JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

Abstract

Homeostasis between pro- and anti- inflammatory responses induced by bacteria is critical for the maintenance of health. In the oral cavity, pro-inflammatory mechanisms induced by pathogenic bacteria are well-established; however, the anti-inflammatory responses that act to restrain innate responses remain poorly characterized. Here, we demonstrate that infection with the periodontal pathogen Porphyromonas gingivalis enhances the activity of Janus kinase 3 (JAK3) in innate immune cells, and subsequently phospho-inactivates Nedd4-2, an ubiquitin E3 ligase. In turn, Wingless-INT (Wnt) 3 (Wnt3) ubiquitination is decreased, while total protein levels are enhanced, leading to a reduction in pro-inflammatory cytokine levels. In contrast, JAK3 or Wnt3a inhibition robustly enhances nuclear factor kappa-light-chain-enhancer of activated B cells activity and the production of pro-inflammatory cytokines in P. gingivalis-stimulated innate immune cells. Moreover, using gain- and loss-of-function approaches, we demonstrate that downstream molecules of Wnt3a signaling, including Dvl3 and β-catenin, are responsible for the negative regulatory role of Wnt3a. In addition, using an in vivo P. gingivalis-mediated periodontal disease model, we show that JAK3 inhibition enhances infiltration of inflammatory cells, reduces expression of Wnt3a and Dvl3 in P. gingivalis-infected gingival tissues, and increases disease severity. Together, our results reveal a new anti-inflammatory role for JAK3 in innate immune cells and show that the underlying signaling pathway involves Nedd4-2-mediated Wnt3a ubiquitination.

Keywords: P. gingivalis; JAK3; Wnt3; inflammation; ubiquitination.

Figure 1.. P. gingivalis challenge robustly enhances the activity of JAK3 in P. gingivalis-stimulated innate immune cells

Human monocytes were stimulated with P. gingivalis at different MOIs (1, 10, 50, and 100) for 24 h, and a trypan blue exclusion assay was performed at various time intervals. (A) The production of proinflammatory cytokines (TNFα, IL-6, and IL-12P40) was determined by ELISA after 24 h of stimulation. (B) Percentage of dead cells after challenge of P. gingivalis with different MOIs. (C to F) Whole-cell lysates of human monocytes (C), THP-1 (D), and RAW264.7 cells (E) challenged with P. gingivalis (MOI of 10) analyzed by Western blot. Immunoblots were probed with antibodies to phospho- and total JAK3 and GAPDH. (F) The mean intensity ratios of phospho- to total- JAK3 were determined by densitometry. (G, H) JAK3 was immunoprecipitated from the cell lysates of P. gingivalis-stimulated human monocytes. Cross-linking and freeze-drying were also used to purify and enrich proteins in immunoprecipitates. Western blotting was used to determine the phosphorylation of JAK3 in regular (G) and enriched (H) immunoprecipitates. All the blots shown are representative of three to five biological replicates. For A and B, the data represent the arithmetic mean±standard deviation (S.D.) of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Figure 2.. JAK3 negatively regulates P. gingivalis-induced proinflammatory cytokine production in innate immune cells

Cells were pretreated with a JAK3 inhibitor (T-1377, 1 nM) for 2 h and then stimulated with P. gingivalis over a 24 h time course. (A to C) Levels of TNFα, IL-6, and IL-12P40 in cell-free supernatants produced by human monocytes (A), THP-1 (B) and RAW 264.7 cells (C). (D) Total cell lysates were collected at the indicated times and probed for phosphorylated STAT5 and STAT6. (E to H) Human monocytes were transfected with specific siRNA or a plasmid to silence or overexpress JAK3, respectively. Nontargeting siRNA and an empty vector were used as controls. After transfection for 72 h, human monocytes were stimulated with P. gingivalis over a 24 h time course. The cell-free supernatant and whole-cell lysates were collected to determine the levels of inflammatory cytokines and the transfection efficiency, respectively. The transfection efficiency was estimated by the expression levels of total JAK3. JAK3 silencing (E) significantly enhances the production of TNFα, IL-6, and IL-12 (F), while the overexpression of JAK3 (G) significantly decreases the levels of TNFα, IL-6, and IL-12 in P. gingivalis-stimulated human monocytes (H). For D, F, and G, the blots shown are representative of three to five biological replicates. Other data represent the arithmetic mean ± S.D. of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Figure 3.. P. gingivalis enhances the activity of Wnt3a-β-catenin signaling through JAK3 in innate immune cells

