Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: The SIV-induced IL-1β production requires receptor-interacting protein kinase 1 (RIPK1) kinase activity. ( A ) PAMs were transfected with a negative control siRNA (siControl) or RIPK1 -targeting siRNA (siRIPK1) for 24 h and infected with Sk02 at an MOI of 1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β was analyzed by Western blotting. RIPK1 knockdown efficiency was determined by real-time PCR. ( B ) PAMs were infected with Sk02 at an MOI of 1 for 20 h in the presence of a vehicle (DMSO) or increasing concentrations (80 and 160 μM) of RIPK1 kinase inhibitor, Nec-1. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β and viral NS1 was analyzed by Western blotting. ( C ) Amino acid sequences of the N-terminal ends of the kinase domains in porcine, murine, and human RIPK1 (based on the GenBank accession numbers XM_005665536, NM_009068, and NM_003804, respectively) were aligned using Clustal Omega. Amino acid positions are based on the human RIPK1 and asterisks indicate conserved residues among the three species. K41/K42 in porcine RIPK1, K45/K46 in murine RIPK1, and K45 in human RIPK1 are underscored. ( D ) For the NLRP3 inflammasome reconstitution assay, HEK293T cells were co-transfected with plasmids expressing porcine NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1β along with Myc-vector or Myc-tagged WT/mutant human RIPK1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Protein expression was analyzed by Western blotting. ( E ) HEK293T cells were transfected as in ( D ) along with the Myc-vector or Myc-tagged porcine RIPK1 WT/mutant for 16 h. ELISA and Western blotting were done as in ( D ). Statistical analysis was done with one-way ANOVA except for the knockdown efficiency data done with an unpaired t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. Results are representative of three independent experiments.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Activity Assay, Transfection, Negative Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Knockdown, Real-time Polymerase Chain Reaction, Reconstitution Assay, Plasmid Preparation, Mutagenesis

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: RIPK1 interacts with DRP1. HEK293T cells were co-transfected with the Flag-vector or Flag-RIPK1 construct along with the DRP1-Myc construct for 24 h. Cell lysates were subjected to co-IP with Flag antibody, and the expression of Flag-RIPK1 and DRP1-Myc in input and IP samples was analyzed by Western blotting. Results are representative of three independent experiments.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Transfection, Plasmid Preparation, Construct, Co-Immunoprecipitation Assay, Expressing, Western Blot

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: A proposed model of RIPK1/DRP1-mediated IL-1β production in SIV-infected PAMs. Recognition of IAV RNA by endosomal TLRs or RIG-I is known to induce pro-IL-1β synthesis. SIV infection turns on the RIPK1/DRP1 signaling for the NLRP3 inflammasome activation in PAMs. SIV seems to utilize the RIPK1 function, while how RIPK1 is activated by SIV or whether an upstream sensor plays a role in its activation is unclear. By interacting with DRP1 and through its kinase activity, RIPK1 induces the phosphorylation of porcine DRP1 at S579. Upon DRP1 translocation to mitochondria, mitochondrial fission occurs and ROS is generated. This promotes the activation of NLRP3 that is required for the NLRP3 inflammasome assembly and caspase-1 activation. Active caspase-1 converts pro-IL-1β into mature IL-1β, which is critical for SIV-induced lung inflammation. Sensing LPS by TLR4 is known to induce the pro-IL-1β expression, and this also induces the DRP1 phosphorylation leading to the NLRP3 inflammasome activation in PAMs, although it is unclear whether LPS activates any upstream regulator of RIPK1.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Infection, Activation Assay, Activity Assay, Phospho-proteomics, Translocation Assay, Generated, Expressing

Journal: Scientific Reports

Article Title: Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses

doi: 10.1038/srep34656

Figure Lengend Snippet: ( a ) HUVEC were left untreated (M: media) or cocultured with 381 MOI 100 for 1, 2, 3, 4, 6, or 24 hr. ( b ) HUVEC were cocultured with 381 MOI 10, 20, 35, 50, 65, 75, or 100 for 2 hr. Densitometry was performed on three independent membranes to calculate RIPK1 R.D.U. relative to GAPDH loading control. ( c ) P. gingivalis 381 or fimbriae mutant strains (major fimbriae mutant -DPG3, minor fimbriae mutant -MF1, or major and minor fimbriae mutant -MFB) were pretreated with DMSO or cytochalasin D (1 μg/mL) for 30 min and then immediately cocultured with HUVEC at an MOI 100 for 2 hr. ( d ) HUVEC were pretreated with vehicle control (DMSO) or MG132 for 2.5 hr (30 μM) and then left untreated or cocultured with P. gingivalis 381 for 2 hr, followed by restimulation with 10 ng/mL TNFα for 15 min. ( e ) P. gingivalis was pretreated with selective gingipain inhibitors (Rgp-specific: 10 μM KYT-1, Kgp-specific: 10 μM KYT-36, 10 μM KYT-1 and 10 μM KYT-36), the general cysteine inhibitor TLCK (1 mM), or vehicle controls for 45 min. HUVEC were then immediately cocultured with medium or pretreated preparations of P. gingivalis for 2 hr. ( f ) HUVEC were cocultured with wild-type strains 381, 33277, or gingipain mutant strains (ΔRgpA, ΔRgpA/B, ΔKgp) at an MOI 100 for 2 hr. ( a–f ) Whole cell lysates were analyzed for RIPK1, TAK1, cIAP2, IKKβ, P- IKKβ, IκBα, P-IκBα, AKT, ubiquitin, ERK, or GAPDH via Western blot analysis.

