Journal: bioRxiv

Article Title: IL-32 drives inflammatory responses in IFN-γ primed human macrophages via a Myddosome-dependent pathway and is elevated in COVID-19

doi: 10.1101/2025.05.29.656795

Figure Lengend Snippet: (A) Heatmap showing signaling pathway activation relative to control ligands, in a panel of HEK-Blue™ reporter cell lines by three IL-32 isoforms (α, β, γ) at the indicated concentrations. (B) NF-κB/AP-1 reporter activation by LPS or IL-32β in HEK-Blue™ TLR4 reporter cells treated for the indicated times. (C) Perturbation of LPS and IL-32β-mediated NF-κB/AP-1 activation in HEK-Blue™ TLR4 reporter cells by TAK-242 and (D) TLR4 or TLR4/MD2 blocking antibodies. (E) NF-κB activation in WT and MyD88 -/- THP-1-Dual™ reporter cell line treated with LPS, IL-32β or TNF-α for 24 h. (F) Perturbation of NF-κB activation with TAK-242 in WT THP1-Dual™ reporter cell line. (G) Effect of TAK-242 on cytokine (TNF-α, IL-6) responses from non-primed macrophages and (H) IFN-γ-primed macrophages treated with LPS or IL-32β. (I) Experimental workflow for the analysis of signaling pathway activation in primary human macrophages by HTRF® assay at the indicated timepoints. For NF-κB and STAT1 phosphorylation normalized signal to total protein is shown. Statistical analysis: data shown in (B-D) are mean values ±SEM of 3 independent experiments with two-way ANOVA with Dunnett’s multiple comparisons test (B) vs duration-matched UT ($ p < 0.05, $$$ p < 0.001), (C) vs 0.01 TAK within LPS/IL-32β treatment groups (++ p < 0.005, +++ p < 0.001), (D) vs concentration-matched IgG control within LPS/IL-32β treatment groups (* p < 0.05, ** p < 0.005, *** p < 0.001). Data shown in (E-F) are mean values ±SEM of 4 independent experiments with two-way ANOVA with Bonferroni’s multiple comparisons test (# p < 0.05, ## p < 0.005, ### p < 0.001). Data shown in (G-H) are mean values ±SEM of n = 3 independent experiments performed on macrophages from one donor, with two-way ANOVA with Dunnett’s multiple comparisons test vs LPS/IL-32β treatment groups (* p < 0.05, ** p < 0.005, *** p < 0.001). Data shown in (I) are mean values ±SEM of n = 3 independent experiments on macrophages from one donor, with two-way ANOVA with uncorrected Fisher’s LSD test versus 0 h within LPS/IL-32β treatment groups (+ p < 0.05, ++ p < 0.005, +++ p < 0.001).

Article Snippet: Neutralizing monoclonal antibodies targeting human TLR4 (InvivoGen, mabg-htlr4) or TLR4/MD2 (InvivoGen, mab-htlr4md) receptors and IgG1 isotype control (InvivoGen, mabg1-ctrlm) were used on HEK-Blue™ hTLR4 cells at the concentrations: 0.1-1-10 μg/mL.

Techniques: Activation Assay, Control, Blocking Assay, HTRF Assay, Phospho-proteomics, Concentration Assay

Journal: bioRxiv

Article Title: IL-32 drives inflammatory responses in IFN-γ primed human macrophages via a Myddosome-dependent pathway and is elevated in COVID-19

