Journal: The Journal of Experimental Medicine

Article Title: Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

doi: 10.1084/jem.20140280

Figure Lengend Snippet: Familial segregation and expression of TYK2 in patients’ cells. (A) Pedigrees of the TYK2-deficient families. Each generation is designated by a Roman numeral (I–II) and each individual by an Arabic numeral. The double lines connecting the parents indicate consanguinity. The probands (P1, P2, P3, P5, P7, and P8) in the six families are indicated by an arrow. Solid shapes indicate disease status. Individuals whose genetic status could not be determined are indicated by “E?”, and “m” indicates a mutated allele. (B) Schematic representations of the TYK2 gene with its 23 coding exons and of the TYK2 protein with its various domains (FERM, SH2, pseudokinase, and kinase). The exons are numbered with Roman numerals (III–XXV). The positions of the TYK2 mutation previously reported for P1 and of the resulting premature STOP codon are indicated by black arrows. The positions of the mutations for P2–P8 and their premature STOP codons are indicated by red arrows. (C) The predicted proteins for P1, P2, P3, P4, P5, P6, P7, and P8 are represented. (D) Levels of TYK2 in the patients’ EBV–B cells. Western blotting was performed with two Abs recognizing the N-terminal epitopes from TYK2 (N-ter1 and N-ter2) and two Abs recognizing the C-terminal epitopes (C-ter1 and C-ter2). Proteins were extracted from EBV–B cells from two healthy controls (C1 and C2) and from the TYK2 -mutated patients (P1, P2, P3, and P5). (E) TYK2 levels in the patients’ HVS–T cells and SV40-fibroblasts from a healthy control and P2. Western blotting was performed with two Abs recognizing two different N-terminal epitopes from TYK2 (N-ter1 and N-ter2) and another Ab recognizing C-terminal epitopes (C-ter1). (F) Western blot showing the detection of phospho-STAT4 (pSTAT4) and STAT4 in HVS–T cells from a healthy control (C), P2, and an IL-12Rβ1–deficient patient (IL-12Rβ1*), not stimulated (−) or stimulated for 30 min with 50 ng/ml IL-12 (+). The results in D–F are representative of at least two independent experiments. (G) Response to BCG alone (MOI = 20) and BCG and IFN-γ (5,000 IU/ml), in terms of IL-12p40 production, as assessed by ELISA on whole blood samples from healthy controls (local controls, LC; travel controls, TC), the TYK2-deficient patients (P2 twice, P3, P4, P5, P6, and P7; TYK2), and IL-12Rβ1–deficient patients (IL-12Rβ1*). Mean values for each set of conditions are indicated by solid lines.

Article Snippet: The following primary Abs were used: mouse anti–phosphorylated STAT1 (BD), mouse anti-STAT1 (BD), rabbit anti–phosphorylated STAT3 (Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti–phosphorylated STAT4 (Abazyme), rabbit anti-STAT4 (Cell Signaling Technology), mouse anti–α-tubulin (Santa Cruz Biotechnology, Inc.), rabbit anti-TYK2 (C-ter1; Santa Cruz Biotechnology, Inc.), mouse anti-TYK2 (C-ter2; Santa Cruz Biotechnology, Inc.), and mouse anti-TYK2 Abs (N-ter1 [BD] and N-ter2 [a gift from S. Pellegrini, Institut Pasteur, CNRS URA 1961, Paris, France]).

Techniques: Expressing, Mutagenesis, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Crosstalk between TBK1/IKKε and the type I interferon pathway contributes to tubulointerstitial inflammation and kidney tubular injury

