial adaptation to environmental cues is often mediated by two-component signal transduction systems (TCS) in which, canonically, a transmembrane sensor histidine kinase (HK) phosphorylates and activates a DNA-binding response regulator (RR). Variations on this theme include hybrid systems in which the HK and RR are in the same molecule, and orphan systems in which the RR lacks a cognate HK (Miller & Lamont, 2019; Stock et al., 2000). Recently, it has become apparent that bacterial serine/threonine and tyrosine kinases can overlay cross-phosphorylation interactions on signal

At mucosal barriers, the virulence of microbial communities reflects the outcome of both dysbiotic and eubiotic interactions with the host, with commensal species mitigating or potentiating the action of pathogens. We examined epithelial responses to the oral pathogen Porphyromonas gingivalis as a monoinfection and in association with a community partner, Streptococcus gordonii. RNA-Seq of oral epithelial cells showed that the Notch signaling pathway, including the downstream effector olfactomedin 4 (OLFM4), was differentially regulated by P. gingivalis alone; however, regulation was overridden by S. gordonii. OLFM4 was required for epithelial cell migratory, proliferative and inflammatory responses to P. gingivalis. Activation of Notch signaling was induced through increased expression of the Notch1 receptor and the Jagged1 (Jag1) agonist. In addition, Jag1 was released in response to P. gingivalis, leading to paracrine activation. Following Jag1-Notch1 engagement, the Notch1 extracellular domain was cleaved by P. gingivalis gingipain proteases. Antagonism by S. gordonii involved inhibition of gingipain activity by secreted hydrogen peroxide. The results establish a novel mechanism by which P. gingivalis modulates epithelial cell function which is dependent on community context. These interrelationships have relevance for innate inflammatory responses and epithelial cell fate decisions in oral health and disease.

Tyrosine phosphatases are often weaponized by bacteria colonizing mucosal barriers to manipulate host cell signal transduction pathways. Porphyromonas gingivalis is a periodontal pathogen and emerging oncopathogen which interferes with gingival epithelial cell proliferation and migration, and induces a partial epithelial mesenchymal transition. P. gingivalis produces two tyrosine phosphatases, and we show here that the low molecular weight tyrosine phosphatase, Ltp1, is secreted within gingival epithelial cells and translocates to the nucleus. An ltp1 mutant of P. gingivalis showed a diminished ability to induce epithelial cell migration and proliferation. Ltp1 was also required for the transcriptional upregulation of Regulator of Growth and Cell Cycle (RGCC), one of the most differentially expressed genes in epithelial cells resulting from P. gingivalis infection. A phosphoarray and siRNA showed that P. gingivalis controlled RGCC expression through Akt, which was activated by phosphorylation on S473. Akt activation is opposed by PTEN, and P. gingivalis decreased the amount of PTEN in epithelial cells. Ectopically expressed Ltp1 bound to PTEN, and reduced phosphorylation of PTEN at Y336 which controls proteasomal degradation. Ltp-1 induced loss of PTEN stability was prevented by chemical inhibition of the proteosome. Knockdown of RGCC suppressed upregulation of Zeb2 and mesenchymal markers by P. gingivalis. RGCC inhibition was also accompanied by a reduction in production of the proinflammatory cytokine IL-6 in response to P. gingivalis. Elevated IL-6 levels can contribute to periodontal destruction, and the ltp1 mutant of P. gingivalis incited less bone loss compared to the parental strain in a murine model of periodontal disease. These results show that P. gingivalis can deliver Ltp1 within gingival epithelial cells, and establish PTEN as the target for Ltp1 phosphatase activity. Disruption of the Akt1/RGCC signaling axis by Ltp1 facilitates P. gingivalis-induced increases in epithelial cell migration, proliferation, EMT and inflammatory cytokine production.