doi: 10.1101/2025.05.29.656795

Figure Lengend Snippet: (A-B) Effect of TAK242 on cytokine (IL-23p19, IL-1β, IL-10, IL-12p70) responses from (A) non-primed macrophages or (B) IFN-γ-primed macrophages treated with LPS or IL-32β for 24 h. (C) Binding of biotinylated IL-32β, biotinylated LPS-EB or positive control biotinylated anti-TGFBR2 to receptor expressing HEK293 cells spotted on a cell microarray. HEK293 cells overexpress components of the TLR4 receptor complex (CD14, MD2, TLR4), a positive control (TGFBR2) or candidate receptors for IL-32 from the literature (ITGAV, ITGB3, PRTN3/PAR3, F2RL1, ITGB6). (D) Activation of NF-κB/AP-1 reporter activity by LPS or IL-32β in HEK-Blue™ TLR4 cells treated for 24 h. (E) Concentration-dependent activation of NF-κB/AP-1 reporter by LPS, IL-32β or IL-32β + LPS in HEK-Blue™ TLR4 cells treated for 24 h. (F) Concentration-dependent activation of NF-κB/AP-1 reporter in HEK-Blue™ TLR4 cells treated with LPS or IL-32β +/- the LPS neutralizing antibiotic polymyxin B (PmB, 10 μg/mL) for 24 h. (G) Cytokine (TNF-α, IL-6) responses from non-primed macrophages treated with different concentrations of LPS or IL-32β +/- the LPS neutralizing antibiotic polymyxin B (PmB, 10 μg/mL) for 24 h. (H-I) Cytokine (TNF-α, IL-6) responses from (H) non-primed and (I) IFN-γ-primed macrophages treated with different concentrations of LPS or IL-32β. Statistical analysis: Data shown in (A-B) are mean values ±SEM of n = 3 independent experiments performed on macrophages from one donor, with two-way ANOVA with Dunnett’s multiple comparisons test vs LPS/IL-32β treatment groups (* p < 0.05, ** p < 0.005, *** p < 0.001). Data shown in (E) are mean values ±SEM of 3 independent experiments, with non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test (*p < 0.05, ***p < 0.001).

Article Snippet: Neutralizing monoclonal antibodies targeting human TLR4 (InvivoGen, mabg-htlr4) or TLR4/MD2 (InvivoGen, mab-htlr4md) receptors and IgG1 isotype control (InvivoGen, mabg1-ctrlm) were used on HEK-Blue™ hTLR4 cells at the concentrations: 0.1-1-10 μg/mL.

Techniques: Binding Assay, Positive Control, Expressing, Microarray, Activation Assay, Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: IL-32 drives inflammatory responses in IFN-γ primed human macrophages via a Myddosome-dependent pathway and is elevated in COVID-19

doi: 10.1101/2025.05.29.656795

Figure Lengend Snippet: (A) Schematic of experimental workflow for genome-wide RNAi screen performed in HEK-Blue™ TLR4 reporter cells to identify receptors and components of the signaling pathway for IL-32β. (B) Average Z-score distribution and associated p-values across 18,301 screened genes and Z-score distribution for each of 2 SMARTpool siRNA technical replicates per gene. Heatmap of the top 10 gene hits selected based on Z-score significance is shown to the right. (C) Z-score distribution for each of 4-siRNA duplexes (dots) from deconvoluted SMARTpools for 68 selected hits in a secondary validation screen; average Z-scores from primary screen are depicted as bars. siRNA duplexes that validated in the deconvolution screen are shown highlighted in bold. (D) Schematic of experimental workflow for validation of 18 selected hits from RNAi screens in human primary macrophages (left). Heatmap showing effect of RNAi-mediated perturbation of 10 selected hits and 4 controls (CD14, LY96, MyD88, TLR4), on fold change in cytokine response from macrophages treated with LPS or IL-32β (right). (E) Variation in effect of RNAi-mediated perturbation on fold change in cytokine response after treatment with LPS or IL-32β for macrophages from individual donors. (F) Co-localization of IL-32 isoforms with hits from RNAi screens in human primary macrophages. Percentage of macrophages with a detectable fluorescent signal with IL-32α or IL-32β pre-coupled to anti-IL-32 antibody (left panel). Co-localization between IL-32 isoform-Ab complexes and Myddosome protein components was quantified with Pearson’s correlation coefficient (middle panels). Cytokine responses from human macrophages used in IF microscopy experiments to IL-32 isoform-Ab complexes (right panel). Representative images of macrophages are shown in the lower panel. Pre-coupled IL-32α (green) or IL-32β (green) co-labelled for Myddosome protein components or downstream signaling mediators (red). Statistical analysis: (F) graphs shown on the left represent mean positive cell counts from 10 random microscopy fields from 2 independent experiments. Graphs on the right represent mean cytokine release from 2 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.005, *** p < 0.001) was used for both analyses.

