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Signaling and innate immune actions of LTB<sub>4</sub> in health and diseas...
Published: 01 February 2023
FIGURE 1. Signaling and innate immune actions of LTB4 in health and disease. Left: LTB4 is generated at homeostatic levels by leukocytes upon encounter with pathogens. Via ligation of BLT1 and reductions in intracellular cAMP, it synergizes with signaling downstream of phagocytic, opsonin, and PRRs to amplify diverse antimicrobial functions that protect the host. These actions include recruitment of PMNs as well as activation of PMNs and resident macrophages to enhance phagocytosis, microbial killing, and generation of proinflammatory cytokines via activation of both the inflammasome and NF-κB-dependent transcription. Right: Excessive and/or prolonged generation of LTB4 during either infectious or noninfectious inflammation leads to BLT1-dependent activation of the inflammasome as well as signaling via JAK-STAT and NF-κB. Collectively, these pathways promote robust cytokine generation and signaling that contribute to exaggerated tissue inflammation and injury. Figure created with BioRender.com. FIGURE 1. Signaling and innate immune actions of LTB4 in health and disease. Left: LTB4 is generated at homeostatic levels by leukocytes upon encounter with pathogens. Via ligation of BLT1 and reductions in intracellular cAMP, it synergizes with signaling downstream of phagocytic, opsonin, and PRRs to amplify diverse antimicrobial functions that protect the host. These actions include recruitment of PMNs as well as activation of PMNs and resident macrophages to enhance phagocytosis, microbial killing, and generation of proinflammatory cytokines via activation of both the inflammasome and NF-κB-dependent transcription. Right: Excessive and/or prolonged generation of LTB4 during either infectious or noninfectious inflammation leads to BLT1-dependent activation of the inflammasome as well as signaling via JAK-STAT and NF-κB. Collectively, these pathways promote robust cytokine generation and signaling that contribute to exaggerated tissue inflammation and injury. Figure created with BioRender.com. More
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Published: 01 February 2023
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Published: 01 February 2023
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Theilovirus 3C protease expression causes specific cleavage of MDA5. ( A ) ...
Published: 01 February 2023
FIGURE 1. Theilovirus 3C protease expression causes specific cleavage of MDA5. ( A ) 293FT cells were transfected with 100 ng of pEFBOS-FLAG-RIG-I or MDA5 and 150 ng of pCAGGS-HA-3C protease plasmid (TMEV, SAFV, EMCV), and the cell lysates were analyzed by Western blot analysis using indicated Abs... More
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Theilovirus 3C protease cleaves two sites in the C-terminal region of human...
Published: 01 February 2023
FIGURE 2. Theilovirus 3C protease cleaves two sites in the C-terminal region of human MDA5. ( A ) Schematic description of the tagged MDA5 construct and predicted cleavage sites. H and F indicate HA-tag and FLAG-tag, respectively. ( B – D ) 293 FT cells were transfected with pCAGGS-HA-MDA5-FLAG (100 ng) and increasing amounts (25–200 ng) of pCAGGS-HA-TMEV 3C or mutant TMEV 3C (B), pCAGGS-HA-SAFV 3C or mutant SAFV 3C (C), or pCAGGS-HA-SAFV 3C (200 ng) or EMCV 3C (200 ng) (D). Afterwards, the cell lysates were analyzed by Western blot analysis using anti-FLAG and HA Abs. ( E ) HeLa cells were transfected with 2.5 μg of empty pCAGGS empty vector (lane 1) or pCAGGS-HA-MDA5-FLAG (lanes 2–4) in a six-well plate and mock-infected (lanes 1 and 2) or infected (MOI of 10) with SAFV (lane 3) or TMEV (lane 4) for 18 h. The cell lysates were analyzed by Western blot analysis using indicated Abs. FIGURE 2. Theilovirus 3C protease cleaves two sites in the C-terminal region of human MDA5. (A) Schematic description of the tagged MDA5 construct and predicted cleavage sites. H and F indicate HA-tag and FLAG-tag, respectively. (B–D) 293 FT cells were transfected with pCAGGS-HA-MDA5-FLAG (100 ng) and increasing amounts (25–200 ng) of pCAGGS-HA-TMEV 3C or mutant TMEV 3C (B), pCAGGS-HA-SAFV 3C or mutant SAFV 3C (C), or pCAGGS-HA-SAFV 3C (200 ng) or EMCV 3C (200 ng) (D). Afterwards, the cell lysates were analyzed by Western blot analysis using anti-FLAG and HA Abs. (E) HeLa cells were transfected with 2.5 μg of empty pCAGGS empty vector (lane 1) or pCAGGS-HA-MDA5-FLAG (lanes 2–4) in a six-well plate and mock-infected (lanes 1 and 2) or infected (MOI of 10) with SAFV (lane 3) or TMEV (lane 4) for 18 h. The cell lysates were analyzed by Western blot analysis using indicated Abs. More
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His<sup>835</sup> and Gln<sup>877</sup> identified as C-terminal cleavage s...
