Journal: Cells

Article Title: Extracellular HSP90α Induces MyD88-IRAK Complex-Associated IKKα/β−NF-κB/IRF3 and JAK2/TYK2−STAT-3 Signaling in Macrophages for Tumor-Promoting M2-Polarization

doi: 10.3390/cells11020229

Figure Lengend Snippet: The canonical CD91/MyD88–IRAK1/4–IKKα/β–NF-κB/IRF3 signaling pathway is involved in eHSP90α-induced macrophage M2-polarization. ( A ) Red fluorescent dots in PLAs showed that the physical associations of HSP90α–TLR4, HSP90α–CD91, TLR4–MyD88, CD91–MyD88, CD91–IRAK1, and MyD88–IRAK1 were induced when THP-1-derived macrophages were treated for 4 h with 15 μg/mL of rHSP90α. The representative data of three independent experiments are shown. ( B ) Levels of IRAK1, phosphorylated IRAK1, IRAK4, phosphorylated IRAK4, IKKα, IKKβ, phosphorylated IKKα/β, NF-κB, phosphorylated NF-κB, IRF3, and phosphorylated IRF3 in the THP-1-derived macrophages treated with PBS, rHSP90α, or rHSP90α plus control IgG or anti-CD91 or TLR4 antibody. The immunoblot data shown are the representative of three independent experiments. The numbers below the image sets are the relative levels of the phosphorylated proteins after normalization to their respective protein total levels. ( C ) Levels of IKKα, IKKβ, phosphorylated IKKα/β, IRF3, phosphorylated IRF3, NF-κB and phosphorylated NF-κB in the THP-1-derived macrophages treated with PBS, rHSP90α, or rHSP90α plus DMSO or 0.1 μM of IKKα/β inhibitor (IKKi; 6-amino-4-(4-phenoxyphenylethylamino)quinazoline, cat. #481406, Merck KGaA, Darmstadt, Germany ). The immunoblot data shown are the representative of three independent experiments. Relative phosphorylated protein levels are indicated below the image sets. ( D ) Secreted levels of IL-1β, IL-10, and TGF-β in the THP-1-derived macrophages treated with PBS, rHSP90α, or rHSP90α plus DMSO, IKKi, control IgG, or anti-CD91 or TLR4 antibody. The mean ± SD of ELISA data were obtained from three independent experiments. a p < 0.01 when compared with “PBS” group. b p < 0.01 when compared with “rHSP90α + DMSO” group. c p < 0.01 when compared with “rHSP90α + IgG” group. ( E ) mRNA levels of TNF-α, IL-1β, CD163, CD204, and IL-10 in the THP-1-derived macrophages treated with PBS, rHSP90α, or rHSP90α plus DMSO or IKKi. The RT-PCR data shown are the representative of three independent experiments. ( F , G ) ChIP assays showed that rHSP90α induced binding of NF-κB ( F ) and IRF3 ( G ) to CD163 , CD204 , and IL-10 gene promoters. The representative data of three independent experiments are shown.

Article Snippet: The primary antibodies used were specific for p-IRAK1 (1:500, cat. #sc-130197, Santa Cruz Biotechnology), IRAK1 (1:500, cat. #sc-5288, Santa Cruz Biotechnology), p-IRAK4 (1:1000, cat. #11927, Cell Signaling), IRAK4 (1:500, Cat. #sc-374349, Santa Cruz Biotechnology), p-IKKα/β (1:1000, cat. #2697, Cell Signaling), IKKα (1:500, cat. #3285, Epitomics, Burlingame, CA, USA), IKKβ (1:500, cat. #sc-7329, Santa Cruz Biotechnology), p-NF-κB (1:1000, cat. #1546-1, Epitomics), NF-κB (1:5000, cat. #sc-516102), p-IRF3 (1:1000, cat. #4947, Cell Signaling), IRF3 (1:500, cat. #sc-33641, Santa Cruz Biotechnology), p-JAK2 (1:1000, cat. #3771, Cell Signaling), JAK2 (1:1000, cat. #3230, Cell Signaling), p-TYK2 (1:1000, cat. #9312, Cell Signaling), TYK2 (1:1000, cat. #9321, Cell Signaling), p-STAT-3 (1:500, cat. #2236, Epitomics), STAT-3 (1:1000, cat. #04-1014, EMD Millipore), and GAPDH (1:20,000, cat. #NB300-221, Novus Biologicals, Littleton, CO, USA).