Article Snippet: Neutralizing monoclonal antibodies targeting human TLR4 (InvivoGen, mabg-htlr4) or TLR4/MD2 (InvivoGen, mab-htlr4md) receptors and IgG1 isotype control (InvivoGen, mabg1-ctrlm) were used on HEK-Blue™ hTLR4 cells at the concentrations: 0.1-1-10 μg/mL.

Techniques: Genome Wide, Biomarker Discovery, Microscopy

Journal: bioRxiv

Article Title: IL-32 drives inflammatory responses in IFN-γ primed human macrophages via a Myddosome-dependent pathway and is elevated in COVID-19

doi: 10.1101/2025.05.29.656795

Figure Lengend Snippet: (A) Donor-specific variation in cytokine (TNF-α, IL-6) responses to LPS (10 ng/mL) or IL-32β (100 ng/mL) from untransfected primary human macrophages used for siRNA experiments (n = 5 donors). (B) Heatmap showing fold change in cytokine (TNF-α, IL-6) responses from macrophages (n = 5 donors) transfected with target-specific siRNA SMARTpools in the validation screen. (C) Binding of biotinylated IL-32β, biotinylated LPS-EB or biotinylated anti-TGFBR2 to HEK293 cells spotted on a cell microarray. Cells overexpress components of the TLR4 receptor complex (CD14, MD2, TLR4), a positive control (TGFBR2) or candidate receptors from the RNAi screen and literature. (D) Representative IF microscopy images of human macrophages incubated for 2 h with pre-coupled IL-32α, or IL-32β (green) to anti-IL-32 Alexa 488 Ab or Alexa 488 Ab alone and nuclear staining with DAPI.

Article Snippet: Neutralizing monoclonal antibodies targeting human TLR4 (InvivoGen, mabg-htlr4) or TLR4/MD2 (InvivoGen, mab-htlr4md) receptors and IgG1 isotype control (InvivoGen, mabg1-ctrlm) were used on HEK-Blue™ hTLR4 cells at the concentrations: 0.1-1-10 μg/mL.

Techniques: Transfection, Biomarker Discovery, Binding Assay, Microarray, Positive Control, Microscopy, Incubation, Staining

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Primer sequences used for reverse transcription-quantitative PCR analysis.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Sulfur compounds inhibit LPS-induced TLR expression. (A) Western blot analysis of TLR2 and TLR4 expression in THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. (B) Reverse transcription-quantitative PCR analysis of the relative expression levels of TLR2 and TLR4 normalized to GAPDH following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ***P<0.001; # P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (C) Flow cytometry showing the inhibition of LPS-induced TLR4 expression by 48-h treatment with NTS (3 µg/ml), MSM (200 mM) and TLR-C34 (40 µM). LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TLR, Toll-like receptor.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Inhibition

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Sulfur compounds induce DNA damage response following LPS-induced DNA damage. (A) Images of the comet assay were captured by fluorescence microscopy at ×10 and ×40 magnification levels, showing the fragmented DNA migrating out of the nucleoid body, which formed a comet tail following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. *P<0.05 and ***P<0.001; $ P<0.05 vs. non-treated control; and $$$ P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (B) Western blot analysis of THP-1 cells; NTS (3 µg/ml) and MSM (200 mM) inhibited the LPS-induced expression of p-ATM, p-ATR, p-Chk2, p-BRCA1, and p-p53. However, the expression levels of p-MDM2 were suppressed by LPS treatment, and were increased by NTS (3 µg/ml), MSM (200 mM) or TLR4-C34 (40 µM). LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; p-, phosphorylated; TLR, Toll-like receptor.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Single Cell Gel Electrophoresis, Fluorescence, Microscopy, Control, Western Blot, Expressing

