Journal: bioRxiv

Article Title: IRG1/itaconate/NRF2/GSH axis in tumor-associated macrophages drives therapy resistance and immune evasion in BRCA1-deficient breast cancer

doi: 10.1101/2025.10.14.682312

Figure Lengend Snippet: a , Schematic representation of the metabolic pathway of IRG1-related metabolites. b, Heatmap indicates the relative levels of itaconate and its related metabolites in CM derived from BP-TAM and control macrophages in the untargeted metabolomic analysis. c, RT-qPCR analysis of IRG1 expression in THP-1-Mφ and 436-TAMs. Data are presented as mean ± s.d.; unpaired Student’s t-test; n = 3. d, Western blot analysis of IRG1 protein expression in THP-1-Mφ and 436-TAMs. e, RT-qPCR analysis of IRG1 expression in BMDMs and BP-TAMs. Data are presented as mean ± s.d.; unpaired Student’s t-test; n = 3. f, Western blot analysis of IRG1 protein expression in BMDMs and BP-TAMs. g, GSEA of RNA-seq data from BMDMs and BP-TAMs. h,i, Western blot analysis of STAT5 and phosphorylated STAT5 (p-STAT5) expression in THP-1-Mφ, 436-TAMs, BMDMs, and BP-TAMs. j, Western blot analysis of IRG1 protein expression in THP-1-Mφ and 436-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. The inhibitors were used at the following concentrations: STAT3-IN-1 (10 μM), STAT5-IN-1 (100 μM), and pyrrolidinedithiocarbamate ammonium (PDTC, 10 μM). k, RT-qPCR analysis of IRG1 mRNA expression in THP-1-Mφ and 436-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. Fold changes and P values in IRG1 mRNA levels in TAMs relative to THP-1-Mφ are shown. The inhibitors were used at the same concentrations as in ( j ). l, Western blot analysis of IRG1 protein expression in BMDMs and BP-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. The inhibitors were used at the same concentrations as in ( j ). m, RT-qPCR analysis of IRG1 mRNA expression in BMDMs and BP-TAMs treated with inhibitors of STAT3, STAT5, and NF-κB. Fold changes and P values in IRG1 mRNA levels in TAMs relative to BMDMs are shown. The inhibitors were used at the same concentrations as in ( j ).

Article Snippet: The following small-molecule compounds, metabolites, inhibitors, and neutralizing antibodies were used in this study: olaparib (HY-10162, MedChemExpress), L-glutathione reduced (GSH; G4251, Sigma-Aldrich, St. Louis, MO, USA), N-acetyl-L-cysteine (NAC; A7250, Sigma-Aldrich), L-(+)-lactic acid (L1750, Sigma-Aldrich), sodium L-lactate (71718, Sigma-Aldrich), itaconic acid (T4837, TargetMol, Wellesley Hills, MA, USA), citraconic acid (C82604, Sigma-Aldrich), 4-octyl itaconate (4-OI; SML2338, Sigma-Aldrich), dimethyl itaconate (DMI; T5377, TargetMol), dimethyl citraconate (DMC; C0346, TCI America, Portland, OR, USA), DL-buthionine-sulfoximine (BSO; 19176, Sigma-Aldrich), sodium oxamate (LDHi; O2751, Sigma-Aldrich), STAT5-IN-1 (S6784, Selleck Chemicals, Houston, TX, USA), STAT3-IN-1 (S0818, Selleck Chemicals), pyrrolidinedithiocarbamate ammonium (PDTC; S3633, Selleck Chemicals), RSL3 (S8155, Selleck Chemicals), Ferrostatin-1 (Fer-1; S7243, Selleck Chemicals), ML385 (T4360, TargetMol), and IRG1-IN-1 (T78525, TargetMol).

Techniques: Derivative Assay, Control, Quantitative RT-PCR, Expressing, Western Blot, RNA Sequencing

Journal: bioRxiv

Article Title: IRG1/itaconate/NRF2/GSH axis in tumor-associated macrophages drives therapy resistance and immune evasion in BRCA1-deficient breast cancer

doi: 10.1101/2025.10.14.682312

Figure Lengend Snippet: STAT5-dependent activation of the IRG1/itaconate pathway in TAMs reprograms mitochondrial metabolism and activates NRF2-driven GSH biosynthesis. The TAM-derived GSH protects tumor cells from ROS-induced DNA damage and ferroptosis, while concurrently suppressing STING-mediated immune activation in both tumor and dendritic cells. Pharmacological inhibition of IRG1 restores tumor sensitivity to PARPi, reactivates anti-tumor immunity, and significantly suppresses tumor growth in BRCA1-deficient preclinical models.

