Journal: iScience

Article Title: Glycolysis maintains effector function of lung Th17 TRM cells in precision cut lung slices

doi: 10.1016/j.isci.2025.114051

Figure Lengend Snippet: Long-lived lung CD4 + Th17 cells are enriched in glycolysis over time (A) Cell number of OVA + cells after tetramer pulldown ( n = 2). (B) Gene set enrichment analysis between day 30 and day 90 OVA + CD4 + T cells. (C) GSEA result comparing day 30 and day 90 OVA + CD4 + T cells. (D) GSEA result comparing day 30 and day 90 OVA + Il17a + CD4 + T cells. (E) GSEA result comparing human lung CD4 + TRM cells and naive CD4 + T cells from lymph nodes. GEO: GSE137967 . (F) Dot plot showing scaled expression of key glycolytic genes ( Hk2, Pfkp, Aldoa, Gapdh, Pgk1, Eno1, Pkm, Ldha, and Galm ) in day 30 vs. day 90 OVA + IL-17A + CD4 + T cells. (G) Heatmap of key glycolytic genes ( HK2, PFKP, ALDOA, GAPDH, PGK1, ENO1, PKM, LDHA, and GALM ) in human lung CD4 + TRM cells and naive CD4 + T cells from lymph nodes.

Article Snippet: Rabbit anti-IL17A antibody , nsj Bioreagents , Cat# RQ4021.

Techniques: Expressing

Journal: iScience

Article Title: Glycolysis maintains effector function of lung Th17 TRM cells in precision cut lung slices

doi: 10.1016/j.isci.2025.114051

Figure Lengend Snippet: Metabolic features of TRM cells are maintained in PCLS (A) In vivo labeling of glucose uptake using 2-NBDG on lung CD4 + CD69 + TRM cells. Mice were immunized with OmpX+LTA1 as described before. On day 30 after prime immunization, mice were given 500 nmol 2-NBDG by intratracheal oropharyngeal aspiration. Mice were sacrificed 30 min later. Lungs were harvested for digestion and flow cytometry. (B) Cartoon depicting immunization schedule with mouse PCLS generation. (C) CD3 + CD69 + IL-17 + TRM cells in PCLS of LTA1/OmpX-immunized C57BL/6 mice, compared with naive mice, were visualized with multicolor immunofluorescence (scale bars: 100 μm). (D–F) RNA was extracted from PCLS. Il17a expression was measured by real-time RT-qPCR. ( n = 4–8 PCLS per group). Data represent two independent experiments. Data are presented as means ± SEM. Significant differences were calculated with Mann-Whitney test or one-way ANOVA. ∗, p ≤ 0.05, ∗∗, p ≤ 0.01, ∗∗∗, p ≤ 0.001, and ∗∗∗∗, p ≤ 0.0001.

Article Snippet: Rabbit anti-IL17A antibody , nsj Bioreagents , Cat# RQ4021.

Techniques: In Vivo, Labeling, Flow Cytometry, Immunofluorescence, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Archives of Microbiology

Article Title: Methanobrevibacter smithii activates immune microenvironment of intestinum tenue in a mouse model

doi: 10.1007/s00203-026-04772-2

Figure Lengend Snippet: Cytokine changes in colon after M. smithii gavage in mouse model. A Western blots showed expressions of TNF-ɑ, IL-22, IL-17 and GM-CSF in colon tissues collected from the control and experimental groups. B-E Quantifications of the relative gray value of western blots analyzed by ImageJ software. F Representative images of expressions of TNF-ɑ, IL-22, IL-17 and GM-CSF in colon sections of PBS and M.smithii groups. G-J Quantifications of staining density of cytokines of TNF-ɑ, IL-22, IL-17 and GM-CSF in colon sections of PBS and M.smithii groups. K-N RNA expression levels of TNF-ɑ, IL-22, IL-17 and GM-CSF in colon of the two groups were analyzed by RT-qPCR. Data are shown as mean ± SEM, two-tailed Student’s t-test was used to perform statistical comparisons between groups, n = 8 in each independent experiment, scale bar: 50 μm, each experiment was repeated three times

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 h at room temperature, then probed overnight at 4 °C with the following primary antibodies (1:2000 dilution): TNF-α (ab215188), IL-17 (13838 S), GM-CSF (505504), and IL-22 (A6212) from respective suppliers (Abcam; CST; BioLegend; ABclonal).

