Fig 2: Osteoclasts are activated by IgA immune complexes. (A) Expression of CD16, CD32, CD64 and CD89 on CD14 negative OCs (upper panels; dark grey histograms) and remaining CD14+ cells (middle panels; light grey histograms) compared to no stain (lower panels; dotted black line histograms). (B) CD89 expression on CD14-aVß3+ OCs. [White (DAPI) = nuclei, blue = CD14, red = aVß3, green = CD89]. (C, D) IL-8 (C) and IL-6 (D) release by OCs stimulated with immune complexes isolated from synovial fluid of RA patients. (E, F) IL-8 (E) and IL-6 (F) release by unstimulated OCs (white bar) and after stimulation with BSA- (light grey bar), IgA- (dark grey bar) or IgG- (black bar) coated beads. (G) Quantification of bone resorption by OCs in presence of M-CSF and RANK-L (unstim, white bar) and added cytokines IL-6 (25 ng/ml, light grey bar) or IL-8 (10 ng/ml, dark grey bar). Data was analyzed using one way ANOVA with Tukey post-hoc; *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001.

Fig 3: THP-1 cells increase CD14 expression during macrophage differentiation. a. THP-1 cells increase the expression of CD14 after treatment for 48 h with the monocyte to macrophage differentiation factor PMA (100 ng/ml) in comparison to the DMSO control. b. Increased MFI values for CD14 expression after treatment with the differentiation factor PMA (100 ng/ml) for 48 h. c. Design of the CRISPR/Cas9 screen in THP-1 cells. A whole-genome library of guide RNAs was built in lentivirus plasmids with a selection marker (resistance to puromycin). THP-1 cells were transduced with lentivirus containing the plasmid, afterwards a culture selection in the presence of puromycin was performed. The remaining cells were treated for 48 h with 100 ng/ml of PMA to induce the differentiation into macrophages or DMSO (undifferentiated cells). Cell sorting was performed based on CD14 expression. The guide RNAs present in either the CD14+ve or CD14-ve cells from both groups were analysed. n = 3 biological replicates for each experiment.

Fig 4: Results from the THP-1 screen partially translate into human primary MCSF macrophages. Effect of the compounds on cytokine production. a. Morphology of human monocytes differentiated with M-CSF (100 ng/ml) into macrophages in the presence of an inhibitor for MAP2K3 (100 nM), PRKCD (100 nM) or DMSO control (0.1%). b. Percentage of live, CD14+ve and CD16+ve cells in the cells differentiated in the presence of the different compounds. c. MFI peaks and fold change values for CD14 and CD16 after treatment with both compounds and DMSO control. d. sCD14 production of MDM differentiated in the presence of MAP2K3 (100 nM), PRKCD (100 nM) or DMSO control (0.1%) was measured by ELISA e. Cells were differentiated in the presence of the two compounds, after differentiation cells were stimulated with LPS (100 ng/ml) for 24 h and IL-1ß, IL-6, TNF levels were measured. Statistical significance was determined by unpaired t-test (p < 0.05). All error bars represent the SEM. n = 4 biological replicates for flowcytometry experiments and n = 3 biological replicates for the stimulation and ELISA experiments.

Fig 5: NLC drive expression of NTSR2-TrkB and pro-survival signaling in B-CLL cells. (a) Brightfield microscopy of co-cultured NLC (red arrows) and B-CLL cells (blue arrows). (b) Confocal microscopy analysis of CD14 (green) and CD68 (red) (left panel), and CD163 (green) and CD68 (red) (right panel), expression by NLC (n = 3). Nuclei are stained with DAPI. (c) Western blot analysis of CD68, CD163, and CD206 expression by NLC (n = 3). (d) Representative western blots showing expression of NTSR2, TrkB, p-Src, Src, and Bcl-2 by B-CLL cells isolated from two independent patients and cultured alone or with autologous NLC for 48 h. (e) Quantification of NTSR2 (n = 15), TrkB (n = 6), p-Src/Src (n = 17) and Bcl2 (n = 16) expression in independent patient samples. Data are presented as the mean ± SEM from at least three independent experiments (*p < 0.05 and **p < 0.01). Blots are cropped for clarity; full-length blots are shown in the Supplementary Fig. S7.