Journal: JCI Insight

Article Title: IFN- γ –induced trained immunity enhances killing of priority pathogens in healthy and genetically vulnerable individuals

doi: 10.1172/jci.insight.195866

Figure Lengend Snippet: Enriched monocytes were left untrained (UT; white) or trained with IFN-γ (10 ng/mL; gray) for 24 hours. Cells were differentiated into MDM. ( A ) Schematic of training assay. ( B – G ) Expression of HLA-DR ( B ), CD14 ( C ), CD40 ( D ), CD80 ( E ), H3K27ac ( F ), or H3K4me3 ( G ) on unstimulated MDM on day 7 measured by flow cytometry. MFI, median fluorescence intensity. ( H and I ) Expression (relative to untrained) of NFKB1 ( H ) or IRF1 ( I ) in unstimulated MDM on day 7 measured by qPCR. ( J – O ) On day 6, MDM were stimulated with LPS (10 ng/mL) or irradiated M.tb (10 μg/mL) for 24 hours, and TNF ( J ), IL-6 ( K ), IL-1β ( L ), IL-10 ( M ), CXCL1 ( N ), or MIP-1α ( O ) was measured by ELISA. Each dot represents an individual donor, n = 6 ( B – E , H , and I ), n = 5 ( F and G ), or n = 7 ( J – O ), with paired data joined by a line. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 determined using a paired t test ( B – I ) or a 2-way ANOVA with Šidák’s multiple-comparison test ( J – O ).

Article Snippet: The concentrations of IL-1β (BioLegend, 437016), IL-10 (BioLegend, 430604), IL-6 (BioLegend, 430516), IFN-γ (BioLegend, 430116), TNF (Invitrogen), MIP-1α (R&D Systems, DY270), and CXCL1 (R&D Systems, DY275) were determined by ELISA following each manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Fluorescence, Irradiation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: JCI Insight

Article Title: IFN- γ –induced trained immunity enhances killing of priority pathogens in healthy and genetically vulnerable individuals

doi: 10.1172/jci.insight.195866

Figure Lengend Snippet: Enriched monocytes were left untrained (UT; white) or trained with IFN-γ (10 ng/mL; gray) for 24 hours. Cells were differentiated into MDM. On day 7, MDM were infected with M . tuberculosis (H37Rv; MOI 1–5) ( A and B ), stimulated with irradiated M.tb (10 μg/mL) ( G ), or infected with S . aureus (USA300; MOI 100:1) ( H – P ) for the indicated times. ( A and B ) Bacterial M . tuberculosis burden within MDM 3 and 48 hours after infection. ( C – F ) Relative expression (to untrained) of CYBA ( C ), CYBB ( D ), NCF1 ( E ), or NCF2 ( F ) in uninfected MDM on day 7 (qPCR). ( G ) MDM were stimulated with irradiated M.tb for 4 hours, and ROS (DHR123; relative to unstimulated MDM) was measured (flow cytometry). ( H – K ) TNF ( H ), IL-6 ( I ), IL-1β ( J ), or IL-10 ( K ) (ELISA) 24 hours after S . aureus infection. ( L ) MDM infected with CFSE-labeled S . aureus (%). ( M ) Intracellular bacterial S . aureus burden within MDM 1, 4, or 24 hours after gentamicin. ( N ) Spearman’s r correlation matrix: correlation of reduced CFU with IL-1β, TNF, IL-6, IL-10, CXCL1, or MIP-1α production. ( O ) MDM were infected with S . aureus alone or in the presence of NAC (10 mM) for 4 hours, and ROS was measured (flow cytometry). ( P ) Fold change in CFU/mL of untrained or IFN-γ–trained (stripes) MDM infected with S . aureus 1, 4, or 24 hours after gentamicin in healthy controls (HC; white) versus MDM from a patient with chronic granulomatous disease (CGD; green). Each dot represents an individual donor, n = 4 ( A – G ), n = 7 ( H – K , M , and N ), n = 5 ( L ), n = 6 ( O ), or HC n = 7, CGD n = 1 ( P ). Data are graphed as paired data joined by a line or the mean value ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, paired t test ( A – L ) or 2-way ANOVA with Šidák’s multiple-comparison test ( M ), uncorrected Fisher’s LSD test ( O ), or Tukey’s multiple-comparison test ( P ).

Article Snippet: The concentrations of IL-1β (BioLegend, 437016), IL-10 (BioLegend, 430604), IL-6 (BioLegend, 430516), IFN-γ (BioLegend, 430116), TNF (Invitrogen), MIP-1α (R&D Systems, DY270), and CXCL1 (R&D Systems, DY275) were determined by ELISA following each manufacturer’s instructions.

Techniques: Infection, Irradiation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Labeling, Comparison