Techniques: Western Blot, Control, Software, Staining, RNA Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Archives of Microbiology

Article Title: Methanobrevibacter smithii activates immune microenvironment of intestinum tenue in a mouse model

doi: 10.1007/s00203-026-04772-2

Figure Lengend Snippet: ILC3s can be activated by M.smithii stimuli. A Representative flow cytometry plots showing the populations of ILCs. B Representative flow cytometry plots of ILC3s cells. C Statistical comparisons of CD117 + cells in the PBS and M.smithii groups. D Representative flow cytometry plots of CD117 + TNF-ɑ+ cells. E Statistical comparisons of CD117 + TNF-ɑ+ percentages in the PBS and M.smithii groups. F Representative flow cytometry plots of CD117 + IL-17 + cells. G. Statistical comparisons of the proportions of CD117 + IL-17 + cells in the groups of PBS and M.smithii . H Representative flow cytometry plots of CD117 + IL-22 + cells. I Statistical comparisons of CD117 + IL-22 + cells proportions in PBS and M.smithii groups. J Representative flow cytometry plots of CD117 + GM-CSF+ cells. K Statistical comparisons of CD117 + GM-CSF+ cells in the PBS and M.smithii groups. Data are shown as mean ± SEM, two-tailed Student’s t-test was used to perform statistical comparisons between groups, n = 8 in each independent experiment, each experiment was repeated three times

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 h at room temperature, then probed overnight at 4 °C with the following primary antibodies (1:2000 dilution): TNF-α (ab215188), IL-17 (13838 S), GM-CSF (505504), and IL-22 (A6212) from respective suppliers (Abcam; CST; BioLegend; ABclonal).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Archives of Microbiology

Article Title: Methanobrevibacter smithii activates immune microenvironment of intestinum tenue in a mouse model

doi: 10.1007/s00203-026-04772-2

Figure Lengend Snippet: Cytokine secretion alterations of CD4 + T cells after M.smithii gavage stimulation. A Representative flow cytometry plots of CD4 + T and CD8 + T cells in colon lamina propria cells. B , C Statistical comparisons of CD4 + T and CD8 + T cells of colon lamina propria in the PBS and M.smithii mice. D Comparisons of CD4 + T/ CD8 + T ratio in PBS and M.smithii groups. E , F Representative flow cytometry plots of CD4 + TNF-ɑ+cells, and statistical comparisons of CD4 + TNF-ɑ+cells in the PBS and M.smithii groups. G , H Representative flow cytometry plots of CD4 + IL-22 + cells, and statistical comparisons of CD4 + IL-22 + cells in the groups of PBS and M.smithii groups. I , J Representative flow cytometry plots of CD4 + IL-17 + cells, and statistical comparisons of CD4 + IL-17 + cells in the groups of PBS and M.smithii groups. K , L Representative flow cytometry plots of CD4 + GM-CSF+ cells, and statistical comparisons of CD4 + GM-CSF+ cells in the groups of PBS and M.smithii . Data are shown as mean ± SEM, two-tailed Student’s t-test was used to perform statistical comparisons between groups, n = 8 in each independent experiment, each experiment was repeated three times

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 h at room temperature, then probed overnight at 4 °C with the following primary antibodies (1:2000 dilution): TNF-α (ab215188), IL-17 (13838 S), GM-CSF (505504), and IL-22 (A6212) from respective suppliers (Abcam; CST; BioLegend; ABclonal).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Archives of Microbiology

Article Title: Methanobrevibacter smithii activates immune microenvironment of intestinum tenue in a mouse model

doi: 10.1007/s00203-026-04772-2

Figure Lengend Snippet: Effect of M.smithii stimuli on CD8 + T cells. A , B Representative flow cytometry plots of CD8 + TNF-ɑ+ cells, and statistical comparisons of CD8 + TNF-ɑ+ cells in the PBS and M.smithii groups. C , D Representative flow cytometry plots of CD8 + IL-22 + cells, and statistical comparisons of CD8 + IL-22 + cells in the groups of PBS and M.smithii . E , F Representative flow cytometry plots of CD8 + IL-17 + cells, and statistical comparisons of CD8 + IL-17 + cells in the groups of PBS and M.smithii . G , H Representative flow cytometry plots of CD8 + GM-CSF+ cells, and statistical comparisons of CD8 + GM-CSF+ cells in the groups of PBS and M.smithii . Data are shown as mean ± SEM, two-tailed Student’s t-test was used to perform statistical comparisons between groups, n = 8 in each independent experiment, each experiment was repeated three times