Article Snippet: The following small-molecule compounds, metabolites, inhibitors, and neutralizing antibodies were used in this study: olaparib (HY-10162, MedChemExpress), L-glutathione reduced (GSH; G4251, Sigma-Aldrich, St. Louis, MO, USA), N-acetyl-L-cysteine (NAC; A7250, Sigma-Aldrich), L-(+)-lactic acid (L1750, Sigma-Aldrich), sodium L-lactate (71718, Sigma-Aldrich), itaconic acid (T4837, TargetMol, Wellesley Hills, MA, USA), citraconic acid (C82604, Sigma-Aldrich), 4-octyl itaconate (4-OI; SML2338, Sigma-Aldrich), dimethyl itaconate (DMI; T5377, TargetMol), dimethyl citraconate (DMC; C0346, TCI America, Portland, OR, USA), DL-buthionine-sulfoximine (BSO; 19176, Sigma-Aldrich), sodium oxamate (LDHi; O2751, Sigma-Aldrich), STAT5-IN-1 (S6784, Selleck Chemicals, Houston, TX, USA), STAT3-IN-1 (S0818, Selleck Chemicals), pyrrolidinedithiocarbamate ammonium (PDTC; S3633, Selleck Chemicals), RSL3 (S8155, Selleck Chemicals), Ferrostatin-1 (Fer-1; S7243, Selleck Chemicals), ML385 (T4360, TargetMol), and IRG1-IN-1 (T78525, TargetMol).

Techniques: Activation Assay, Derivative Assay, Inhibition

Journal: Cells

Article Title: Pathologic Th1–Treg Cells Exacerbate Acute Lung Injury and Lethality in Sepsis

doi: 10.3390/cells15060521

Figure Lengend Snippet: eCIRP activates STAT1 and STAT5 via TLR4 to induce Th1-Treg cells. ( A , B ) CD4 + T cells from WT and TLR4 −/− mice were cultured with PBS or 2.5 μg/mL of eCIRP for 48 h. The frequency of Th1-Treg cells of PBS-treated CD4 + T cells and eCIRP-treated CD4 + T cells was evaluated by flow cytometry. ( C , D ) CD4 + T cells from WT and TLR4 −/− mice were cultured with 2.5 μg/mL of eCIRP for 2 h. The frequency of pSTAT1 + pSTAT5 + cells of PBS-treated CD4 + T cells and eCIRP-treated CD4 + T cells was evaluated by flow cytometry. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 6, 7 samples/group) and compared by one-way ANOVA and Tukey’s multiple comparison test for multiple groups. * p < 0.05 vs. WT PBS, # p < 0.05 vs. WT eCIRP. ( E , F ) CD4 + T cells from WT mice were cultured with 2.5 μg/mL of eCIRP for 48 h in the presence and absence of 1 μM of STAT1 inhibitor and 10 μM of STAT5 inhibitor. The frequency of Th1-Treg cells was analyzed by flow cytometry. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 6 samples/group) and compared by one-way ANOVA and Tukey’s multiple comparison test for multiple groups. * p < 0.05 vs. PBS, # p < 0.05 vs. eCIRP alone.

Article Snippet: CD4 + T cells were treated with 1.0, 2.5 μg/mL of eCIRP for the indicated time periods on CD3/CD28 coated plates (Ultra-LEAF Purified anti-mouse CD3ε, Cat. No.: 100340; Ultra-LEAF Purified anti-mouse CD28, Cat. No.: 102116, Biolegend, San Diego, CA, USA) in the presence and absence of 1 μM of STAT1 inhibitor (Fludarabine; Cat. No.: HY-B0069, MedChemExpress, Monmouth Junction, NJ, USA) and/or 10 μM of STAT5 inhibitor (Cat. No.: HY-101853, MedChemExpress), followed by stimulation with Cell Activation Cocktail (with Brefeldin A) (Cat. No.: 423304, Biolegend) for 4 h for cytokine evaluation.

Techniques: Cell Culture, Flow Cytometry, Comparison

Journal: Cells

Article Title: Pathologic Th1–Treg Cells Exacerbate Acute Lung Injury and Lethality in Sepsis

doi: 10.3390/cells15060521

Figure Lengend Snippet: Finding summary. During sepsis, eCIRP signals through TLR4 to activate STAT1 and STAT5, driving the differentiation of pathogenic Th1-Treg cells. These cells accumulate in the lungs, exacerbate acute lung injury, and ultimately increase mortality in sepsis.

