Fig 2: 3′,5′-Cyclic diguanylic acid (cyclic di-GMP, CDG) mediated lung TNFR2+ cDC2, or monocyte-derived dendritic cell (moDC) activation is impaired in aged mice. (A–C) Flow cytometry analysis and quantification of lung DC subsets of adult and aged C57BL/6 mice at steady state. n = 4 mice/group. Data are representative of three independent experiments. (D,E) Flow cytometry analysis of CD86 expression in lung TNFR2+ cDC2 and moDC of adult and aged C57BL/6 mice treated intranasally (i.n.) with PBS or CDG (5 μg) for 16 h. n = 4 mice/group. Data are representative of three independent experiments. (F) Quantification of TNF production in the lung homogenates of adult and aged mice treated (i.n.) with PBS or CDG (5 μg) for 5 h. n = 4 mice/group. Data are representative of three independent experiments. Significance value, p, was calculated using one-way ANOVA followed by Tukey's multiple comparison test. Error bars represent mean ± SEM. Statistical significance is represented by p; *p < 0.05, **p < 0.01.

Fig 3: Effect of XPa on DC functions. a and b DC maturation. DCs were stimulated with saline, XPa (10 MOI) and LPS (100 ng), XPa sup (same volume of XPa), and XPa + DNase I (10 MOI) for 24 h, and then collected for flow cytometry analysis. b The percentages of CD86+, CD80+, and MHCII+ among CD11c+ cells. c Uptake of FITC–dextran by BMDCs subjected to the different treatments. Scale bar=50 μm. d Data analysis of c. e The stimulated DCs (saline; XPa (10 MOI) and LPS (100 ng); XPa sup (same volume as XPa) and XPa + DNase I (10 MOI) for 24 h) were cocultured with the CFSE-stained T cells at a ratio of 1:10 after 72 h, with CD3/CD28 as a positive control. * indicates significant from the saline control. f The IFN-γ concentration in the culture medium. Data were analyzed via two-way ANOVA. No letter is present if no significant difference was observed. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: The loss of cortical ARPC1B-containing ARP2/3 complexes increases spontaneous, oscillatory calcium signaling, phospho-Akt, and CD86 expression.(A and B) Control (CK-689) or CK-666–treated Ramos B cells loaded with the Fluo-8 calcium sensor were imaged every 10 seconds for 5 minutes; they were then treated with the PLC inhibitor U73122 and imaged for another 5 minutes (middle panel). Ensemble traces are shown in A. (B) The variance (deviation from the mean) was determined as a measure of [Ca2+]cytosol flux. Greater than 50 cells were used; n = 3. (C and D) WT and ARPC1B-KO Ramos cells imaged and quantified as in A. Cells were treated with the SYK inhibitor BAY 61-3606 for 10 minutes and imaged for another 5 minutes. (D) The variance was determined as a measure of [Ca2+]cytosol flux. Greater than 50 cells were used; n = 3. (E) Ramos cells blotted for AKT and ERK phosphorylation at indicated times after addition of CK-666. Results were assessed using a 1-way ANOVA and Dunnett’s test comparing all groups to the DMSO control; n = 6. (F) Unstimulated WT and ARPC1B-KO Ramos cells blotted for ERK phosphorylation. (G) Primary B cells given Control (CK-689) or CK-666 overnight together with 100 ng/mL LPS. Representative surface CD86 by FACS is shown; n = 3. *P < 0.05; ****P < 0.0001.

Fig 5: Galactose inhibits IFNγ-induced JAK-STAT-1 and HIF-1α pathway activation in M1 macrophages by modulating ATP and mitochondrial ROS production.(a) Cytokine production of BMDMs after stimulation with IFN-γ for 24 h in medium containing glucose or galactose.(b) FACS analysis of the M1 macrophage surface marker CD86 of BMDMs after stimulation with IFN-γ for 24 h in medium containing glucose or galactose.(c, d, and e) Immunoblot analysis of specified proteins in BMDMs stimulated with IFN-γ for indicated time intervals in medium containing glucose or galactose. Data are representative of two independent experiments.(f) Immunoblot analysis of specified proteins in BMDMs stimulated with IFN-γ for indicated time intervals with or without mito-tempo (1 mM) pretreatment for 2 h. Data are representative of two independent experiments.(g) Nitric oxide (NO) production by BMDMs after stimulation with IFN-γ for 24 h in medium containing glucose or galactose. Data are representative of two independent experiments (n = 4, mean ± sem).