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 h at room temperature, then probed overnight at 4 °C with the following primary antibodies (1:2000 dilution): TNF-α (ab215188), IL-17 (13838 S), GM-CSF (505504), and IL-22 (A6212) from respective suppliers (Abcam; CST; BioLegend; ABclonal).

Techniques: Flow Cytometry, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Itaconate Modulates Neutrophil Homeostasis to Ameliorate Airway Inflammation in Diesel Exhaust Particles-exacerbated Asthma via Inhibiting NETs Formation

doi: 10.7150/ijbs.124927

Figure Lengend Snippet: DEP exposure results in corticosteroid-resistant allergic airway inflammation. (A) HDM-induced asthma and HDM/DEP-induced corticosteroid-resistant asthma mouse models were established. DEX was administrated intraperitoneally. (B) Representative H&E and PAS staining pictures of lung tissues (n = 5 mice per group). Scale bar, 100 μm. (C) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 5 mice per group). (D and E) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 5 mice per group). (F) Levels of inflammatory cytokines in BALF measured by ELISA (n = 5 mice per group). (G) For neutrophil depletion in HDM/DEP-induced corticosteroid-resistant asthma, 0.5 mg of anti-Ly6G antibody or the isotype control antibody was administered intraperitoneally on days 10-13, 1 hour prior to each challenge. (H) Representative H&E and PAS staining pictures of lung tissues (n = 6 mice per group). Scale bar, 100 μm. (I) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 6 mice per group). (J and K) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 6 mice per group). (L) Levels of inflammatory cytokines in BALF measured by ELISA (n = 4 mice per group). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. i.t., intratracheally; i.p., intraperitoneally; HDM, house dust mite; DEP, diesel exhaust particles; DEX, dexamethasone; BALF, bronchoalveolar lavage fluid.

Article Snippet: Anti-IL-17A monoclonal antibody (BioXcell, USA) or the isotype control antibody was administered intraperitoneally at the dose of 100 μg/mouse on days 10-13, 1 hour prior to each challenge.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Biological Sciences

Article Title: Itaconate Modulates Neutrophil Homeostasis to Ameliorate Airway Inflammation in Diesel Exhaust Particles-exacerbated Asthma via Inhibiting NETs Formation

doi: 10.7150/ijbs.124927

Figure Lengend Snippet: ACOD1/ITA alleviates airway inflammation in corticosteroid-resistant asthma. (A) WT or Acod1 -/- mice were sensitized with HDM, followed by challenge with HDM and DEP. (B) Representative H&E and PAS staining pictures of lung tissues (n = 6 mice per group). Scale bar, 100 μm. (C) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 6 mice per group). (D and E) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 5 mice per group). (F) Levels of inflammatory cytokines in BALF measured by using a cytometric bead array (n = 6 mice per group). (G) Levels of inflammatory cytokines in lung homogenates measured by using a cytometric bead array (n = 6 mice per group). (H) HDM/DEP-exposed asthmatic mice were established. DEX or ITA was administered 1 hour before HDM and DEP challenge. (I) Representative H&E and PAS staining pictures of lung tissues (n = 6 mice per group). Scale bar, 100 μm. (J) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 6 mice per group). (K and L) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 6 mice per group). (M) Levels of inflammatory cytokines in BALF measured by using a cytometric bead array (n = 6 mice per group). (N) Levels of inflammatory cytokines in lung homogenates measured by using a cytometric bead array (n = 6 mice per group). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. i.t., intratracheally; i.p., intraperitoneally; i.n. intranasally; HDM, house dust mite; DEP, diesel exhaust particles; ACOD1, aconitate decarboxylase 1; DEX, dexamethasone; ITA, itaconate; BALF, bronchoalveolar lavage fluid.

