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. Author manuscript; available in PMC: 2023 Oct 1.
Published in final edited form as: J Immunol. 2022 Aug 26;209(7):1370–1378. doi: 10.4049/jimmunol.2200338

Figure 5. C3a-induced IL-6 release in human gingival epithelial cells.

Figure 5.

(A) Human immortalized gingival keratinocytes (HIGK) were stimulated, or not, with heat-killed P. gingivalis (MOI=10:1), C3a, or C5a and combinations thereof (at the indicated concentrations). Culture media were collected after 24-h incubation and assayed for IL-6 by ELISA. (B) HIGK were stimulated for 24 h with heat-killed P. gingivalis (MOI=10:1) or Pam3Cys lipopeptide (1 μg/ml) and C3aR expression was measured by FACS. Representative histogram (left) and bar graphs for MFI of C3aR expression (right). (C) HIGK were stimulated, or not, with C3a (500 ng/ml), Pam3Cys (1 μg/ml), or both, and IL-6 was measured in collected culture supernatants after 24-h incubation. (D) HIGK were stimulated for 24 h, or not, with P. gingivalis (MOI=10:1) alone or with C3a (500 ng/ml), in the presence or absence of 10 μg/ml anti-TLR2 neutralizing Ab or isotype control (IC), which were added 2 h prior to stimulation. IL-6 release was assayed by ELISA. (E) HIGK were pretreated with PD98059 (10 μM; MEK/ERK inhibitor), SP600125 (50 μM; JNK inhibitor), SB202190 (20 μM; p38 MAPK inhibitor), or SN50 (50 μM, NF-κB inhibitor). After 1 h, Pam3Cys (1 μg/ml) was added in the cultures and C3aR expression (MFI) was determined by FACS after 24 h-incubation. (F) HIGK were stimulated with Pam3Cys (1 μg/ml) for the indicated time lengths. Total protein was extracted and immunoblot analysis was performed with specific Abs against phosphorylated and total ERK1/2, JNK, and p38 MAPK as well as against GAPDH (loading control). (G) HIGK were pretreated with Pam3Cys (1 μg/ml) for 4 h, and then exposed to PD98059 (10 μM; MEK/ERK inhibitor), SP600125 (50 μM; JNK inhibitor), SB202190 (20 μM; p38 MAPK inhibitor), or SN50 (50 μM, NFκB inhibitor). After 1 h, C3a (500 ng/ml) was added in the cultures. Culture media were collected after 24 h and assayed for IL-6 by ELISA. Data are means ± SD (n = 6 cultures per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA and Tukey’s (A,D,E,G) or Dunnett’s (B,C) multiple comparisons tests. NS, not significant.