Techniques: Derivative Assay, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Binding Assay

Journal: Cells

Article Title: Extracellular HSP90α Induces MyD88-IRAK Complex-Associated IKKα/β−NF-κB/IRF3 and JAK2/TYK2−STAT-3 Signaling in Macrophages for Tumor-Promoting M2-Polarization

doi: 10.3390/cells11020229

Figure Lengend Snippet: Schematic summary of eHSP90α-induced CD91 and TLR4-associated signal transduction pathways for tumor-promoting M2-polarization in macrophages. Once macrophages are exposed to eHSP90α, CD91 is enhanced to be physically associated with TLR4 to recruit IRAK1/4 and MyD88. Besides the canonical CD91/MyD88–IRAK1/4–IKKα/β–NF-κB/IRF3 signaling pathway, JAK2 and TYK2 are recruited onto MyD88 to turn on a MyD88−JAK2/TYK2−STAT-3 pathway. Both signaling axes act together leading to downregulation of inflammatory cytokines TNF-α and IL-1β as well as upregulation of M2-associated TGF-β, CD163, CD204, IL-10, and VEGF expressions. Following our previous report that the eHSP90α-induced MyD88−JAK2/TYK2−STAT-3 pathway is involved in a feedforward loop of HSP90α expression and secretion, this signal transduction pathway is also responsible for the induction of Sec22b expression to decrease the phagocytotic activity of macrophages. Green ↓, down-regulated. Red ↑, up-regulated.

Article Snippet: The primary antibodies used were specific for p-IRAK1 (1:500, cat. #sc-130197, Santa Cruz Biotechnology), IRAK1 (1:500, cat. #sc-5288, Santa Cruz Biotechnology), p-IRAK4 (1:1000, cat. #11927, Cell Signaling), IRAK4 (1:500, Cat. #sc-374349, Santa Cruz Biotechnology), p-IKKα/β (1:1000, cat. #2697, Cell Signaling), IKKα (1:500, cat. #3285, Epitomics, Burlingame, CA, USA), IKKβ (1:500, cat. #sc-7329, Santa Cruz Biotechnology), p-NF-κB (1:1000, cat. #1546-1, Epitomics), NF-κB (1:5000, cat. #sc-516102), p-IRF3 (1:1000, cat. #4947, Cell Signaling), IRF3 (1:500, cat. #sc-33641, Santa Cruz Biotechnology), p-JAK2 (1:1000, cat. #3771, Cell Signaling), JAK2 (1:1000, cat. #3230, Cell Signaling), p-TYK2 (1:1000, cat. #9312, Cell Signaling), TYK2 (1:1000, cat. #9321, Cell Signaling), p-STAT-3 (1:500, cat. #2236, Epitomics), STAT-3 (1:1000, cat. #04-1014, EMD Millipore), and GAPDH (1:20,000, cat. #NB300-221, Novus Biologicals, Littleton, CO, USA).

Techniques: Transduction, Expressing, Activity Assay

Journal: Molecular Cancer

Article Title: IRAK1-regulated IFN-γ signaling induces MDSC to facilitate immune evasion in FGFR1-driven hematological malignancies