Article Snippet: Anti-IL-17A monoclonal antibody (BioXcell, USA) or the isotype control antibody was administered intraperitoneally at the dose of 100 μg/mouse on days 10-13, 1 hour prior to each challenge.

Techniques: Staining, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Itaconate Modulates Neutrophil Homeostasis to Ameliorate Airway Inflammation in Diesel Exhaust Particles-exacerbated Asthma via Inhibiting NETs Formation

doi: 10.7150/ijbs.124927

Figure Lengend Snippet: ACOD1/ITA inhibits DEP-induced inflammatory responses in neutrophils and Th17 cell differentiation in vitro . (A) Real-time qPCR analysis of inflammatory cytokines ( Il-1β , Tnf-α , and Il-6 ) in BMDNs stimulated with DEP and treated with ITA. ** P < 0.01, *** P < 0.001, compared with the control group. # P < 0.05, ## P < 0.01, ### P < 0.001, compared with the DEP group. (B) Real-time qPCR analysis of inflammatory cytokines ( Il-1β , Tnf-α , and Il-6 ) in DEP-stimulated BMDNs from WT or Acod1 -/- mice. (C) Cellchat analysis of intercellular communication networks from scRNA-seq data. (D) Identification of T cell clusters. (E) The intercellular communication between neutrophils and Th1, Th2, Th17, and Treg cells. (F) Naive CD4 + T cells from mouse spleen were co-cultured with BMDNs in U-bottom 96-well plates in Th17 differentiation condition. After 6 hours, ITA (6 mM) was added to the appropriate wells. One hour later, cells were stimulated with DEP (100 μg/mL). After 72-96 h, cells were harvested for flow cytometry. (G and H) The percentage of IL-17A + (Th17) cells in CD4 + T cells measured using flow cytometry to detect the effect of ITA on Th17 cell differentiation under different stimulation conditions. (I and J) The percentage of IL-17A + (Th17) cells in CD4 + T cells measured using flow cytometry under the co-culture system of naive CD4 + T cells from WT mice and BMDNs from WT or Acod1 -/- mice. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. DEP, diesel exhaust particles; BMDNs, bone marrow-derived neutrophils; ITA, itaconate; NEU, neutrophils; ACOD1, aconitate decarboxylase 1.

Article Snippet: Anti-IL-17A monoclonal antibody (BioXcell, USA) or the isotype control antibody was administered intraperitoneally at the dose of 100 μg/mouse on days 10-13, 1 hour prior to each challenge.

Techniques: Cell Differentiation, In Vitro, Control, Cell Culture, Flow Cytometry, Co-Culture Assay, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: Itaconate Modulates Neutrophil Homeostasis to Ameliorate Airway Inflammation in Diesel Exhaust Particles-exacerbated Asthma via Inhibiting NETs Formation

