Fig 2: CMTM6 suppresses inflammation through the NF-κB/p65 pathway in HCE-T cells under hyperosmotic stress. (A, B) The effect of CMTM6 on the activation of p38 (A), Erk (A), and p65 (B) was examined using western blot analysis. (C–G) For the treatment of p65 inhibition, 50 µM JSH-23 was added to the cell culture medium, followed by incubation with 500-mOsM media for 8 or 24 hours. DMSO was used as a control. (C) The effect of JSH-23 was detected using western blot analysis. (D–F) The mRNA levels of TNFA (D), IL1B (E), and IL6 (F) were examined using RT-PCR. (G) The protein level of IL-6 in cell culture supernatants was detected using CBA. Six groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: CMTM6 reduces DLNs Th1 and Th17 cell infiltration and proinflammatory cytokine production in DE mice. (A) Representative flow cytometry plots and quantitative summary of Th1, Th17, and Treg percentages among the DLNs. (B–D) RT-PCR was used to examine the production of proinflammatory cytokines Tnfa (B), Il1b (C), and Il6 (D) in corneas. (E) The expression of TNF-α in tears was measured using CBA. Results are shown as mean ± SD (n = 3–4 in each group). A two-group statistical analysis was conducted using the unpaired Student's t-test; “n” represents bilateral draining lymph nodes (A) or both eyes (B–E) from a single mouse. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: CMTM6 attenuates inflammation stimulated by hyperosmolarity in HCE-T cells. A TG006-CMTM6-IRES-EGFP lentiviral vector was used to overexpress CMTM6. The TG006 lentiviral vector was used as a negative control. (A, B) Overexpression of CMTM6 in HCE-T cells was validated using RT-PCR (A) and western blot analyses (B). (C–E) RT-PCR was used to detect the mRNA levels of TNFA (C), IL1B (D), and IL6 (E). (F) The protein level of IL-6 in the culture supernatant was measured using CBA. Two-group statistical analysis was conducted using the unpaired Student's t-test (A). Four groups were compared using one-way ANOVA followed by Tukey's multiple comparisons test (B–F). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Association of treatment of Siltuximab with an accumulation of hIL-6-anti-hIL-6 IgG immune complexes (ICs). Comparison of circulating levels of IL-6 between Siltuximab-treated mice and IgG1 isotype control-treated mice (a), and between Tocilizumab-treated mice and IgG1 isotype control-treated mice (b). Schematic diagram illustrating the experimental design to measure hIL-6-anti-hIL-6 IgG ICs (c). Representative histogram from flow cytometric analysis depicting the detection of hIL-6-anti-hIL-6 IgG ICs in plasma of mice treated with Siltuximab, Tocilizumab, or IgG1 isotype controls (d). Comparison of levels of circulating hIL-6-anti-hIL-6 IgG ICs between mice treated with human IgG1 isotype control and those treated with Siltuximab (e), and between mice treated with human IgG1 isotype control and those treated with Tocilizumab (f). Correlation analysis between levels of hIL-6 and levels of hIL-6-anti-hIL-6 IgG ICs (g). Correlation analysis between levels of the hIL-6-anti-hIL-6 IgG ICs and neutrophil infiltrations (h). Spearman’s rank correlation coefficient (R) and associated P values are indicated. Statistical significance of comparisons was determined using the Wilcoxon matched-pairs signed rank test. ns, not significant, **P < 0.01, P values < 0.05 is considered as significance.