doi: 10.3389/fphar.2022.987979

Figure Lengend Snippet: Type I interferons promote inflammation in renal tubular cells and kidney tissue. (A) Quantitative reverse transcription PCR (qRT-PCR) analysis of ISGs ( Oasl2, Usp18 ) in MCT cells stimulated with increasing concentrations of IFNβ for 6 and 24 h. Values for mRNA were normalized to GAPDH expression. Data are expressed as the Mean ± SEM of three independent experiments. * p ≤ 0.05 vs untreated control cells. (B) Time-course (minutes, m) for the activation of the TI-IFN pathway. Representative western blot of phosphorylated isoforms of STAT1, JAK1 and TYK2 in MCT cells stimulated with 1.0 mUI/ml IFNβ. Tubulin was used as loading control. (C,D) Activation of the NF-κB pathway. (C) Cells were stimulated as indicated in A and the mRNA expression of cytokines ( Cxcl10, Ccl2, IL-6 ) assessed by PCR. Data are Mean ± SEM of three independent experiments. * p ≤ 0.05 vs untreated control cells. (D) Representative western blot of phosphorylated IκBα (upper panel) and detection of nuclear p65 expression by immunofluorescence and confocal microscopy (green signal) (lower panel) in MCT cells stimulated with IFNβ for the indicated times (minutes, m). Original magnification ×400. Scale bar 10 µM. (E,F) Inhibition of the TI-IFN pathway by functional blockade of IFNAR (E) or pharmacological inhibition of TYK2/JAK1 (F) . MCT cells were preincubated with 10 μg/ml neutralizing anti-IFNAR antibody (IFNAR-Ab) or with 2.5 μM PF-06700,841 (PF) for 1 h before the addition of IFNβ for 6 h or 24 h Oasl2 mRNA expression was assessed by RT-PCR after 24 h whereas Cxcl10, Ccl2, and Il-6 gene expression were measured after 6 h. Results are shown as Mean ± SEM ( n = 3). * p ≤ 0.05 vs untreated control cells; # p ≤ 0.05 vs IFNβ treatment. (G,H) Activation and functional analysis of TBK1/IKKε signaling. (G) MCT cells were stimulated with 1.0 mUI/ml IFNβ for the indicated times (minutes, m). Activation of TBK/IKKε was detected by their phosphorylated isoforms by western blot (pTBK1, upper panel) or confocal microscopy (pIRF3, lower panel). Original magnification ×400. Scale bar 10 µM. (H) Transcriptional response in MCT cells pretreated for 1 h with 50 µM amlexanox (AMX) before 1.0 mUI/ml IFNβ addition was assessed by qRT-PCR following 6 h ( Cxcl10, Ccl2, Il-6 ) or 24 h ( Oasl2 ). Data are Mean ± SEM of three independent experiments. * p ≤ 0.05 vs untreated control cells; # p ≤ 0.05 vs IFNβ-treated cells. (I) Molecular routes activated by IFNβ in tubular cells. IFNβ activates the canonical TI-IFN pathway leading to ISG expression by binding to IFNAR and recruiting TYK2/JAK1/STAT1 signaling (blue pathway). Downstream IFNAR or TYK2/JAK1, IFNβ also promotes the synthesis of proinflammatory cytokines and ISGs by activating the NF-κB (red pathway) and IRF3 (green pathway) transcription factors, respectively. Inhibiting TI-IFN signaling at IFNAR (IFNAR-Ab) or TYK2/JAK1 (PF-06700,841: PF) downmodulates the direct (blue pathway) or indirect (green pathway) pathways driving cytokine and IFNα/β gene expression, consistent with crosstalk between the TI-IFN and NF-κB pathways (discontinued blue line). Likewise, interfering with TBK1/IKKε activation (AMX) decreases both ISG and cytokine gene expression, disclosing a second crosstalk between the TBK1/IKKε signaling node and NF-κB. (J-L) Murine model of systemic IFNβ injection for evaluation of the renal response. (J) Experimental design for IFNβ administration to mice. (K) Time-course of mRNA expression of canonical ISGs ( Oasl2, Usp18, Ifit1 ) and chemokines and cytokines ( Cxcl10, Ccl2, Il-6 ) in kidneys from control or IFNβ-injected mice. Bar charts represent the Mean ± SEM for each gene (n = 5-6 mice/group). * p ≤ 0.05 vs control mice. (L) Immunohistochemical analysis and quantification of markers for mononuclear phagocytes (F4/80) (upper panel) and lymphocytes (CD3) (lower panel) in kidneys from control and IFNβ-injected mice. Representative microphotographs of immune cells in kidney tissue from control and IFNβ-injected mice at 24 h. The number of cells per high power field (hpf) was quantified and results were expressed as Mean ± SEM. * p ≤ 0.05 vs control untreated mice (n = 4-6 mice/group). Original magnification ×200. Scale bar 100 µm.