Article Snippet: Plasmids pDONR223-MAP3K7 (#23693) and PRK5-Myc RIPK1 (#44159) (RIPK1-myc) were purchased from Addgene.

Techniques: Control, Mutagenesis, Ubiquitin Proteomics, Western Blot

Journal: Scientific Reports

Article Title: Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses

doi: 10.1038/srep34656

Figure Lengend Snippet: Bacterial supernatants of overnight grown broth cultures (1x equivalent to supernatant harvested from OD 660 = 1 culture) were diluted 1.35, 2, 4, 8, 16, or 32x. ( a ) HUVEC were left untreated (M: media), cocultured with either P. gingivalis strain 381 MOI 100 (381), or incubated with bacterial supernatants harboring a range of decreasing Kgp-X activity (381 supernatant) for 2 hr. Whole cell lysates were collected and analyzed for RIPK1, TAK1, or AKT expression compared to GAPDH loading control. ( b–d ) Each bacterial supernatant was assayed for lysine-specific proteolytic activity by measuring the hydrolysis kinetics of N-Tosylglycl-L-prolyl-L-lysine-4-nitroanilide. Supernatant Kgp-X activities were calculated at 1.44, 2.07, 4.26, 8.04, 14.43, 22.97, and 33.75 mOD/min, respectively. Densitometry was performed on three independent Western blots in order to calculate the average percent degradation of ( b ) RIPK1, (c) TAK1, and (d) AKT, compared to Kgp-X activity. A Michaelis-menton model was used to calculate the enzyme activity required to degrade 50% of the host protein. ( e ) Protein abundance of RIPK1, TAK1, and AKT was calculated relative to GAPDH and plotted against untreated (M), MOI 100 (381), or a range of P. gingivalis dilutions and is shown as the ratio of target expression mean + SEM (n = 3).

Article Snippet: Plasmids pDONR223-MAP3K7 (#23693) and PRK5-Myc RIPK1 (#44159) (RIPK1-myc) were purchased from Addgene.

Techniques: Incubation, Activity Assay, Expressing, Control, Western Blot, Quantitative Proteomics

Journal: Scientific Reports

Article Title: Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses

doi: 10.1038/srep34656

Figure Lengend Snippet: ( a , b ) HUVEC were transfected with either negative control or RIPK1 specific siRNA for 20 hr. Cultures were then left uninfected or cocultured with P. ginigivalis strain 381 or ΔKgp (MOI 35: hatched bars, 70: grey bars, 100: black bars) for 2 hr, followed by stimulation with TNFα (10 ng/mL) for 60 min in order to monitor cellular activation. ( a ) Whole cell lysates were probed for P-IκBα, RIPK1, and ERK. A representative blot for only MOI100 is depicted. ( b ) Densitometry was performed (n = 3) while comparing each of the three different multiplicities of infection (35, 70, 100) and the ratio of P-IκBα/ERK was calculated by setting negative control + TNFα = 1. ****p < 0.0001, **p < 0.01, *p < 0.05, two-way ANOVA with Bonferroni post test comparing each MOI across all conditions. ( c ) The relationship between P-IκBα and RIPK1 R.D.U. are graphed for each representative phosphoblot for TNFα stimulated samples at each MOI (35, 70, 100), with or without RIPK1 siRNA knockdown. A linear correlation with R 2 = 0.5633 demonstrates that RIPK1 protein abundance dictates endothelial responsiveness to TNFα. ( d–h ) HUVEC were transfected with either negative control or RIPK1 specific siRNA for 20 hr, followed by coculturing with P. gingivalis 381 MOI 100 for 2 hr and then restimulation with TNFα (10 ng/mL) for 15 min. ( d ) Whole cell lysates were examined via Western blot analysis to determine TNFα-dependent activation status. Densitometry of independent membranes (n = 3–5) was used to determine ( e ) P-IKKα/β, ( f ) P-ERK, ( g ) P-IκBα, and ( h ) RIPK1 R.D.U. expression. RIPK1 protein abundance was calculated as % of media. ****p < 0.0001, **p < 0.01, *p < 0.05, one-way ANOVA with Bonferroni post test.