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Molecular mechanism of LPS-induced regulation of the inflammatory response through iron/heme metabolism and TLR4/NF-κB expression through the canonical NF-κB and PKC-mediated inflammatory pathways. The anti-inflammatory activities of NTS and MSM were achieved by inhibiting iron/heme metabolism and suppressing the expression of TLR4/NF-κB signaling molecules, thus blocking the binding of NF-κB to the gene promoters of proinflammatory cytokines. DMT1, divalent metal transporter 1; Fe 2+ , ferrous ion; FPN, ferroportin; TfR, transferrin receptor; TLR4, Toll-like receptor 4.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Expressing, Blocking 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 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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: The proinflammatory activity of the PYRIN domain is conserved among PYHIN family members and across species Equimolar concentrations (111 nM) of the PYDs from various human and mouse family members, as well as from the unrelated NLRP3 and ASC proteins, were used to stimulate human (A and B) or mouse (C) macrophages for 24 h, with or without CLI-095 (TLR4 inhibitor, 5 μM). (A) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h. Values are normalized to GAPDH mRNA and plotted as fold induction over untreated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001, ∗∗ p < 0.01; one-way ANOVA followed by Dunnett’s test). (B and C) Protein concentration of TNF-α and IL-8 in the supernatants were evaluated by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗∗∗ p < 0.001, ### p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis showing the binding of various PYDs to TLR4/MD2. The TLR4/MD2 complex was immobilized on a CM5 sensor chip surface, followed by the injection of increasing concentrations of the indicated PYDs (20–800 nM) in running buffer. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: The following antibodies were used: rabbit polyclonal anti-IFI16 N-term and C-term (produced as described in ), mAb anti-human TLR4 (sc-293072, Santa Cruz Biotechnologies), mAb anti-human TLR4 (sc-13593, Santa Cruz Biotechnologies), mAb anti-β-actin (A1978, Sigma-Aldrich), rabbit IgG-HRP (A6154, Sigma-Aldrich), and mouse IgG-HRP (A16072, Thermo Fisher Scientific). pET30a expression vectors encoding the human and murine proteins used in this study were purchased from GenScript.

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: 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: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

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: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

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: The proinflammatory activity of the PYRIN domain is conserved among PYHIN family members and across species Equimolar concentrations (111 nM) of the PYDs from various human and mouse family members, as well as from the unrelated NLRP3 and ASC proteins, were used to stimulate human (A and B) or mouse (C) macrophages for 24 h, with or without CLI-095 (TLR4 inhibitor, 5 μM). (A) qRT-PCR analysis of TNF-α, IL-8, and IL-1β mRNA expression levels in human macrophages stimulated for 24 h. Values are normalized to GAPDH mRNA and plotted as fold induction over untreated cells. qRT-PCR data are presented as mean values ±SEM of biological triplicates (∗∗∗ p < 0.001, ∗∗ p < 0.01; one-way ANOVA followed by Dunnett’s test). (B and C) Protein concentration of TNF-α and IL-8 in the supernatants were evaluated by ELISA. Data are representative of three independent experiments, shown as the mean ± SEM (∗∗∗ p < 0.001, ### p < 0.001; one-way ANOVA followed by Dunnett’s test). (D) SPR analysis showing the binding of various PYDs to TLR4/MD2. The TLR4/MD2 complex was immobilized on a CM5 sensor chip surface, followed by the injection of increasing concentrations of the indicated PYDs (20–800 nM) in running buffer. Data are representative of three different experiments, and for each analysis the dissociation constant (K D ) value is shown.

Article Snippet: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

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

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: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

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: Mouse anti-human TLR4 monoclonal antibody , Santa Cruz Biotechnology , Cat# sc-13593; RRID: AB_628366.

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

Journal: Immunology

Article Title: Human Alveolar Macrophages Detect SARS‐CoV‐2 Envelope Protein Through TLR2 and TLR4 and Secrete Cytokines in Response

doi: 10.1111/imm.13922

Figure Lengend Snippet: SARS‐CoV‐2 envelope protein, but not spike protein, induces the secretion of cytokines from human alveolar macrophages. AMs were isolated from BAL fluid of patients undergoing routine bronchoscopy by adherence purification. AMs were pre‐treated with an isotype control antibody (control IgG) (10 μg/mL) or untreated for 1 h and then stimulated with SARS‐CoV‐2 spike protein or envelope protein (both 1 μg/mL). Untreated AMs that were stimulated with LPS (100 ng/mL) or unstimulated were used as positive and negative controls, respectively. Supernatant was collected at 24 h poststimulation. The concentration of cytokines was measured using an MSD multiplex assay ( n = 9). Statistical comparisons were made using the Friedman test with Dunn's multiple comparisons test.