Article Snippet: The following primary antibodies were used to detect specific proteins of interest: rabbit polyclonal anti-p-STAT1 (Tyr701) (Invitrogen, 44-376G), p-TYK2 (pTyr1054) (Origene, TA333304), and p-IKKε (Ser172) (Sigma Aldrich, 06-1340); rabbit monoclonal anti-p-TBK1/NAK (S172) (D52C2) XP ® (Cell Signaling Technology, 1,675,483), TBK1/NAK (E8I3G) (Cell Signaling Technology, 38,066), IKKε (D61F9) XP ® (Cell Signalling Technology, 3416), pIRF3 (Ser396) (4D4G) (Cell Signalling Technology, 4,947) and IRF-3 (D83B9) (Cell Signalling Technology, 4,302); monoclonal mouse anti-pIKBα (Santa Cruz, sc-8404).

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Control, Activation Assay, Western Blot, Immunofluorescence, Confocal Microscopy, Inhibition, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Binding Assay, Injection, Immunohistochemical staining

Journal: Frontiers in Pharmacology

Article Title: Crosstalk between TBK1/IKKε and the type I interferon pathway contributes to tubulointerstitial inflammation and kidney tubular injury

doi: 10.3389/fphar.2022.987979

Figure Lengend Snippet: TWEAK and LPS activate the TBK1/IKKε and TI-IFN pathways in cultured kidney tubular cells and in vivo in the kidneys. (A,B) TWEAK and LPS activated the TBK1/IKKε/IRF3 pathway as assessed by phosphorylated TBK1 and IKKε detected by western blot (A) and phosphorylated IRF3 detected by western blot (B, upper panel) or immunofluorescence (B, lower panel) as compared with vehicle-treated control cells (Cont). Tubular MCT cells were stimulated with 100 ng/ml TWEAK or 1 μg/ml LPS for 15-60 min (m). Figures are representative of experiments repeated at least thrice. Original magnification ×400 in B (lower panel). Scale bar 10 µM. (C–E) TWEAK and LPS activated the TI-IFN pathway. (C) Quantitative RT-PCR for mRNA expression of Ifna , Ifnb1 , and Ifnar1/2 in MCT cells stimulated with TWEAK or LPS for 6 and 24 h, and TWEAK-dependent IFNβ secretion assessed in cell culture supernatants (24 h) by ELISA (right upper panel). Results are expressed as the Mean ± SEM of at least three individual experiments. * p ≤ 0.05 vs control untreated cells . (D) Representative western blots of increased levels of phosphorylated STAT1 (pSTAT1) and TYK2 (pTYK2) in total protein extracts from MCT cells stimulated with 100 ng/ml TWEAK or 1 μg/ml LPS. Stimulation times are expressed in hours (H) . (E) Extended gene expression study of samples analyzed in panel C shows that TWEAK and LPS also upregulated ISG genes ( Ifit1, Usp18, Oasl2, Cxcl10 ). Results are the Mean ± SEM. * p ≤ 0.05 vs control untreated cells. (F) The contribution of the autocrine/paracrine recruitment of the TI-IFN pathway to TWEAK- and LPS-elicited responses in MCT cells was studied by blocking IFNAR with 10 μg/ml neutralizing anti-IFNAR antibody before stimulation with 100 ng/ml TWEAK or 1 μg/ml LPS. Gene expression was evaluated by q-RT-PCR after 6 h ( Cxcl10, Ccl2 ) or 24 h ( Oasl2 ). Results are expressed as Mean ± SEM of four or five experiments. * p ≤ 0.05 vs control untreated cells, # p ≤ 0.05 vs TWEAK- or LPS-stimulated cells. (G) Pharmacological inactivation of canonical IKKs limits TBK1/IKK-dependent ISG transcription in TWEAK- or LPS-stimulated MCT cells. Cultured MCT cells were pretreated for 1 h with chemical inhibitors of IKKα/β, namely 10 µM parthenolide (Parth) or 2.5 µM IKK16, before the addition of TWEAK or LPS for 24 h. Gene expression of ISGs ( Usp18, Oasl2 ) was assessed by q-RT-PCR. Bar chart represents the Mean ± SEM. * p ≤ 0.05 vs control vehicle-stimulated cells and # p ≤ 0.05 vs TWEAK- or LPS-stimulated cells ( n = 3/4).