Purified human monocytes (A to C) and THP-1 cells (D, E) were pretreated with a JAK3 inhibitor (1 nM T-1377) for 2 h and then stimulated with P. gingivalis (MOI of 10) for the indicated time. (A to E) Total cell lysates were probed for Wnt3a, Dvl3, p-GSK3β, p-β-catenin, and GAPDH (A, D) and mRNA levels of Wnt3a and Dvl3 were also detected after challenge with P. gingivalis for 2 h using qRT-PCR (C). (F to H) Western blot of human monocytes transfected with nontargeting or JAK3-specific siRNA for 72 h and then stimulated with P. gingivalis for the indicated time. Blots were probed with antibodies against Wnt3a, Dvl3, p-GSK3β, p-β-catenin, and GAPDH (G). The intensity ratio of each protein relative to that of GAPDH was determined by densitometry (B, E, F, H). (I, J) THP-1 cells were stimulated with Streptococcus sanguinis and Streptococcus gordonii (MOI of 10) for the indicated time. Total cell lysates were probed for p-JAK3, Wnt3a, Dvl3, and GAPDH. All the blots shown are representative of three to five biological replicates.

Figure 4.. JAK3-mediated Wnt3a-β-catenin signaling suppresses the production of inflammatory cytokines in P. gingivalis-stimulated innate immune cells

Purified human monocytes were transfected with nontargeting control, Wnt3a, Dvl3, or GSK3β siRNAs, or a plasmid encoding β-catenin or a serine 9 to alanine constitutively active GSK3β mutant for 72 h and then stimulated with P. gingivalis over a 24 h time course in the absence or presence of a JAK3 inhibitor (1 nM T-1377). Cell-free supernatants and total cell lysates were harvested to determine the levels of TNFα, IL-6, and IL-12P40 and the transfection efficiency, respectively. (A to C) The silencing of Wnt3a or Dvl3 (A) significantly enhances the production of TNFα, IL-6, and IL-12P40 (B, C) in P. gingivalis-stimulated monocytes. The silencing of GSK3β or overexpression of β-catenin (D) abrogates the influence of JAK3 inhibition on the production of inflammatory cytokines and significantly reduces the levels of TNFα, IL-6, and IL-12P40 in P. gingivalis-stimulated monocytes (F). (G, H) Total cell lysates were probed with antibodies against β-catenin and the inserted HA tag to determine the transfection efficiency and its effect on the expression of β-catenin (G). (H) The production of TNFα, IL-6, and IL-12P40 in P. gingivalis-stimulated human monocytes overexpressing the GSK3β mutant. All the blots shown are representative of three to five experiments. For B, C, E, F, H, all the data were generated in the same experiment and represent the arithmetic mean±S.D. of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Figure 5.. JAK3 controls the expression of Wnt3a through the phospho-inactivation of Nedd4-2 and enhances the ubiquitination of Wnt3a

Purified human monocytes were stimulated with P. gingivalis and total cell lysates were probed for the levels of Wnt3a with or without pretreatment of JAK3 inhibitor, T-1377 (1 nM) (A, B), or proteasome inhibitor, MG-132 (10 μM) (B, D). (C) The intensity ratio of Wnt3a to GAPDH was determined by densitometry. JAK3- specific siRNA was used to silence JAK3 in human monocytes (D, E) and THP-1 cells (F), and the total lysate of P. gingivalis-stimulated human monocytes was probed for the levels of immunoprecipitated Wnt3a and K48 ubiquitinated Wnt3a (D), the phosphorylation of Nedd4-2, and the levels of total Wnt3a and GAPDH (E, F). (F) JAK3 inhibitor (1nM T-1377) was also used to determine the effect of JAK3 on the phosphorylation of Nedd4-2 in P. gingivalis-stimulated THP-1 cells. (G to H) Purified human monocytes were transfected with nontargeting control siRNA or Nedd4-2-specific siRNA for 72 h and then stimulated with P. gingivalis over a 24 h time course. (G, H) Silencing of Nedd4-2 (G) significantly reduces the levels of TNFα, IL-6, and IL-12P40 in P. gingivalis-stimulated monocytes (H). All the blots shown are representative of three to five biological replicates. The data in (H) represent the arithmetic mean±S.D. of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Figure 6.. Inhibition of JAK3 or Wnt3 signaling increases P. gingivalis-induced NF-κB activity