doi: 10.7150/ijbs.124927

Figure Lengend Snippet: ACOD1/ITA inhibits the formation of NETs to attenuate airway inflammation in corticosteroid-resistant asthma. (A) The concentration of dsDNA in BMDNs stimulated with DEP and treated with ITA. (B) Representative immunofluorescence staining of CitH3 and MPO in BMDNs stimulated with DEP and treated with ITA. Scale bar, 25 μm. (C) The concentration of dsDNA in BALF of HDM/DEP-induced asthma with or without treatment of ITA. (D) Western blot analysis of CitH3 in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. (E) Representative immunofluorescence staining of CitH3 and MPO in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. The arrows indicate the colocalization of CitH3 and MPO. (F) The concentration of dsDNA in BALF of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (G) Western blot analysis of CitH3 in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (H) Representative immunofluorescence staining of CitH3 and MPO in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. The arrows indicate the colocalization of CitH3 and MPO. (I) Western blot analysis of PAD4 in the lung tissues of HDM/DEP-induced asthma with or without treatment of ITA. (J) Immunohistochemical staining of PAD4 in lung tissue of HDM/DEP mice and treated with or without ITA. Scale bar, 20μm. (K) Western blot analysis of PAD4 in the lung tissues of HDM/DEP-induced asthma in WT or Acod1 -/- mice. (L) Immunohistochemical staining of PAD4 in lung tissue of HDM/DEP mice in WT or Acod1 -/- mice. Scale bar, 20μm. (M) Human blood samples were collected. Plasma, PBMCs, and neutrophils were isolated for further study. (N) The concentration of dsDNA in supernatant of PBNs stimulated with DEP for different time points and treated with ITA. *** P < 0.001, compared with the control group. (O) The concentration of dsDNA in plasma of healthy control, mild-to-moderate asthma patients, and severe asthma patients. (P) Correlation analysis of dsDNA and lung function index FEV 1 %pred, FEV 1 , and FEV 1 /FVC. (Q) HDM/DEP-induced asthmatic mice were established. NETs inhibitor DNase I was administered 1 hour before HDM and DEP challenge. (R) Representative H&E and PAS staining pictures of lung tissues (n = 5 mice per group). Scale bar, 100 μm. (S) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 5 mice per group). (T-U) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 4-5 mice per group). (V) Levels of inflammatory cytokines in BALF measured by using a cytometric bead array (n = 5 mice per group). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. CitH3, citrulline histone H3; dsDNA, double-stranded DNA; i.t., intratracheally; i.p., intraperitoneally; HDM, house dust mite; DEP, diesel exhaust particles; BALF, bronchoalveolar lavage fluid; BMDNs, bone marrow-derived neutrophils; ITA, itaconate; ACOD1, aconitate decarboxylase 1; PBMCs, peripheral blood mononuclear cells; PBNs, peripheral blood neutrophils; PAD4, peptidylarginine deiminase 4; NETs, neutrophil extracellular traps.

Article Snippet: Anti-IL-17A monoclonal antibody (BioXcell, USA) or the isotype control antibody was administered intraperitoneally at the dose of 100 μg/mouse on days 10-13, 1 hour prior to each challenge.

Techniques: Concentration Assay, Immunofluorescence, Staining, Western Blot, Immunohistochemical staining, Clinical Proteomics, Isolation, Control, Flow Cytometry, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: Itaconate Modulates Neutrophil Homeostasis to Ameliorate Airway Inflammation in Diesel Exhaust Particles-exacerbated Asthma via Inhibiting NETs Formation

doi: 10.7150/ijbs.124927

Figure Lengend Snippet: ITA inhibits Th17 cell differentiation by suppressing the formation of NETs in corticosteroid-resistant asthma. (A) Neutrophils were treated with PMA and the NETs were collected to stimulate naive CD4 + T cell in vitro . (B) Naive CD4 + T cells were isolated from mouse spleens and cultured under Th17 differentiation conditions. The cells were stimulated with NETs and treated with ITA. Flow cytometry was used to detect Th17 cell differentiation. (C) Naive CD4 + T cells were isolated from PBMCs and cultured under Th17 differentiation conditions. The cells were stimulated with NETs and treated with ITA. Flow cytometry was used to assess Th17 cell differentiation. (D) Representative H&E and PAS staining pictures of lung tissues (n = 5 mice per group). Scale bar, 100 μm. (E) Total cell, neutrophil, and eosinophil counts in BALF measured by flow cytometry (n = 5 mice per group). (F and G) The percentage of IL-4 + (Th2) and IL-17A + (Th17) cells in CD4 + T cells (gated on Live, CD45 + CD3 + CD4 + ) measured using flow cytometry (n = 5 mice per group). (H) Levels of inflammatory cytokines in BALF measured by using a cytometric bead array (n = 5 mice per group). (I) Levels of inflammatory cytokines in lung homogenates measured by using a cytometric bead array (n = 5 mice per group). Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. PMA, phorbol 12-myristate 13-acetate; HDM, house dust mite; DEP, diesel exhaust particles; ACOD1, aconitate decarboxylase 1; BALF, bronchoalveolar lavage fluid; NETs, neutrophil extracellular traps; ITA, itaconate.

Article Snippet: Anti-IL-17A monoclonal antibody (BioXcell, USA) or the isotype control antibody was administered intraperitoneally at the dose of 100 μg/mouse on days 10-13, 1 hour prior to each challenge.

Techniques: Cell Differentiation, In Vitro, Isolation, Cell Culture, Flow Cytometry, Staining