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. 2022 Apr 28;18(4):e1010407. doi: 10.1371/journal.ppat.1010407

Fig 4. Chitinase induces NOS and ROS generation in phagocytic cells and inhibits antigen presentation.

Fig 4

(A) % invasion and (B) fold proliferation of STM WT and STM ΔchiA strains, and (C) % invasion and (D) fold proliferation of STY WT, STY ΔchiA, STY ΔchiA:chiA and STY ΔchiA:pQE60 strains in U937 derived monocytes by gentamicin protection assay. (N = 3, n = 3). (E) % invasion and (F) fold proliferation of STM WT and STM ΔchiA strains, and (G) % invasion and (H) fold proliferation of STY WT, STY ΔchiA, STY ΔchiA:chiA and STY ΔchiA:pQE60 strains in BMDCs by gentamicin protection assay. (N = 3, n = 3). (I) Extracellular NO was estimated by Greiss assay from spent media obtained from STM WT, STM ΔchiA, STY WT, STY ΔchiA and STY ΔchiA:chiA infected BMDCs. (UI- Uninfected). (N = 3, n = 3). Two-way ANOVA was used to analyze the data. Intracellular survival of (J) STM WT and STM ΔchiA and (K) STY WT and STY ΔchiA strains was calculated in WT (NOS2+/+) and NOS2-/- BMDCs by gentamicin protection assay. (N = 3, n = 3). (L) Representative flow cytometry plot for ROS estimation by DCFDA assay from STM WT, STM ΔchiA, STY WT, STY ΔchiA and STY ΔchiA:chiA infected and lipopolysaccharide (LPS; control) treated PMs. (UI US- Unstained and uninfected, UI- Uninfected). (M) 3H thymidine incorporation assay to assess CD8+ T cell proliferation 20 hpi with STM WT, STM ΔchiA, STY WT, STY ΔchiA and STY ΔchiA:chiA (UI- Uninfected, OVA- Ovalbumin, ConA- Concanavalin A). (N = 3, n = 6). (N) Quantification of the MHC-II density per unit length of the cell membrane of STM WT, STM ΔchiA, STY WT, STY ΔchiA and STY ΔchiA:chiA infected PMs after the indicated time (UI- Uninfected). (N = 2). (O) Representative flow cytometry plot showing surface MHC-II level on PMs infected with STM WT and STM ΔchiA for 20 hours (UI US- Unstained and uninfected, UI- Uninfected). Unpaired Student’s t test was used to analyze the data for A, B, E, F, J, K and one-way ANOVA was used to analyze the data for C, D, G, H, M.