Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized TLR4. 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Article Snippet: pET30a IFI16 FL -K34A , Genscript , N/A.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Incubation, Protein Concentration, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Article Snippet: pET30a IFI16 FL -K34A , Genscript , N/A.

Techniques: Binding Assay, Activation Assay, Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Injection, Immunoprecipitation, Western Blot, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information.

Article Snippet: pET30a IFI16 FL -K34A , Genscript , N/A.

Techniques: Software, Sequencing, Mutagenesis, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: pET30a IFI16 FL -K34A , Genscript , N/A.

Techniques: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: IFI16 induces neutrophil recruitment, macrophage activation and fibrosis in vivo (A–C) C57BL/6J mice were i.p. injected with 5 mg/kg IFI16 FL , 5 mg/kg IFI16 PYD , 5 mg/kg IFI16ΔPYD or vehicle as control. After 24h peritoneal exudated cells (PECs) were harvested and neutrophil recruitment and macrophage activation were analyzed by FACS. (A) Representative FACS plots showing the gating strategy for FACS analysis of neutrophils and inflammatory activated macrophages in PEC samples. The bar graphs depict the frequency of (B) neutrophils (CD11b+Ly6G + Ly6C- cells) among total live PECs and (C) macrophages (CD11b+F4/80+Ly6G-Ly6C- cells) expressing the activation markers I-A/I-E (MHC II) and CD86 among total macrophages (∗ p < 0.05, ∗∗ p < 0.01 by two-tailed one-way ANOVA). (D and E) C57BL/6J mice received every other day for 6 weeks s.c. injections of PBS (vehicle), bleomycin (Bleo, 0.1 U/ml), or IFI16 FL (50 μg). Mice were sacrificed at day 22, and skin was harvested for analysis. (D) Dermal collagen deposition was assessed by Masson’s trichrome staining and analyzed for dermal thickness, as indicated by the lines in the representative photomicrographs on the left (scale bar: 200 μm). Results are shown in the right histograms where each dot indicate the mean of three different measurements per skin sample. Error bars indicate SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test; n = 4 per group). (E) Mast cells recruitment was assessed by toluidine blue staining, as shown in the representative photomicrographs on the left (scale bar: 200 μm) where black arrows indicate representative mast cells. Results are shown in the right histograms as mean values of two different measurements per skin sample. Error bars indicate SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test; n = 4 per group).

Article Snippet: pET30a IFI16 FL -K34A , Genscript , N/A.

Techniques: Activation Assay, In Vivo, Injection, Control, Expressing, Two Tailed Test, Staining

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized TLR4. 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Article Snippet: pET30a IFI16 FL -I17A , Genscript , N/A.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Incubation, Protein Concentration, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Article Snippet: pET30a IFI16 FL -I17A , Genscript , N/A.

Techniques: Binding Assay, Activation Assay, Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Injection, Immunoprecipitation, Western Blot, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information.

Article Snippet: pET30a IFI16 FL -I17A , Genscript , N/A.

Techniques: Software, Sequencing, Mutagenesis, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: pET30a IFI16 FL -I17A , Genscript , N/A.

Techniques: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: IFI16 induces neutrophil recruitment, macrophage activation and fibrosis in vivo (A–C) C57BL/6J mice were i.p. injected with 5 mg/kg IFI16 FL , 5 mg/kg IFI16 PYD , 5 mg/kg IFI16ΔPYD or vehicle as control. After 24h peritoneal exudated cells (PECs) were harvested and neutrophil recruitment and macrophage activation were analyzed by FACS. (A) Representative FACS plots showing the gating strategy for FACS analysis of neutrophils and inflammatory activated macrophages in PEC samples. The bar graphs depict the frequency of (B) neutrophils (CD11b+Ly6G + Ly6C- cells) among total live PECs and (C) macrophages (CD11b+F4/80+Ly6G-Ly6C- cells) expressing the activation markers I-A/I-E (MHC II) and CD86 among total macrophages (∗ p < 0.05, ∗∗ p < 0.01 by two-tailed one-way ANOVA). (D and E) C57BL/6J mice received every other day for 6 weeks s.c. injections of PBS (vehicle), bleomycin (Bleo, 0.1 U/ml), or IFI16 FL (50 μg). Mice were sacrificed at day 22, and skin was harvested for analysis. (D) Dermal collagen deposition was assessed by Masson’s trichrome staining and analyzed for dermal thickness, as indicated by the lines in the representative photomicrographs on the left (scale bar: 200 μm). Results are shown in the right histograms where each dot indicate the mean of three different measurements per skin sample. Error bars indicate SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test; n = 4 per group). (E) Mast cells recruitment was assessed by toluidine blue staining, as shown in the representative photomicrographs on the left (scale bar: 200 μm) where black arrows indicate representative mast cells. Results are shown in the right histograms as mean values of two different measurements per skin sample. Error bars indicate SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test; n = 4 per group).