Published: 01 February 2023
FIGURE 3. His835 and Gln877 identified as C-terminal cleavage sites of MDA5 by Theilovirus protease. ( A ) Schematic representation of the cleavage site identification strategy. ( B ) 293FT cells were transfected with pCAGGS-HA-MDA5-FLAG and empty pCAGGS vector (lane 1) or pCAGGS-HA-SAFV 3C (lanes 2 and 3), and the cell lysates were immunopurified with anti-FLAG beads (lanes 1 and 3) or anti-HA beads (lane 2). Eluates were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Analyzed nonspecific bands, IgG, and protease-cleaved fragments are indicated. ( C ) 293FT cells were transfected with pCAGGS-HA-MDA5-FLAG plasmid and the indicated mutation in MDA5 as well as with SAFV or TMEV protease plasmid and analyzed by Western blot analysis using the indicated Abs. FIGURE 3. His835 and Gln877 identified as C-terminal cleavage sites of MDA5 by Theilovirus protease. (A) Schematic representation of the cleavage site identification strategy. (B) 293FT cells were transfected with pCAGGS-HA-MDA5-FLAG and empty pCAGGS vector (lane 1) or pCAGGS-HA-SAFV 3C (lanes 2 and 3), and the cell lysates were immunopurified with anti-FLAG beads (lanes 1 and 3) or anti-HA beads (lane 2). Eluates were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Analyzed nonspecific bands, IgG, and protease-cleaved fragments are indicated. (C) 293FT cells were transfected with pCAGGS-HA-MDA5-FLAG plasmid and the indicated mutation in MDA5 as well as with SAFV or TMEV protease plasmid and analyzed by Western blot analysis using the indicated Abs. More
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Differential cleavage in the C-terminal domain of human and mouse MDA5 by T...
Published: 01 February 2023
FIGURE 4. Differential cleavage in the C-terminal domain of human and mouse MDA5 by Theilovirus 3C protease. ( A ) Alignments of C-terminal sequences of MDA5 from several species. ( B ) 293FT cells were transfected with the human MDA5 expression vector used in ( Fig. 3 or the same version of the ... More
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Theilovirus 3C protease inhibits MDA5-mediated IFN responses. ( A – D ) 293...
Published: 01 February 2023
FIGURE 5. Theilovirus 3C protease inhibits MDA5-mediated IFN responses. ( A – D ) 293FT cells were transfected with empty or MDA5 expression plasmid and increasing amounts of WT or mutant SAFV 3C plasmid (25–100 ng) (A and C), or increasing amounts of WT, mutant TMEV 3C, or EMCV 3C plasmid (25–100 ng) (B and D), along with IFN-β reporter plasmid and Renilla luciferase plasmid. ( E ) 293FT cells were transfected with empty, full-length MDA5, or deletion MDA5 expression plasmid along with IFN-β reporter plasmid and Renilla luciferase plasmid. These cells were transfected with poly(I:C) (pIC) for 12–14 h, if indicated (C–E), and the cell lysates were subjected to the Dual-Luciferase assay. The data are representative of three independent experiments with similar results, and the bars indicate the average (n = 3) ± SD. The Student t test was used for statistical analysis. *p > 0.05, **p = 0.05–0.025, ***p < 0.025. A p value >0.05 is considered as not significant. FIGURE 5. Theilovirus 3C protease inhibits MDA5-mediated IFN responses. (A–D) 293FT cells were transfected with empty or MDA5 expression plasmid and increasing amounts of WT or mutant SAFV 3C plasmid (25–100 ng) (A and C), or increasing amounts of WT, mutant TMEV 3C, or EMCV 3C plasmid (25–100 ng) (B and D), along with IFN-β reporter plasmid and Renilla luciferase plasmid. (E) 293FT cells were transfected with empty, full-length MDA5, or deletion MDA5 expression plasmid along with IFN-β reporter plasmid and Renilla luciferase plasmid. These cells were transfected with poly(I:C) (pIC) for 12–14 h, if indicated (C–E), and the cell lysates were subjected to the Dual-Luciferase assay. The data are representative of three independent experiments with similar results, and the bars indicate the average (n = 3) ± SD. The Student t test was used for statistical analysis. *p &gt; 0.05, **p = 0.05–0.025, ***p &lt; 0.025. A p value &gt;0.05 is considered as not significant. More