Journal: Frontiers in Immunology

Article Title: Inflammasome Priming Mediated via Toll-Like Receptors 2 and 4, Induces Th1-Like Regulatory T Cells in De Novo Autoimmune Hepatitis

doi: 10.3389/fimmu.2018.01612

Figure Lengend Snippet: Damage-associated molecular patterns (DAMPs) providing inflammatory signal to the receptors toll-like receptor (TLR) 2/4 and 9, are significantly increased in sera of patients with de novo autoimmune hepatitis (DAIH). Sera from liver transplanted patients: with DAIH ( n = 10), without DAIH (LTC) ( n = 19); healthy, non-transplanted children (HC) ( n = 29), and non-transplanted children with autoimmune hepatitis (AIH) ( n = 9), was subjected to (i) qPCR; (ii) ELISA. HEK cells that stably co-express a human TLR2, TLR4, or TLR9 gene and an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were used to determine activation of TLRs from patient sera. (A,B) significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of cytosolic proteins associated with TLR 2/4 activation in sera of patients with DAIH compared to sera of LTC subjects (qPCR: p < 0.001 for ACTB; p < 0.001 for MT-ATP/complex I; p < 0.001 for Fibrinogen; p < 0.001 for HMGB1, p < 0.001 for HSP70; and p < 0.001 for HSP90, p = 0.05 for HSP60) (ELISA: p = 0.003 for Fibrinogen; p = 0.37 for HMGB1). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of mitochondrial, cytosolic, and functional proteins in sera of patients with DAIH compared to sera of HC subjects (qPCR: p < 0.001 for Fibrinogen, HMGB1, HSP70, HSP90, ACTB, and MT-ATP6, respectively, and p = 0.64 for HSP60). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of functional proteins in sera of patients with AIH compared to sera of HC subjects. [qPCR: p < 0.001 for ACTB (nuclear DNA); p = 0.002 for MT-ATP6 (mitochondrial DNA); p = 0.013 for Fibrinogen; p = 0.008, p = 0.005, and p = 0.008 for HSPs 60, 70 and 90, respectively] (ELISA: p = 0.043 for HMGB1; p = 0.11 for fibrinogen). *** p < 0.001, ** p < 0.01, * p < 0.05. (C,D) Activation of TLRs 2, 4, and 9 reporter cell lines by sera of patients with DAIH. DAIH vs. LTC: ( p = 0.47; TLR2) ( p = 0.008; TLR4) ( p = 0.02; TLR9). DAIH vs. HC: ( p = 0.17; TLR2) ( p < 0.001; TLR4) ( p = 0.001; TLR9). Minus sign: negative control, plus sign: positive control. Representative plate for three patients from each subject group and summary data. (E) Protein inhibition targeting heat shock protein, fibrinogen, and complexes I–III significantly reduced activation of TLR2/4 and TLR9 reporter cell lines, respectively [for TLR 9 reporter cell line inhibition: p = 0.03 for Rotenone (Complex 1), p = 0.03 for Thenoyltrifluoroacetone, TTFA (Complex II), p = 1.0 for Antimycin A (Complex III), p = 0.03 for Complexes I and II, p = 0.03 for Complexes I and III, p = 0.03 for Complexes II and III, p = 0.03 for Complexes I, II, and III; for TLR 4 reporter cell line inhibition: p = 0.06 for fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17- N -Allylamino-17-demethoxygeldanamycin, 17AAG, and p = 0.03 for both HSP90 and fibrinogen inhibitors; for TLR 2 reporter cell line inhibition: p = 0.09 for Fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17AAG, p = 0.06 for both HSP90 and fibrinogen inhibitors]. (HSP—heat shock protein; positive controls: FSL-1—synthetic diacylated lipoprotein, LPS—lipopolysaccharide, ODN 2006—CpG oligonucleotide). Summary data.

Article Snippet: Three Milliplex Map kits (EMD Millipore, Bilerica, MA, USA) were used to measure mitochondrial proteins in one assay (Complexes I–IV, Human Oxidative Phosphorylation Magnetic Bead Panel); heat shock proteins in another (Heat Shock Protein Magnetic Bead 5-Plex Kit); Fibrinogen protein in the third (Human Cardiovascular Disease Magnetic Bead Panel 3—Fibrinogen 1 Plex); and S100A8 and S100A12 in the fourth and fifth kit, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Stable Transfection, Activation Assay, Functional Assay, Negative Control, Positive Control, Inhibition

