Activin a

Neutrophil migration assays were conducted using Pseudomonas aeruginosa grown in LB broth to an OD₆₀₀ of 0.25 and then washed and resuspended in EGM-2 cell culture media.
Activin A blocking experiments were carried out using 100 μg/mL of either Purified Mouse IgG2b, κ Isotype Ctrl Antibody (BioLegend, #401212), Ultra-LEAF^™ Purified anti-Activin A Antibody (BioLegend, # 693603), or 200 ng/mL recombinant human follistatin (BioLegend, #776004) supplemented into the EGM-2 used for the experiment.
The Activin A/Follistatin migration experiments were conducted using 200 ng/mL recombinant human Activin A (BioLegend, #592002) or 200 ng/mL recombinant human follistatin supplemented into the EGM-2 used in the experiment.
The Activin A addition experiments were conducted using 200 ng/mL recombinant human Activin A (BioLegend, #592002) supplemented into EGM-2 and added into the lumens.
Follistatin:Activin A ratio experiments were conducted using a combined 1 μg/mL of recombinant human follistatin (BioLegend, #776004), and recombinant human Activin A (BioLegend, #592002) supplemented into the EGM-2 used in the experiment.

1×10⁶ CD4⁺ T cells were isolated and activated in 24 well-plates pre-coated with 10 μg/ml anti-CD3 (145–2C11, BioXCell) and 10 μg/ml anti-CD28 mAb (37.51, BioXCell) in serum-free X-VIVO 20 medium (Lonza). For pathogenic-Th17 cell differentiation, 20 ng/ml IL-1β (Biolegend), 40 ng/ml IL-6 (Biolegend), 50 ng/ml IL-23 (Biolegend) and 20 µg/ml anti-IFN-γ (XMG1.2, BioXcell) were added to the culture. For TGF-β1+IL-6 induced Th17 cell differentiation, 1 ng/ml TGF-β1 (Biolegend), 40 ng/ml IL-6 (Biolegend) and 20 µg/ ml anti-IFN-γ (XMG1.2, BioXcell) were added to the culture. For Activin-A+IL-6 promoted Th17 cells, 30ng/ml Activin-A (Biolegend), 40 ng/ml IL-6 (Biolegend) and 20µg/ml anti-IFN-γ mAb (XMG1.2, BioXcell) were added to the culture. Anti-Activin-A (clone 69403, R&D), anti-TGF-β1 (1D11, BioXcell), Follistatin (R&D) and MEK inhibitor PD98059 (Selleckchem) were added when needed as indicated in the figure legends. To assess proliferation, isolated CD4⁺ T Cells were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, AnaSpec) for 5 minutes at the room temperature. Labelled T cells were washed with PBS twice and activated under various conditions as indicated in the figure legends. The T cell proliferation was assessed 72 hours post activation based on CFSE dilution by flow-cytometry.