doi: 10.1186/s12943-021-01460-1

Figure Lengend Snippet: Relative expression levels ( A , left) of miR-146b-5p in BBC2 cells relative to normal pre/pro-B cells, ZNF112 relative to normal CD4 + CD8 + cells, BCRF8C relative to normal Sca1+ cells and KG1 relative to peripheral blood mononuclear cells ( N = 3 in all cases) shows consistent downregulation. When the same cell lines are treated with the BGJ398 FGFR1 inhibitor ( N = 3) miR-146-5p levels increase compared with DMSO treated cells ( A , right). Analysis of primary leukemic cells from a BCR-FGFR1 SCLL mouse model (N = 3) shows reduced levels of miR-146b-5p compared with normal BALB/c spleen cells ( B , left). The same reduced levels of miR-146b-5p are seen in primary leukemic cells from the ZMYM2-FGFR1 mouse model (N = 3) of SCLL ( B , right). Analysis of expression levels in primary human GSE79547 B-ALL samples ( N = 20) shows a reverse correlation between miR-146b-5p and FGFR1 expression levels ( C ). When BBC2 and KG1 cells (N = 3) are treated with the 5-aza-2’deoxycytidine (DAC) methylation inhibitor ( D , left) there is a dose-dependent increase in miR-146b-5p expression levels compared with DMSO treated controls. In BBC2 cells in which Dnmt1 is knocked down using two different shRNAs ( D , right) there is ~ 10 fold increase in miR-146b-5p expression compared with BBC2 cells treated with a scrambled control (shSCR). Location of the miR-146b-5p target sites within the murine ( E , above) and human ( F , above) IRAK1 mRNA. Western blot analysis of IRAK1 expression in primary leukemic cells from the syngeneic BCR-FGFR1 model of SCLL shows significantly increased IRAK1 expression levels ( E , left below). In BBC2 cells forced to overexpress (OE) miR-146b-5p ( E , right below), there is also a highly significant decrease in IRAK1 expression levels compared with cells expressing the empty vector (EV). IRAK1 expression levels are also increased in leukemic cells from a human SCLL model for BCR-FGFR1 transformed CD34+ cells propagated in immunocompromised (NSG) mice ( F , left below). In human KG1 cells forced to overexpress miR-146b-5p, IRAK1 level is reduced ( F , right below). Analysis of the GSE79547 expression data set from B-ALL patients shows an inverse correlation between IRAK1 expression levels and expression of miR-146b-5p ( G ). Kaplan-Meyer analysis of an AML cohort ( N = 422) demonstrates a highly significant decrease in overall survival in patients showing high level expression of IRAK1 compared with those showing low level expression ( H , left). A similar, although slightly less significant relationship between IRAK1 expression and survival is also observed ( H , right) in a B-Cell lymphoma ( N = 414) data set . In each case the cut off is determined using the SCAN algorithm within the R2 Gemome Analysis and Visualization Platform. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Antibodies used for western blotting (dilution 1:1000): β-Actin (Cell signaling, #5125), IRAK1 (Cell signaling, # 4504), p-IRAK1 (Sigma-Aldrich, SAB4504246), IFN-γ (Abclonal, # A12450), CXCL9 (Abclonal, # A19135), p-AKT (Cell Signaling, # 9272), AKT (Cell Signaling, # 9271), p- p38α (Cell Signaling, # 9211), p38α (Cell Signaling, # 9218), p-Stat3 (Cell Signaling, # 9134), Stat3 (Cell Signaling, # 4904).

Techniques: Expressing, Methylation, Western Blot, Plasmid Preparation, Transformation Assay

Journal: Molecular Cancer

Article Title: IRAK1-regulated IFN-γ signaling induces MDSC to facilitate immune evasion in FGFR1-driven hematological malignancies