Purified human monocytes were pretreated with T-1377 for 2 hours or transfected with nontargeting control, or JAK3-, Wnt3a- or Dvl3-specific siRNA for 72 h and then stimulated with P. gingivalis for the time indicated. Total cell lysates and nuclear lysates were harvested from separate groups. (A to D) Western blot of total cell lysates probed for the levels of phospho-NF-κB (Ser536) and GAPDH (D). (E, F) Ten micrograms of nuclear lysate were used to determine the transcription factor binding levels of NF-κB in human monocytes stimulated with P. gingivalis for 6 h. For A-D, the blots shown are representative of three to five experiments. For E and F, the data represent the arithmetic mean±S.D. of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Figure 7.. JAK3 restrains inflammation in periodontal tissues and aggravates the severity of bone loss in P. gingivalis-infected mice

(A, B) Eight- to 12-week-old C57BL/6 mice were divided randomly into one sham control group and two experimental groups (n=5 per group). The sham control group was treated with cellulose and 0.01% DMSO. The experimental groups were orally infected with P. gingivalis with or without pretreatment with the JAK3 inhibitor T-1377 (15 mg/kg) (A). Samples from the mouse oral cavity were examined by quantitative nested PCR to confirm P. gingivalis infection. (B) Representative electrophoresis images showing P. gingivalis DNA obtained from oral samples. (C, D) mRNA levels of TNFα, IL-6, and IL-12P40 (C), as well as Wnt3a and Dvl3 (D) were determined by RT-qPCR. (E) Total lysates of gingival tissue from upper jaws were probed for TNFα, IL-6, IL-12P40, and GAPDH. (F) Alveolar bone loss visualized by methylene blue/eosin staining and typical maxillae from sham-infected; P. gingivalis-infected; and T-1377-treated P. gingivalis-infected mice are presented. (G) Quantification of P. gingivalis-induced bone loss representing the distance from the CEJ to the ABC. Data are presented as the mean CEJ-ABC distance in mm±S.D.; n=5 mice per group. Error bars represent the S.D. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively. Data represent the arithmetic mean±S.D. of three independent experiments.

Figure 8.. JAK3 inhibition reduces the expression of Wnt3 and Dvl3 in periodontal tissue in P. gingivalis-infected mice

Immunohistochemistry staining of serial sections of gingival tissues from experimental mice treated with P. gingivalis, or P. gingivalis plus JAK3 inhibitor (T-1377, 15mg/kg), or sham control mice treated with DMSO and Cellulose with or without T-1377, showing the expression of Wnt3a (A to F) and Dvl3 (I to N). (G, O, H, P) The mean pixel intensity of Wnt3a (G) and Dvl3 (H), and the inflammatory cells infiltrated in the gingival tissue of P. gingivalis-infected mice (H, P) observed from more than 20 microscope fields of view are presented. Error bars represent the S.D. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively. Data represent the arithmetic mean±S.D. of three independent experiments.

Figure 9.. Schematic signaling model of JAK3 restrains P. gingivalis-induced inflammation through Wnt3-Dvl3 signaling in innate immune cells

(A) P. gingivalis infection activates proinflammatory signaling (not shown here) and thus induces the production of proinflammatory cytokines. Concurrently, JAK3 is phosphorylated upon the challenge of P. gingivalis and in turn phospho-inactivates Nedd4-2, leading to the decreased ubiquitination of Wnt3a and a subsequent increase in Wnt3a, Dvl3, and phospho-GSK3β levels, and the accumulation of β-catenin, which diminishes NF-κB signaling and ultimately constrains the proinflammatory immune response. In contrast, JAK3 inhibition promotes the activity of Nedd4-2, decreases the amount of Wnt3a, Dvl3, phospho-GSK3β, and β-catenin, which ultimately increases NF-κB activity, leading to increased proinflammatory cytokine production and inflammatory cell infiltration and the consequent exacerbation of inflammation-induced alveolar bone loss. (B) Briefly, activation of JAK3 is essential to restrain the production of pro-inflammatory cytokines (green) while inhibition of JAK3 leads to a robust increase of these cytokines (pink) in innate immune cells.