Article Snippet: Monoclonal anti‐human TLR2 IgG2a kappa (anti‐TLR2) (ThermoFisher; 16‐9922), monoclonal anti‐human TLR4 IgG2a kappa (anti‐TLR4) (ThermoFisher; 16‐9917) and isotype control anti‐mouse IgG2a kappa (ThermoFisher; 16‐4724) were reconstituted in sterile PBS and stored at 4°C prior to experimentation.

Techniques: Isolation, Purification, Control, Concentration Assay, Multiplex Assay

Journal: Immunology

Article Title: Human Alveolar Macrophages Detect SARS‐CoV‐2 Envelope Protein Through TLR2 and TLR4 and Secrete Cytokines in Response

doi: 10.1111/imm.13922

Figure Lengend Snippet: Blocking antibodies against TLR2 and TLR4 alter the cytokine responses of human alveolar macrophages to SARS‐CoV‐2 envelope protein. AMs were isolated and treated with blocking antibodies against TLR2, TLR4, or an isotype control antibody (control IgG) (all 10 μg/mL) and stimulated as previously described. Supernatant was collected at 24‐h post stimulation. The concentration of cytokines was measured using an MSD multiplex assay ( n = 9). Statistical comparisons were made using the Friedman test with Dunn's multiple comparisons test.

Article Snippet: Monoclonal anti‐human TLR2 IgG2a kappa (anti‐TLR2) (ThermoFisher; 16‐9922), monoclonal anti‐human TLR4 IgG2a kappa (anti‐TLR4) (ThermoFisher; 16‐9917) and isotype control anti‐mouse IgG2a kappa (ThermoFisher; 16‐4724) were reconstituted in sterile PBS and stored at 4°C prior to experimentation.

Techniques: Blocking Assay, Isolation, Control, Concentration Assay, Multiplex Assay

Journal: Immunology

Article Title: Human Alveolar Macrophages Detect SARS‐CoV‐2 Envelope Protein Through TLR2 and TLR4 and Secrete Cytokines in Response

doi: 10.1111/imm.13922

Figure Lengend Snippet: Alveolar macrophages from donors aged over 70 years had higher cytokine responses to SARS‐CoV‐2 envelope protein than younger donors. AMs were isolated, treated,and stimulated as previously described. Supernatant was collected at 24 h poststimulation. The concentration of cytokines was measured using an MSD multiplex assay. The statistical comparison presented here is younger ( n = 5) and older ( n = 4) donor AMs treated with control IgG, then stimulated with envelope protein, or unstimulated. Planned statistical comparisons were made between stimulated cells from donors aged < 70 years versus those from donors aged > 70 years using two‐way ANOVA with Šídák's multiple comparison test.

Article Snippet: Monoclonal anti‐human TLR2 IgG2a kappa (anti‐TLR2) (ThermoFisher; 16‐9922), monoclonal anti‐human TLR4 IgG2a kappa (anti‐TLR4) (ThermoFisher; 16‐9917) and isotype control anti‐mouse IgG2a kappa (ThermoFisher; 16‐4724) were reconstituted in sterile PBS and stored at 4°C prior to experimentation.

Techniques: Isolation, Concentration Assay, Multiplex Assay, Comparison, Control

Journal: Immunology

Article Title: Human Alveolar Macrophages Detect SARS‐CoV‐2 Envelope Protein Through TLR2 and TLR4 and Secrete Cytokines in Response

doi: 10.1111/imm.13922

Figure Lengend Snippet: Alveolar macrophages from smokers had lower cytokine responses to SARS‐CoV‐2 envelope protein than non‐smokers. AMs were isolated, treated, and stimulated as previously described. Supernatant was collected at 24‐h poststimulation. The concentration of cytokines was measured using an MSD multiplex assay. The statistical comparison presented here is smokers' ( n = 5) and non‐smokers' ( n = 4) AMs treated with control IgG, then stimulated with envelope protein, or unstimulated. Planned statistical comparisons were made between stimulated cells from never/ex‐smokers versus those from current smokers using two‐way ANOVA with Šídák's multiple comparison test.