Article Snippet: pET30a IFI16 FL -I17A , Genscript , N/A.

Techniques: Activation Assay, In Vivo, Injection, Control, Expressing, Two Tailed Test, Staining

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The proinflammatory activity of IFI16 lies within its N-terminal region (A) qRT-PCR analysis for TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with IFI16 alone (10 μg/mL) or pre-incubated for 1 h with the indicated amounts of anti-IFI16 polyclonal antibodies directed against either the N- or C-terminal region of the protein. Values are normalized to GAPDH mRNA and plotted as fold of induction over untreated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SEM (∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of TNF-α and IL-8 determined by ELISA in the culture supernatants harvested from human macrophages stimulated for 24 h as described in (A). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analysis of IFI16 binding to immobilized TLR4. 500 nM of IFI16 diluted in running buffer, alone or pre-incubated for 1 h at RT with increasing concentrations (62.5–500 nM) of the anti-N-term-IFI16 antibody (red bars) or with 500 nM of anti-C-term antibody (green bar), were flowed over a TLR4/MD2-coated chip. Data are representative of two independent experiments, shown as the mean ± SEM (∗∗∗∗ p < 0.0001; one-way ANOVA followed by Dunnett’s test).

Article Snippet: pET30a IFI16 FL -K86A , Genscript , N/A.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Incubation, Protein Concentration, Enzyme-linked Immunosorbent Assay, SPR Assay, Binding Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: The PYRIN domain of IFI16 is involved in TLR4/MD2 binding and activation (A) Schematic representation of the full-length IFI16 (IFI16 FL ), the truncated forms IFI16ΔHINB and IFI16ΔPYD, and the single domains used in this study. Numbers represent the amino acid positions based on the NCBI Reference Sequence GenBank: NP_005522 . (B) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h with or without the recombinant proteins described in A (111 nM). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Protein concentration of TNF-α and IL-8 evaluated by ELISA in supernatants derived from human macrophages stimulated for 24 h as described in the legend (B), with or without CLI-095 (TLR4 inhibitor, 5μM). Data are expressed as mean values ±SEM of three independent experiments (∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis of IFI16 mutants or domains binding to immobilized TLR4/MD2. After immobilization of TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of IFI16 FL , IFI16ΔHINB, IFI16ΔPYD, IFI16 PYD , HINA, or HINB domains (20–800 nM), diluted in running buffer, were injected over the immobilized complex. Data are representative of three different experiments, with the dissociation constant (K D ) value provided where applicable. In the sensogram for IFI16ΔPYD, the asterisk (∗) denotes the mass transport effect observed at the highest concentration (2 μM). (E) Human macrophages were stimulated for 1 h with IFI16 FL , IFI16 PYD , IFI16ΔPYD, or left untreated (25 μg/mL). Total cellular extracts were then subjected to immunoprecipitation using an anti-TLR4 monoclonal antibody. Immunoprecipitates (left, IP:TLR4) and whole-cell lysates (right, Input) were analyzed by immunoblotting using antibodies against N-term-IFI16, C-term-IFI16 or TLR4. β-actin protein expression served as a protein loading control. Data are representative of three independent experiments with similar results.

Article Snippet: pET30a IFI16 FL -K86A , Genscript , N/A.

