1. Volume 21, Issue 1, Pages 47-58 (January 2017)
The Bacterial T6SS Effector EvpP Prevents NLRP3 Inflammasome Activation by Inhibiting the Ca2+-Dependent MAPK-Jnk Pathway 
Hao Chen, Dahai Yang, Fajun Han, Jinchao Tan, Lingzhi Zhang, Jingfan Xiao, Yuanxing Zhang, Qin Liu 
Cell Host & Microbe 
Volume 21, Issue 1, Pages (January 2017)
DOI: /j.chom
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2. Cell Host & Microbe 2017 21, 47-58DOI: (10.1016/j.chom.2016.12.004)
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3. Figure 1 E. tarda Activates NLRC4 and NLRP3 Inflammasomes via T3SS
BMDMs from wild-type or the indicated KO mice were primed with LPS (100 ng/mL, 6 hr) and then infected with wild-type (WT) E. tarda, ΔesaI, ΔfliC, or ΔethA for the indicated time periods at an MOI of 10.
(A, D, and G) Caspase-1 activation in the BMDMs infected by WT E. tarda (A and D) or E. tarda mutants (G) was analyzed by immunoblotting.
(B, E, and H) IL-1β secretion from the BMDMs infected by WT E. tarda (B and E) or E. tarda mutants (H) was determined by ELISA.
(C, F, and I) Supernatants from the BMDMs infected by WT E. tarda (C and F) or E. tarda mutants (I) were analyzed for cell death measured by LDH release.
Data are presented as the mean ± SD of triplicate samples. Results are representative of three separate experiments. ∗p < 0.05, ∗∗p < 0.01 by unpaired two-tailed Student’s t test. p45, pro-caspase-1; p10, activated caspase-1; hpi, hour post-infection. See also Figures S1 and S2.
Cell Host & Microbe  , 47-58DOI: ( /j.chom )
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4. Figure 2 T6SS Negatively Regulates NLRP3 Inflammasome Activation
BMDMs from wild-type or the indicated KO mice were primed with LPS (100 ng/mL, 6 hr) and then infected with WT E. tarda or ΔevpAB for the indicated time periods at an MOI of 10.
(A) The activation of caspase-1 was analyzed by immunoblotting.
(B and D) IL-1β (B) and IL-6 (D) secretion from the infected BMDMs into the supernatants at the indicated time was determined by ELISA.
(C) Supernatants from the infected BMDMs were analyzed for cell death measured by LDH release.
Data are presented as the mean ± SD of triplicate samples. Results are representative of three separate experiments. ∗p < 0.05, ∗∗p < 0.01 by unpaired two-tailed Student’s t test. See also Figure S2.
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5. Figure 3
EvpP Is a T6SS Effector that Inhibits NLRP3 Inflammasome Activation during Infection
(A) HeLa cells were infected with WT E. tarda or ΔevpAB, expressing effector-TEM fusion protein, at an MOI of 100. Eight hours after infection, cells were loaded with CCF4-AM. Translocation of effector-TEM into cell cytosol results in cleavage of CCF4-AM, causing emission of blue fluorescence. Un-cleaved CCF4-AM emits green fluorescence. Scale bar, 50 μm. TEM, TEM-1-β-lactamase. Statistics of percentages of cells emitting blue fluorescence are listed on the right. (Approximately 200 cells were counted in each sample. Mean ± SD of triplicate samples.)
(B) HeLa cells were infected as in (A) with EvpP-HA fusion-expressing WT E. tarda or ΔevpAB. The translocation of EvpP-HA was examined by immunofluorescence using anti-HA antibody. Statistics of percentages of cells showing signals for intracellular EvpP are listed on the right. (Approximately 200 cells were counted in each sample. Mean ± SD of triplicate samples.)
(C) HeLa cells and J774A.1 cells were infected with EvpP-HA fusion-expressing WT E. tarda or ΔevpAB. After infection, cells were subjected to differential centrifugation to separate subcellular fractions. These fractions were analyzed by immunoblotting as indicated. Calnexin and β-tubulin, markers of host cell membrane and cytosolic proteins, respectively. RnaP, bacterial cytosolic protein.
(D) BMDMs from the indicated KO mice were primed with LPS (100 ng/mL, 6 hr) and then infected with WT E. tarda, ΔevpP, evpP-complemented ΔevpP (ΔevpP+pEvpP), or empty vector-complemented ΔevpP (ΔevpP+Vector) for the indicated time periods at an MOI of 10. The activation of caspase-1 was analyzed by immunoblotting.
(E and G) IL-1β (E) and IL-6 (G) secretion from the infected Nlrc4−/− BMDMs into the supernatants at the indicated time was determined by ELISA.
(F) Supernatants from the infected Nlrc4−/− BMDMs were analyzed for cell death measured by LDH release.
Data are presented as the mean ± SD of triplicate samples. Results are representative of three separate experiments. ∗p < 0.05, ∗∗p < 0.01 by unpaired two-tailed Student’s t test. pEvpP, a complementation plasmid encoding evpP. See also Figures S3 and S4.
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6. Figure 4
Overexpression of EvpP in Macrophages Suppresses Activation of NLRP3 Inflammasome
J774A.1 cells transduced with or without (Mock) EvpP-encoding vector (EvpP) or empty vector (Vector) by lentiviral infection were primed with LPS (1 μg/mL, 5 hr) and then stimulated with or without the indicated concentration of ATP or nigericin (Nig) for 50 min or MSU crystals for 6 hr.
(A–C) Caspase-1 activation by ATP (A), Nig (B), or MSU (C) was analyzed by immunoblotting.
(D–I) IL-1β (D–F) and IL-6 (G–I) secretion from the stimulated cells into the supernatants was determined by ELISA.
Data are presented as the mean ± SD of triplicate samples. Results are representative of at least three separate experiments. ∗p < 0.05, ∗∗p < 0.01 by unpaired two-tailed Student’s t test.
Cell Host & Microbe  , 47-58DOI: ( /j.chom )
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7. Figure 5
EvpP Inhibits NLRP3 Inflammasome Activation by Suppressing Jnk-Dependent ASC Oligomerization
(A) LPS-primed J774A.1 cells were pretreated with or without DMSO or Jnk inhibitor (SP600125, 40 μM, 1 hr) and then infected with or without WT E. tarda, ΔevpP, ΔevpP+pEvpP, or ΔevpP+Vector for 3 hr at an MOI of 10. The activation of caspase-1 and phosphorylated JNK (P-Jnk) was analyzed by immunoblotting.
(B, D, and F) IL-1β secretion from the BMDMs infected by E. tarda (B and D), or stimulated by ATP or Nig (F), was determined by ELISA.
(C) J774A.1 cells transduced with or without EvpP-encoding vector (EvpP) or empty vector (Vector) by lentiviral infection were primed with LPS and then infected with or without WT E. tarda, ΔevpP, or ΔevpAB for 3 hr at an MOI of 10. The activation of caspase-1 and P-Jnk was analyzed by immunoblotting.
(E) J774A.1 cells transduced with or without EvpP-encoding vector (EvpP) were primed with LPS and then pretreated as in (A). Cells were then stimulated with or without ATP (2.5 mM) or Nig (20 μM) for 50 min. The activation of caspase-1 and P-Jnk was analyzed by immunoblotting.
(G) LPS-primed J774A.1 cells were pretreated with DMSO or SP and then infected with or without WT E. tarda or ΔevpP. ASC foci (arrowheads) formation was detected by immunostaining. Scale bar, 25 μm. High-magnification images of the area marked by white boxes are arrayed at the lower part of the panel. Arrows indicate bacteria. Scale bars, 10 μm. Statistics of percentages of cells containing ASC foci are listed at the bottom. (Approximately 200 cells were counted in each sample. Mean ± SD of duplicate samples.) ASC, red; DAPI, blue.
(H) Cell lysates and crosslinked pellets from J774A.1 cells treated as indicated were analyzed by immunoblotting for ASC oligomerization.
Data are presented as the mean ± SD of triplicate samples. Results are representative of three separate experiments. ∗p < 0.05, ∗∗p < 0.01 by unpaired two-tailed Student’s t test. See also Figures S5 and S6.
Cell Host & Microbe  , 47-58DOI: ( /j.chom )
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8. Figure 6
EvpP Inhibits NLRP3 Inflammasome Activation by Preventing Ca2+-Dependent Jnk-ASC Axis
J774A.1 cells transduced with or without EvpP-encoding vector (EvpP) or empty vector (Vector) by lentiviral infection were primed with LPS and then pretreated with or without DMSO, SP (40 μM, 1 hr), or BAPTA-AM (50 μM, 1 hr). Cells were then infected with or without WT E. tarda or ΔevpP for 3 hr at an MOI of 10 or stimulated with or without ATP (2.5 mM) or nigericin (20 μM) for 50 min.
(A, D, and G) Intracellular calcium mobilization induced by E. tarda (A), ATP (D), or Nig (G) was analyzed by a SpectraMax plate reader to measure fluorescence intensity.
(B, E, and H) The activation of caspase-1 and P-Jnk, induced by E. tarda (B), ATP (E), or Nig (H), was analyzed by immunoblotting.
(C, F, and I) IL-1β secretion from the BMDMs infected by E. tarda (C), or stimulated by ATP (E) or Nig (H), was determined by ELISA.
Data are presented as the mean of duplicate samples (A, D, and G) or mean ± SD of triplicate samples. Results are representative of three separate experiments. ∗p < 0.05, ∗∗p < 0.01 by unpaired two-tailed Student’s t test. See also Figure S7.
Cell Host & Microbe  , 47-58DOI: ( /j.chom )
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9. Figure 7
EvpP Inhibits Inflammasome-Mediated Bacterial Clearance In Vivo
Wild-type, caspase-1, NLRP3, or NLRC4 KO mice were injected intraperitoneally with PBS, WT E. tarda, or ΔevpP at the indicated dose of bacteria (6 × 108 CFU/mouse in B, C, and D).
(A and B) Serum IL-1β from the infected mice at 8 hpi was determined by ELISA (n = 4 in A and n = 6 in B).
(C and D) Spleen (C) and liver (D) homogenates were plated to determine the bacterial CFU per gram of organs (n = 6).
Data are presented as the mean (C and D) or mean ± SD (A and B). Results are representative of two (A) or three (B, C, and D) separate experiments. ∗p < 0.05, ∗∗p < 0.01 by unpaired two-tailed Student’s t test.
Cell Host & Microbe  , 47-58DOI: ( /j.chom )
Copyright © 2017 Elsevier Inc. Terms and Conditions