Article Snippet: Monoclonal anti‐human TLR2 IgG2a kappa (anti‐TLR2) (ThermoFisher; 16‐9922), monoclonal anti‐human TLR4 IgG2a kappa (anti‐TLR4) (ThermoFisher; 16‐9917) and isotype control anti‐mouse IgG2a kappa (ThermoFisher; 16‐4724) were reconstituted in sterile PBS and stored at 4°C prior to experimentation.

Techniques: Isolation, Concentration Assay, Multiplex Assay, Comparison, Control

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: The percentage of the neutrophil extracellular trap (NET) area in the SS and FS plots of flow cytometry was compared among untreated neutrophils and samples following NET induction with E. coli DH5α or PMA. (a) Expression intensities of Cit-H3, flavocytochrome b558, SYTOX™ Green, CD14, TLR2, TLR4, and LL-37 were compared between untreated neutrophil and NET areas after E. coli DH5α stimulation. (b) NET ratios were compared among untreated neutrophils and samples treated with E. coli DH5α or PMA using enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. ** p < 0.01. (c) Western blotting was performed to examine CD14 and Cit-H3 protein levels in untreated neutrophils and those stimulated with E. coli DH5α or PMA. Band intensities were quantified using ImageJ and normalized to β-actin. Data are presented as mean ± standard deviation (SD) (n = 3). Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s HSD test. ** p < 0.01

Article Snippet: Details of the antibodies used in this study and their sources are as follows: FITC-conjugated anti-human PR3 mouse monoclonal antibody (Cat. No. ab65255; Abcam, Cambridge, UK), PE-conjugated anti-human cathepsin D mouse monoclonal antibody (Cat. No. sc-13148 PE; Santa Cruz Biotechnology, Dallas, TX, USA), FITC-conjugated anti-human neutrophil elastase mouse monoclonal antibody (Cat. No. sc-55549 FITC; Santa Cruz Biotechnology, Dallas, TX, USA), PE-conjugated anti-human flavocytochrome b558 mouse monoclonal antibody (Cat. No. D162-5; Medical & Biological Laboratories, Tokyo, Japan), PE/Cyanine7-conjugated anti-human CD14 mouse monoclonal antibody (Cat. No. 367111; BioLegend, San Diego, CA, USA), FITC-conjugated anti-human CD68 mouse monoclonal antibody (Cat. No. F7135; DAKO, Glostrup, Denmark), FITC-conjugated anti-human CD282 (TLR2) mouse monoclonal antibody (Cat. No. 309705; BioLegend, San Diego, CA, USA), PE-conjugated anti-human CD284 (TLR4) mouse monoclonal antibody (Cat. No. 312805; BioLegend, San Diego, CA, USA), anti-citrullinated histone H3 (Cit-H3) (citrulline R2 + R8 + R17) rabbit polyclonal antibody (Cat. No. ab5103; Abcam, Cambridge, UK), anti-human LL-37 mouse monoclonal antibody (Cat. No. sc-166770; Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin rabbit monoclonal antibody (Cat. No. 4970; Cell Signaling Technology, Danvers, MA, USA), anti-human CD14 mouse monoclonal antibody (Cat. No. 14-0149-82; Invitrogen, Waltham, MA, USA), anti-PR3 mouse monoclonal antibody (Cat. No. sc-74534; Santa Cruz Biotechnology, Dallas, TX, USA), anti-human presepsin mouse monoclonal antibody (F1106-13–3; Mochida Pharmaceutical, Tokyo, Japan), anti-human presepsin rabbit monoclonal antibody (S68; Mochida Pharmaceutical, Tokyo, Japan), tetramethylrhodamine (TRITC)-conjugated goat anti-mouse IgG (H+L) secondary antibody (Cat. No. SA00007-1; Cosmo Bio, Tokyo, Japan), TRITC-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. SA00007-2, Cosmo Bio, Tokyo, Japan), Alexa Flour 405-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. ab175652; Invitrogen, Waltham, MA, USA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) secondary antibody (Cat. No. 7076; Cell Signaling Technology, Danvers, MA, USA), HRP-conjugated gort anti-rabbit IgG (H+L) secondary antibody (Cat. No. 7074; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot