Fig 2: CD11c deficiency leads to uncontrolled immature neutrophil mobilization upon LPS challenge. Both WT and CD11c KO mice were IV injected with LPS. (A) BM neutrophils were analyzed by CyTOF, 6 hours after LPS injection. Shown are viSNE plots of 1 of 3 biological samples with the same pattern. The color indicates expression level of labeled marker. (B) Violin plot of CD11b, CD11c, Ki67, and cleaved caspase-3 expressions. (C) The number of preneutrophils and immature/mature neutrophils in the BM. (D) Cleaved caspase-3 and Ki67 expression of preneutrophils and immature/mature neutrophils. Left panel: representative flow cytometry picture; right panel: statistical analysis. (E) Cell number and CD11b expression of blood neutrophils at indicated time points after LPS injection (3-5 mice in each time point). For panels C and D, Student t test was used; *P < .05, **P < .01, ***P < .001. For panel E, we used two-way analysis of variance. **P < .01, ***P < .001. Experiments were repeated 3 times with a similar pattern. n.s., not significant.

Fig 3: Constitutive activation of CD11c enhances neutrophil maturation and functions. (A) Scheme showing the strategy for creating the constitutively active CD11c KI mice (CD11cI334G). (B) Rosetting assay confirming the constitutive activation of CD11c molecule in CD11cI334G mice. Magnesium/calcium ions (1 mM) nonactivating condition, 1 mM manganese ions activating condition. (C) Giemsa staining of BM neutrophils. (D) Total BM neutrophil count. (E) Ratios of preneutrophils and immature and mature neutrophils. (F) Cell number and CXCR2 expression of blood neutrophils. (G) Cell number and CXCR2 expression of splenic neutrophils. (H) ROS detection in immature and mature neutrophils from naive WT and CD11c I334G mice. Left: representative flow cytometry overlay plot; right: data from 3 independent experiments. Cells from 3 to 4 mice were pooled together as 1 biological sample. (I) Neutrophil counts in the blood, the spleen, and the BM from WT and CD11cI334G mice, 6 hours after IV LPS stimulation. (J-K) WT and CD11c I334G mice were challenged intraperitoneally with E coli (1 × 108 colony-forming units) and sacrificed 6 hours later. (J) Giemsa staining of peritoneal neutrophils. (K) Bacterial loads of peritoneal cavity. *P < .05, **P < .01.

Fig 4: CD11c is expressed in neutrophils intracellularly. (A) Flow cytometry gating strategy identifying preneutrophils and immature and mature neutrophils in mouse BM. From the side scatter–A and forward scatter–A plot, live cells were selected (supplemental Figure 1A). Here, Lin includes CD3e, B220, and NK1.1. (B) Surface and intracellular CD11c expression detected by flow cytometry. Shown are representative data of at least 5 independent experiments with the same pattern. (C) Fluorescence microscopic examination (×60) of BM neutrophils. MPO, red; 4',6-diamidino-2-phenylindole, blue; CD11c, green. Images show representative images of 3 independent experiments. c, conventional; p, plasmacytoid.

Fig 5: IQGAP1 interacts with CD11c. (A) Predicted interaction of top-hit proteins pulled down by immunoprecipitation (supplemental Figure 13) by STRING software. (B) Immunoprecipitation assay. Cell lysates of HL-60 cells were incubated with anti-human CD11c mAb (clone: CBR p150 2c1) for immunoprecipitation followed by blotting with anti-IQGAP-1 antibody or anti-CD11c polyclonal antibody. Cell lysates of 32D cl3 cells were incubated with anti-mouse CD11c mAb (clone: N417) for immunoprecipitation, followed by blotting with anti-IQGAP-1 antibody. What is shown is representative of 3 independent experiments. (C-E) CD11b (C), ROS (D), and phagocytosis (E) analyzed on HL-60 cells at 4 days under differentiation toward neutrophils. Three independent cell pools were analyzed in both WT and IQGAP1-KO groups. Shown are 1 of 3 independent experiments with the same pattern. (F) Apoptosis analysis by detecting cleaved caspase-3 at day 4 under differentiation toward neutrophils. HL-60 cells were treated with medium (control) or LPS (10 µg/mL) for 6 hours. Shown are 1 of 3 independent experiments with the same pattern. (G) Scheme of the role of CD11c-IQGAP1 interaction in neutrophil maturation. mAb, monoclonal antibody.