Fig 2: Silencing of IL22RA2 in immature moDCs reduces GRP78 expression. (A) CD14+ monocytes were stained with DAPI (blue), trans-GOLGI (red) and IL-22BP (green, IL-22BP Ab 4). (B) Overall (best coverage, BC) and isoform-specific IL22RA2 expression levels in classical, intermediate and non-classical monocyte subpopulations (top) from four healthy donors (average ± SEM). Percentages of IL-22BP+ monocytes found in each subpopulation by immunofluorescence microscopy (bottom) (mean ± SEM; n = 4; *p < 0.05, Kruskal-Wallis test for comparison of differences in IL22RA2 mRNA levels or IL-22BP+ counts in the three subpopulations). (C) IL22RA2 expression levels in classical and intermediate immature moDCs at day 6 of cultivation. moDCs derived from non-classical monocytes showed high levels of cell death and had not experienced any changes in the shape of the cells. High Ct values of HK gene HPRT1 in qPCR compromised correct interpretation of IL22RA2 levels in this subgroup, which was therefore excluded from the graph. (D) Fold change relative to non-targeting siRNA control of IL22RA2 expression in CD14+ monocyte.-derived immature DCs on day 5 of cultivation following IL22RA2 silencing with Viromer Green at two concentrations of siRNA. (E) Silencing as described in (D) was not variant specific and both IL22RA2v1 and v2 were silenced to similar extent. (F) Scheme comparing efficiencies of four distinct silencing strategies on suppression of IL22RA2 mRNA (qPCR) and intracellular IL-22BP (ELISA) in immature CD14+ moDCs using 100 nM IL22RA2 siRNA or non-targeting siRNA in combination with Viromer Green. S, silencing, H, harvest. Bar diagrams represent fold change relative to non-targeting siRNA. (G) Effect of IL22RA2 silencing on GRP78 and ERp44 mRNA levels in CD14+ monocyte-derived immature DCs from healthy donors using silencing strategy B in (F). CD14+ monocytes were cultivated in DM medium in the absence or presence of AM580 starting on day 0 up to the day of harvest. Average of fold change (±SEM) relative to non-silencing control over 6 (control) and 5 (AM580) independent measurements relative to the housekeeping gene HPRT1. *p < 0.03 by Wilcoxon test.

Fig 3: CRISPR-Cas9-mediated gene knockout preserves key aspects of differentiation and function in targeted myeloid cells(A) Principal-component analysis of RNA sequencing (RNA-seq) from the indicated cell types.(B) Normalized transcript abundance (Z score) for selected markers of MDM or MDDC differentiation (Lehtonen et al., 2007).(C) Dendrogram of hierarchical clustering of the data in (B) by Euclidean distance.(D and E) Among cells subjected to CRISPR-Cas9 RNP nucleofection, cell surface protein levels of CD16, CD14, and CD206 were compared between the cells that bear the desired ß2 m knockout (pink) and those that do not (teal) by flow cytometry after 7 days of MDM differentiation. (D) shows gating, while (E) shows the expression of the indicated markers.(F) Representative images of unperturbed (left) and RNP-nucleofected (right) MDMs infected with GFP-expressing M. tuberculosis (Mtb-GFP) show that CRISPR-Cas9-targeted cells remain competent to phagocytose living pathogens. Top, membrane staining with Cell Mask Far Red; CENTER, Mtb-GFP; bottom, composite. Scale bars represent 100 µm.See also Figure S2.

Fig 4: Macrophage surface marker expression of osteomacs isolated by MACS®. The immunofluorescence (IF) images show staining for the macrophage markers CD14, CD45 and markers for M1-like (CD86 and CD68) and M2-like macrophage polarization (CD163, CD209) together with nuclei staining by using DAPI (blue). Arrows indicate thread-like configuration of some osteomacs. Shown here are representative IF images. IF staining was performed with n = 2 donors.

Fig 5: Gating strategy for cell sorting of osteomacs (CD45+ ALP− CD14+). After Gating of single cells, using the width of the forward and side-scatter signal (FSC-W, SSC-W), living cells were selected (Zombie-Aqua-). CD45+ ALP− cells were then selected to identify the osteomac population by CD14+. Those cells were selected for sorting. Shown here is a representative FACS run. This FACS protocol was established with n = 3 donors.