Article Snippet: CD4 + T cells were treated with 1.0, 2.5 μg/mL of eCIRP for the indicated time periods on CD3/CD28 coated plates (Ultra-LEAF Purified anti-mouse CD3ε, Cat. No.: 100340; Ultra-LEAF Purified anti-mouse CD28, Cat. No.: 102116, Biolegend, San Diego, CA, USA) in the presence and absence of 1 μM of STAT1 inhibitor (Fludarabine; Cat. No.: HY-B0069, MedChemExpress, Monmouth Junction, NJ, USA) and/or 10 μM of STAT5 inhibitor (Cat. No.: HY-101853, MedChemExpress), followed by stimulation with Cell Activation Cocktail (with Brefeldin A) (Cat. No.: 423304, Biolegend) for 4 h for cytokine evaluation.

Techniques:

Journal: Infection and Immunity

Article Title: GM-CSF engages multiple signaling pathways to enhance pro-inflammatory cytokine responses in human monocytes during Legionella infection

doi: 10.1128/iai.00565-24

Figure Lengend Snippet: GM-CSF-dependent JAK2-STAT5 signaling is required to enhance inflammatory cytokine expression during Legionella infection. ( A ) THP-1 human monocytes were pretreated with PBS or rGM-CSF for 1 h. Cells were harvested at 6 h after infection to perform immunoblot analysis for phospho-STAT5, total STAT5, or β-actin as loading control. Lanes from one membrane have been cropped to depict the appropriate conditions, as indicated by the dashed lines. No changes were made to the original image during the editing. (B to C) THP-1 monocytes were pretreated with vehicle control, ( B ) the JAK2 inhibitor NVP-BSK805, or ( C ) the STAT5 inhibitor SH-4-54 for 1 h. Cells were then treated with PBS or rGM-CSF for 30–60 min and left uninfected or infected with L.p . Cells were harvested at 6 h post-infection (hpi) to measure IL1A , IL1B , and IL6 transcript levels by qPCR. Data represent the mean ± SEM of triplicate wells from at least two ( C ) or three ( B ) independent experiments. Data were analyzed by two-way ANOVA with Šidák’s multiple comparisons test; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.

Article Snippet: For inhibitor treatments, cells were pretreated 1 h before GM-CSF stimulation with either 250 nM IKK inhibitor (BMS-345541; Selleck Chemicals S8044), 2 μM JAK2 inhibitor (NVP-BSK805; Selleck Chemicals S2686), 5 μM STAT5 inhibitor (SH-4-54; Selleck Chemicals S7337), 10 μM PI-3K inhibitor (Ly294002; Selleck Chemicals S1105), 5 μM Akt inhibitor (MK-2206; Selleck Chemicals S1078), or 100 nM rapamycin (AY-22989; Selleck Chemicals S1039).

Techniques: Expressing, Infection, Western Blot, Control, Membrane

Journal: Infection and Immunity

Article Title: GM-CSF engages multiple signaling pathways to enhance pro-inflammatory cytokine responses in human monocytes during Legionella infection

doi: 10.1128/iai.00565-24

Figure Lengend Snippet: Multistep regulation of GM-CSF-enhanced cytokine gene expression in Legionella -infected human monocytes. Graphical model depicting the findings of this study. GM-CSF-enhanced cytokine production requires an initial bacterial PAMP that activates TLR-dependent NF-κB signaling in Legionella -infected human monocytes. Cytokine gene expression is then enhanced by GM-CSF-dependent JAK2/STAT5 signaling. PI-3K/Akt/mTORC1 signaling, glycolysis, and amino acid metabolism are required for upregulation of cytokine responses by GM-CSF. Dashed lines represent putative links between signaling pathways that need to be further dissected in future studies. Figure created in https://BioRender.com .

Article Snippet: For inhibitor treatments, cells were pretreated 1 h before GM-CSF stimulation with either 250 nM IKK inhibitor (BMS-345541; Selleck Chemicals S8044), 2 μM JAK2 inhibitor (NVP-BSK805; Selleck Chemicals S2686), 5 μM STAT5 inhibitor (SH-4-54; Selleck Chemicals S7337), 10 μM PI-3K inhibitor (Ly294002; Selleck Chemicals S1105), 5 μM Akt inhibitor (MK-2206; Selleck Chemicals S1078), or 100 nM rapamycin (AY-22989; Selleck Chemicals S1039).

Techniques: Gene Expression, Infection, Protein-Protein interactions