PLCβ2 negatively regulates the inflammatory response to virus infection by inhibiting phosphoinositide-mediated activation of TAK1

Figures

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  Fig. 1. RNA virus infection specifically induces PLCβ2 expression. a Protein expression ratio of white blood cells mixture from control and HFMD group patients detected by mass spectrometry. b Immunoblot (IB) and analysis of PLCβ2 in PBMC from control and HFMD patients. c, d IB of PLCβ2 in lysates of mouse peritoneal macrophages infected with CVA16 virus (c) or stimulated with poly(I:C) (d) for the indicated times. e IB of PLCβ2 in wild-type or Tlr3−/− mouse peritoneal macrophages stimulated with poly(I:C) for the indicated times. f IB of PLCβ2 in mouse peritoneal macrophages pretreated for 1 h with the p38 inhibitor SB203580 before poly(I:C) stimulation for the indicated times. *p < 0.05 by unpaired t-test (b). Data are representative of three experiments with at least three independent biological replicates (mean and s.e.m. of n = 6 per group in b and n = 3 cultures in c–f)

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  Fig. 2. PLCβ2 suppresses RNA virus-induced inflammation. a–c Q-PCR analysis of relative Tnf (a), Il6 (b), and Il12p40 (c) mRNA levels in skeletal muscle tissue from 14-day-old wild-type or Plcb2−/− mice infected with 50 μl of CVA16 virus (1.5 × 104 PFU/mouse) for 0 or 5 days. d, e Q-PCR analysis of relative Tnf (d) and Il6 (e) mRNA in wild-type or Plcb2−/− macrophages treated with poly(I:C) for the indicated times. f IB of lysates from wild-type or Plcb2−/− macrophages treated with poly(I:C) for the indicated times using anti-phospho-antibodies. g Q-PCR analysis of relative Plcb1-4 mRNA levels in mice peritoneal macrophages. h Q-PCR analysis of relative Tnf (h) and Il6 (i) mRNA in Plcb1-4 silence macrophages stimulated with poly(I:C) for the indicated times. *p < 0.05; **p < 0.01 and ****p < 0.0001 by unpaired t-test (a–e, h and i). Data are representative of three experiments with at least three independent biological replicates (mean and s.e.m. of n = 3 cultures in d–i or n = 3 mice per group in a–c)

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  Fig. 3. PLCβ2 interacts with TAK1. a–c IB or immunoprecipitates (IP) of lysates from HEK293T cells transfected with various plasmids as indicated. d In vitro GST precipitation assay of TAK1 and PLCβ2. e Precipitation of endogenous PLCβ2 from RAW264.7 cells expressing GST or GST-TAK1. f Endogenous interaction of PLCβ2 with TAK1 in poly(I:C)-treated primary macrophages. g Confocal analysis of colocolization of TAK1 and PLCβ2 in peritoneal macrophages stimulated with poly(I:C) for 4 h. Scale bar, 5 μm. h IB or IP of lysates from HEK293T cells transfected with FLAG-tagged PLCβ1-3 and HA-tagged TAK1. Data are representative of three experiments with at least three independent biological replicates (n = 3 cultures in f, g)

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  Fig. 4. PLCβ2 inhibits TAK1 activation. a IB of cell lysates from HEK293 cells expressing TAK1 and TAB1 in the presence or absence of PLCβ2. b, c Reporter assay of NF-κB (b) and AP-1 (c) activation in HEK293T cells expressing TAK1 and TAB1 with increasing concentrations of PLCβ2. d Immunoblot analysis of lysates from wild-type or Plcb2−/− macrophages treated with poly(I:C) for the indicated times using phospho-TAK1 antibody. e IB of lysates from wild-type or Plcb2−/− macrophages pretreated with TAK1 inhibitor, (5Z)-7-oxozeaenol, for 1 h, then stimulated with poly(I:C) for the indicated times using anti-phospho-antibodies. f, g IB of PLCβ2 and p-TAK1 in PBMC from HFMD patients. Both PLCβ2 and p-TAK1 levels were classified as low, medium, or high based on the intensities of each band; the percentages of patients classified in each category are depicted the histogram in (f) and the representative IB is shown in (g). **p < 0.01 and ***p < 0.001 by unpaired t-test (b, c). Data are representative of three experiments with at least three independent biological replicates (n = 3 cultures in d, e)

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  Fig. 5. PLCβ2 inhibits TAK1 activation via PIP2. a, b Reporter assay of NF-κB (b) and AP-1 (c) activation in HEK293T cells expressing TAK1 and TAB1 in the presence or absence of PLCβ2 (WT) or phospholipase-inactive mutant (M). c IB of cell lysates from HEK293 cells expressing TAK1 and TAB1 in the presence or absence of PLCβ2 (WT) or phospholipase-inactive mutant (M). d–g IB of cell lysates from macrophages treated with PIP2 in a lipid carrier for 10 min or 10 mM neomycin for 2 h before stimulating with poly(I:C) for the indicated times using anti-phospho-antibodies. h, i Q-PCR analysis of relative Tnf (h) and Il6 (i) mRNA levels in macrophages pretreated with neomycin before stimulating with poly(I:C) for the indicated time. j IB of cell lysates from wild-type or Plcb2−/− macrophages treated with 10 mM neomycin for 2 h before stimulating with poly(I:C) for the indicated times using phospho-antibodies. k, l Q-PCR analysis of relative Tnf (k) and Il6 (l) mRNA levels in wild-type or Plcb2−/− macrophages pretreated with 10 mM neomycin for 2 h before stimulating with poly(I:C) for 6 h. m Phospholipid-binding assays with purified FLAG-TAK1 protein from HEK293 cells. PI phosphatidyl- inositol,…

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  Fig. 6. PLCβ2-deficient mice are more susceptible to CVA16 infection. Fourteen-day-old wild-type or Plcb2−/− mice were infected intraperitoneally with 50 μl of CVA16 (1.5 × 104 PFU/mouse) for 0 or 5 days. Survival (a), clinical score (b), or histopathology of lung (c), or skeletal muscle (d) tissue of wild-type or Plcb2−/− mice (n = 6) treated as indicated. Scale bar, 50 μm. Data are representative of three experiments with at least three independent biological replicates (mean and s.e.m. of n = 6 mice per group). Gehan–Breslow–Wilcoxon test (a) and two-way analysis of variance plus Bonferroni’s posttest (b) were used to calculate p value

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  Fig. 7. PLCβ2 activator alleviates the pathogenesis of CVA16 infection. a, b Immunoblot analysis of lysates from macrophages pretreated with DMSO or m-3M3FBS, and then stimulated with poly(I:C) for the indicated times using different phospho-antibody. c–e Q-PCR analysis of relative Tnf (c), Il6 (d) and Il12p40 (e) mRNA in macrophages pretreated with DMSO or m-3M3FBS, then stimulated with poly(I:C) for the indicated times. Fourteen-day-old wild-type mice were pretreated with DMSO or m-3M3FBS and subsequently infected with CVA16 for 0 or 5 days. f–i Survival rate (f), clinical score (g) and histopathology in lung (h) or skeletal muscle (i) tissue of mice treated as above at either day 0 (not infected) or day 5. Scale bar, 50 μm. j Diagram. *p < 0.05; **p < 0.01 and ***p < 0.001 by unpaired t-test (c–e). Gehan–Breslow–Wilcoxon test (f) and two-way analysis of variance plus Bonferroni’s posttest (g) were used to calculate p value. Data are representative of three experiments with at least three independent biological replicates (mean and s.e.m. of n = 3 cultures in a–e or n = 6 mice per group in f–h)