doi: 10.1186/s12943-021-01460-1

Figure Lengend Snippet: Analysis of relative cell number increase using the trypan blue exclusion assay ( N = 3) over 4 days in the two Irak1 KO clones shows no difference compared with the mock control (MC) BBC2 cells ( A , left). Analysis of cell viability, relative to that seen in the MC cells, after 4 days (N = 3), using the CellTiter Glow assay ( A , right), shows no difference between the KO and MC cells. Flow cytometric analysis ( B , left) shows no difference in the distribution of cells in the S/G2/M phases of the cell cycle when the two KO clones were compared individually with the MC cells (N = 3). There is also no difference in levels of apoptosis as determined by annexin V staining ( B , right). Analysis of BALB/c mice xenografted with either 10,000 ( C , left) or 100,000 ( C , center) cells ( N = 6) from KO clones #5 and #7 and MC cells that show survival times of 14–17 days in mice xenografted with the MC BBC2 cells. In contrast, mice xenografted with the KO clones did not develop leukemia during the observation period up to 120 days regardless of inoculated tumor cell burden. When 10,000 cells from the MC and KO lines were engrafted into immunocompromized NSG mice ( C , right) however, all mice ( N = 5) developed disease and died within 13–19 days. The survival data shown in ( C ) was reflected in a significant reduction in spleen size and the proportion of GFP+ cells in the spleens from mice xenografted with the KO clones when compared individually with mice xenografted with the MC cells at autopsy ( D ). Spleen size in the NSG mice ( E ) was enlarged in all cohorts and levels of GFP+ cells were equally high. Treatment of mice ( N = 5) with an anti-CD4 or anti-CD8 antibody shows a significant reduction in CD4+ or CD8+ cells, respectively ( F and G ) when compared individually with the isotype control. Conbination treatment with anti-CD4/CD8 antibodies led to significant loss of both cell types as shown in F and G . When IRAK KO #7 cells are xenografted into BALB/c mice treated with the various antibodies to deplete T-cells, while the isotype treated mice did not develop leulemia, mice experiencing depletion of CD4 or CD8 or both types of cells showed robust development of leukemia when each is compared with the isotype control ( H , left). The development of leukemia was paralleled with increased spleen and liver weight ( H , right). ns = not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: Antibodies used for western blotting (dilution 1:1000): β-Actin (Cell signaling, #5125), IRAK1 (Cell signaling, # 4504), p-IRAK1 (Sigma-Aldrich, SAB4504246), IFN-γ (Abclonal, # A12450), CXCL9 (Abclonal, # A19135), p-AKT (Cell Signaling, # 9272), AKT (Cell Signaling, # 9271), p- p38α (Cell Signaling, # 9211), p38α (Cell Signaling, # 9218), p-Stat3 (Cell Signaling, # 9134), Stat3 (Cell Signaling, # 4904).

Techniques: Trypan Blue Exclusion Assay, Clone Assay, Staining

Journal: Molecular Cancer

Article Title: IRAK1-regulated IFN-γ signaling induces MDSC to facilitate immune evasion in FGFR1-driven hematological malignancies

doi: 10.1186/s12943-021-01460-1

Figure Lengend Snippet: Treatment of MC BBC2 cells and IRAK1 KO #5 and #7 cells (N = 3) with pacritinib shows no differential effects on cell growth inhibition at high doses as determined using CellTiter-Glo Cell Viability Assays ( A ). Pacritinib treatment of the MC cells which express IRAK1 leads to suppression of its phosphactivation and loss of IFN-γ production ( B ). Flow cytometry analysis of annexin V staining (N = 3) of the same cells from ( B ) shows no differential induction of apoptosis in either the MC of KO Cells ( C ). When BBC2 cells were xenografted into BALB/c mice (N = 8) and then treated with the pacritinib IRAK1 inhibitor, there was a significant increase in survival time ( D ), which was reflected in a significantly reduced spleen size and reduction in white blood cell count in the treated cohort. Representative flow cytometry analysis of peripheral blood from the treated and control cells ( E ) at the time of sacrifice shows reduced levels of GFP+ cells in the drug-treated mice (N = 5) and a significantly higher level of CD4+/CD8+ T-cells ( F ). Levels of MDSC in the drug treated mice was also reduced compared with mice treated with vehicle alone. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Antibodies used for western blotting (dilution 1:1000): β-Actin (Cell signaling, #5125), IRAK1 (Cell signaling, # 4504), p-IRAK1 (Sigma-Aldrich, SAB4504246), IFN-γ (Abclonal, # A12450), CXCL9 (Abclonal, # A19135), p-AKT (Cell Signaling, # 9272), AKT (Cell Signaling, # 9271), p- p38α (Cell Signaling, # 9211), p38α (Cell Signaling, # 9218), p-Stat3 (Cell Signaling, # 9134), Stat3 (Cell Signaling, # 4904).

Techniques: Inhibition, Flow Cytometry, Staining, Cell Counting