Article Snippet: Plasmids pDONR223-MAP3K7 (#23693) and PRK5-Myc RIPK1 (#44159) (RIPK1-myc) were purchased from Addgene.

Techniques: Transfection, Negative Control, Activation Assay, Infection, Knockdown, Quantitative Proteomics, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses

doi: 10.1038/srep34656

Figure Lengend Snippet: ( a ) Normal TNFα-dependent signal propagation is capable of proceeding through both AKT and RIPK1-dependent pathways, which culminates in the activation of either ERK1/2 or NFκB and thus promotes inflammatory mediator induction. RIPK1 and AKT can both interact with MEKK3, providing cross-talk for signal flux between these pathways. Additionally, RIPK1 indirectly activates AKT via its role in reducing expression of PTEN, a phosphatase that inhibits the PI3K/AKT pathway. During TNFα stimulation, signal flux can be monitored through these pathways by the detection of high phosphorylation status of MEK1/2, IKKα/β and IκBα. ( b ) Here we demonstrated that P. gingivalis dysregulates TNFα-dependent signaling via degradation of host cell RIPK1, TAK1 and AKT kinases. Degradation of host kinases would limit the required interactions of RIPK1 and AKT with MEKK3, in addition to IKK recruitment, effectively blunting ERK1/2 and NFκB activation and proinflammatory induction. Cross-talk between pathways would be reduced due to a lack of RIPK1 initiating an increase in PTEN levels, allowing for inhibition of AKT activity. Attenuated signal flux was observed within downstream signaling proteins (reduced phosphorylation of MEK1/2, IKKα/β and IκBα). Of note, Nakayama et. al. have reported that P. gingivalis infection impairs PI3K/AKT activity through degradation of an unidentified membrane protein, creating a secondary assault to the signaling capacity of the AKT pathway. Dashed boxes represent host proteins whose protein abundance and/or activity are disrupted by P. gingivalis. Circled proteins are internal signaling proteins whose phosphorylation status are reduced and those in triangles are terminal signaling proteins identified to have reduced phosphorylation and/or activity during P. gingivalis induced dysregulated TNFα signaling.

Article Snippet: Plasmids pDONR223-MAP3K7 (#23693) and PRK5-Myc RIPK1 (#44159) (RIPK1-myc) were purchased from Addgene.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Inhibition, Activity Assay, Infection, Membrane, Quantitative Proteomics

Journal: Scientific Reports

Article Title: Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses

doi: 10.1038/srep34656

Figure Lengend Snippet: Immune activation of the endothelium during microbial infection involves a number of critical events that are influential in facilitating resolution of infection. (1). Recognition of pathogens by the endothelium occurs through pattern recognition receptors, effectively driving specific genetic programs, leading to mRNA induction (2) and endothelial activation. This is primarily observed as secretion of cytokines and recruitment factors (3), in addition to the up regulation of cell adhesion molecules (4). Inflammatory mediator output activates (5) and recruits (6) immune cells to the site of the infection in order to help mediate clearance and recovery of the tissue, while cell adhesion molecules allow for enhanced transendothelial migration of professional immune cells into deeper tissues. Recruited inflammatory cells can produce large amounts of TNFα (7), which can act in an autocrine (8) or paracrine fashion (9). Endothelial responsiveness to TNFα functions as an amplification step to further activate the endothelium itself (10), while also promoting increased immune cell recruitment and/or activation due to further increases in inflammatory mediator output. We have demonstrated that P. gingivalis gingipains modulate endothelial innate immunity at several key points. Degradation of RIPK1, TAK1 and AKT kinases ( a ) results in attenuated responsiveness to TNFα. This was observed as reduced activation of NFκB, ERK and AKT pathways ( b ), in addition to impaired mRNA induction of both cell adhesion molecules ( c ) and secreted cytokines and chemokines ( d ). Analogous to TNFα-dependent responses, we propose that host immune kinase expressional changes ( a ) mediate reduced mRNA induction downstream of PRR-dependent responses to P. gingivalis . Significantly, as opposed to simply subverting the ability of the endothelial cell to properly induce inflammatory mediator secretion, gingipain activity can directly degrade already produced and secreted effectors ( e ).

Article Snippet: Plasmids pDONR223-MAP3K7 (#23693) and PRK5-Myc RIPK1 (#44159) (RIPK1-myc) were purchased from Addgene.

Techniques: Activation Assay, Infection, Migration, Amplification, Activity Assay, Produced