Journal: Frontiers in Immunology

Article Title: Inflammasome Priming Mediated via Toll-Like Receptors 2 and 4, Induces Th1-Like Regulatory T Cells in De Novo Autoimmune Hepatitis

doi: 10.3389/fimmu.2018.01612

Figure Lengend Snippet: Damage-associated molecular patterns (DAMPs) providing inflammatory signal to the receptors toll-like receptor (TLR) 2/4 and 9, are significantly increased in sera of patients with de novo autoimmune hepatitis (DAIH). Sera from liver transplanted patients: with DAIH ( n = 10), without DAIH (LTC) ( n = 19); healthy, non-transplanted children (HC) ( n = 29), and non-transplanted children with autoimmune hepatitis (AIH) ( n = 9), was subjected to (i) qPCR; (ii) ELISA. HEK cells that stably co-express a human TLR2, TLR4, or TLR9 gene and an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene were used to determine activation of TLRs from patient sera. (A,B) significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of cytosolic proteins associated with TLR 2/4 activation in sera of patients with DAIH compared to sera of LTC subjects (qPCR: p < 0.001 for ACTB; p < 0.001 for MT-ATP/complex I; p < 0.001 for Fibrinogen; p < 0.001 for HMGB1, p < 0.001 for HSP70; and p < 0.001 for HSP90, p = 0.05 for HSP60) (ELISA: p = 0.003 for Fibrinogen; p = 0.37 for HMGB1). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of mitochondrial, cytosolic, and functional proteins in sera of patients with DAIH compared to sera of HC subjects (qPCR: p < 0.001 for Fibrinogen, HMGB1, HSP70, HSP90, ACTB, and MT-ATP6, respectively, and p = 0.64 for HSP60). Significant abundance of DAMPs that activate TLRs 2/4 and 9 and levels of functional proteins in sera of patients with AIH compared to sera of HC subjects. [qPCR: p < 0.001 for ACTB (nuclear DNA); p = 0.002 for MT-ATP6 (mitochondrial DNA); p = 0.013 for Fibrinogen; p = 0.008, p = 0.005, and p = 0.008 for HSPs 60, 70 and 90, respectively] (ELISA: p = 0.043 for HMGB1; p = 0.11 for fibrinogen). *** p < 0.001, ** p < 0.01, * p < 0.05. (C,D) Activation of TLRs 2, 4, and 9 reporter cell lines by sera of patients with DAIH. DAIH vs. LTC: ( p = 0.47; TLR2) ( p = 0.008; TLR4) ( p = 0.02; TLR9). DAIH vs. HC: ( p = 0.17; TLR2) ( p < 0.001; TLR4) ( p = 0.001; TLR9). Minus sign: negative control, plus sign: positive control. Representative plate for three patients from each subject group and summary data. (E) Protein inhibition targeting heat shock protein, fibrinogen, and complexes I–III significantly reduced activation of TLR2/4 and TLR9 reporter cell lines, respectively [for TLR 9 reporter cell line inhibition: p = 0.03 for Rotenone (Complex 1), p = 0.03 for Thenoyltrifluoroacetone, TTFA (Complex II), p = 1.0 for Antimycin A (Complex III), p = 0.03 for Complexes I and II, p = 0.03 for Complexes I and III, p = 0.03 for Complexes II and III, p = 0.03 for Complexes I, II, and III; for TLR 4 reporter cell line inhibition: p = 0.06 for fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17- N -Allylamino-17-demethoxygeldanamycin, 17AAG, and p = 0.03 for both HSP90 and fibrinogen inhibitors; for TLR 2 reporter cell line inhibition: p = 0.09 for Fibrinogen inhibitor, p = 0.03 for the HSP90 inhibitor, 17AAG, p = 0.06 for both HSP90 and fibrinogen inhibitors]. (HSP—heat shock protein; positive controls: FSL-1—synthetic diacylated lipoprotein, LPS—lipopolysaccharide, ODN 2006—CpG oligonucleotide). Summary data.

Article Snippet: Three Milliplex Map kits (EMD Millipore, Bilerica, MA, USA) were used to measure mitochondrial proteins in one assay (Complexes I–IV, Human Oxidative Phosphorylation Magnetic Bead Panel); heat shock proteins in another (Heat Shock Protein Magnetic Bead 5-Plex Kit); Fibrinogen protein in the third (Human Cardiovascular Disease Magnetic Bead Panel 3—Fibrinogen 1 Plex); and S100A8 and S100A12 in the fourth and fifth kit, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Stable Transfection, Activation Assay, Functional Assay, Negative Control, Positive Control, Inhibition