Article Snippet: The following primary antibodies were used to detect specific proteins of interest: rabbit polyclonal anti-p-STAT1 (Tyr701) (Invitrogen, 44-376G), p-TYK2 (pTyr1054) (Origene, TA333304), and p-IKKε (Ser172) (Sigma Aldrich, 06-1340); rabbit monoclonal anti-p-TBK1/NAK (S172) (D52C2) XP ® (Cell Signaling Technology, 1,675,483), TBK1/NAK (E8I3G) (Cell Signaling Technology, 38,066), IKKε (D61F9) XP ® (Cell Signalling Technology, 3416), pIRF3 (Ser396) (4D4G) (Cell Signalling Technology, 4,947) and IRF-3 (D83B9) (Cell Signalling Technology, 4,302); monoclonal mouse anti-pIKBα (Santa Cruz, sc-8404).

Techniques: Cell Culture, In Vivo, Western Blot, Immunofluorescence, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression, Blocking Assay, Reverse Transcription Polymerase Chain Reaction

Journal: International immunopharmacology

Article Title: Regulation of IL-10 expression and function by JAK-STAT in CD8 + T cells.

doi: 10.1016/j.intimp.2024.111563

Figure Lengend Snippet: Fig. 1. Effect of JAK kinase inhibitors on IL-10 expression by CD8+ T cells. A. Freshly isolated lymphocytes from Tiger mice were cultured in antibody-coated wells with fresh medium containing 100 U/ml IL-2 and 10 nM Jak3 inhibitor Tofacitinib was added at 48hr, and 72hr after T cell activation, respectively. The expression of IL-10eGFP in CD8+ T cells was analyzed by flow cytometry. B. The enzyme-linked immunosorbent assay (ELISA) was used to detect the inhibition rate of JAK inhibitor. The inhibition rates of JAK1, JAK2, and JAK3 by 10 nM Tofacitinib were determined by comparing the phosphorylation levels of the treatment group to those of the control group. C. T cells were obtained and activated as in Fig. A, and 10 nM Jak1,2 inhibitor Ruxolitinib, 10 nM Jak3 inhibitor Tofacitinib, 10 nM Tyk2 inhibitor Deucravacitinib were added to the activated T cells 48hrs after T cell activation. The expression of IFN-γ and IL-10eGFP in CD8+ T cells were analyzed by flow cytometry. D. T cells were obtained and activated as in Fig. A, and 100 nM Jak1 inhibitor Solcitinib, 10 nM Jak2 inhibitor CEP-33779, 10 nM Jak1&2 inhibitor Ruxolitinib, 100 nM Jak1 inhibitor Solcitinib plus 10 nM Jak2 inhibitor CEP-33779 were added to the activated T cells 48hr after T cell activation. The expression of IFN-γ and IL-10eGFP in CD8+ T cells were analyzed by flow cytometry. ns indicates statistical results are not significant, * P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the IL-10 group, a fresh complete medium containing 100U/ mL IL-2(PEPROTECH#200-02) and 100U/mL IL-10 (PEPROTECH#20010) was used, and different concentrations of inhibitors (10 nM Jak1&2 inhibitor Ruxolitinib (Selleck), 10 nM Tofacitinib (Selleck), 10 nM Tyk2 inhibitor Deucravacitinib (Selleck), 1 μM STAT1 inhibitor Fludarabine (AbMole), 100 nM STAT3 inhibitor Niclosamide (AbMole), 1 μM STAT5 inhibitor AC-4-130 (MCE), 100 nM STAT6 AS1517499 (MCE), 100 nM STAT6 inhibitor AS1810722 (MCE)) were added.

Techniques: Expressing, Isolation, Cell Culture, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Inhibition, Phospho-proteomics, Control