Techniques: Binding Assay, Activation Assay, Sequencing, Quantitative RT-PCR, Expressing, Recombinant, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay, Injection, Immunoprecipitation, Western Blot, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Structural analysis of the interaction moiety between the IFI16 PYD and the TLR4/MD2 complex (A) Predicted 3D structure of the IFI16 PYD using the Robetta software. (B) Molecular docking analysis of the IFI16 PYD (green) and the extracellular portion of TLR4 (blue, PDB: 3FXI ) using High Ambiguity Driven protein-protein DOCKing software. (C) Alignment of the primary sequence of PYHIN PYDs along with those of NLRP3 and ASC performed using MEGAX and ClustalW algorithm to identify PYHIN-specific amino acids with similar chemical properties. Green arrows identify amino acids conserved across the different proteins, whereas light blue arrows identify amino acids that are conserved only across the PYHIN family members, which were used in mutagenesis experiments (see Figure 5 ). The red arrow identifies an amino acid conserved in all the proteins, which was mutated and used as control. The six α helices of the PYDs with known structures are also indicated. (D) Identification of the IFI16 PYD residues (green) potentially involved in the interaction with TLR4 (Lys34, Lys64, and Lys86) obtained by integrating the alignment and molecular docking information.

Article Snippet: pET30a IFI16 FL -K86A , Genscript , N/A.

Techniques: Software, Sequencing, Mutagenesis, Control

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: Three amino acids located within the IFI16 PYD are critically involved in its TLR4-mediated proinflammatory activity (A) Schematic representation of the polar residues identified in Figures 4 E and 4F, highlighting their positions within the IFI16 PYD and the specific amino acid substitutions used in the mutagenesis experiments. (B) Human macrophages were stimulated for 24 h with equimolar concentrations (111 nM) of the IFI16 mutants described in (A ) and the IFI16 PYD−TM , alongside the wild type IFI16 FL . The levels of TNF-α released in the culture supernatants were measured by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test). (C) SPR analysis assessing the binding of IFI16 FL and its mutants to immobilized TLR4/MD2 on a CM5 sensor chip. Increasing concentration of IFI16 FL or the mutants (20–800 nM), diluted in running buffer, were flowed over the immobilized complex. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: pET30a IFI16 FL -K86A , Genscript , N/A.

Techniques: Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay

Journal: iScience

Article Title: The PYRIN domain is required for TLR4-mediated inflammation by PYHIN family members

doi: 10.1016/j.isci.2025.112413

Figure Lengend Snippet: IFI16 induces neutrophil recruitment, macrophage activation and fibrosis in vivo (A–C) C57BL/6J mice were i.p. injected with 5 mg/kg IFI16 FL , 5 mg/kg IFI16 PYD , 5 mg/kg IFI16ΔPYD or vehicle as control. After 24h peritoneal exudated cells (PECs) were harvested and neutrophil recruitment and macrophage activation were analyzed by FACS. (A) Representative FACS plots showing the gating strategy for FACS analysis of neutrophils and inflammatory activated macrophages in PEC samples. The bar graphs depict the frequency of (B) neutrophils (CD11b+Ly6G + Ly6C- cells) among total live PECs and (C) macrophages (CD11b+F4/80+Ly6G-Ly6C- cells) expressing the activation markers I-A/I-E (MHC II) and CD86 among total macrophages (∗ p < 0.05, ∗∗ p < 0.01 by two-tailed one-way ANOVA). (D and E) C57BL/6J mice received every other day for 6 weeks s.c. injections of PBS (vehicle), bleomycin (Bleo, 0.1 U/ml), or IFI16 FL (50 μg). Mice were sacrificed at day 22, and skin was harvested for analysis. (D) Dermal collagen deposition was assessed by Masson’s trichrome staining and analyzed for dermal thickness, as indicated by the lines in the representative photomicrographs on the left (scale bar: 200 μm). Results are shown in the right histograms where each dot indicate the mean of three different measurements per skin sample. Error bars indicate SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test; n = 4 per group). (E) Mast cells recruitment was assessed by toluidine blue staining, as shown in the representative photomicrographs on the left (scale bar: 200 μm) where black arrows indicate representative mast cells. Results are shown in the right histograms as mean values of two different measurements per skin sample. Error bars indicate SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA followed by Dunnett’s test; n = 4 per group).

Article Snippet: pET30a IFI16 FL -K86A , Genscript , N/A.

Techniques: Activation Assay, In Vivo, Injection, Control, Expressing, Two Tailed Test, Staining