Volume 21, Issue 1, Pages 126-140 (October 2017) M. tuberculosis-Initiated Human Mannose Receptor Signaling Regulates Macrophage Recognition and Vesicle Trafficking by FcRγ-Chain, Grb2, and SHP-1  Murugesan V.S. Rajaram, Eusondia Arnett, Abul K. Azad, Evelyn Guirado, Bin Ni, Abigail D. Gerberick, Li-Zhen He, Tibor Keler, Lawrence J. Thomas, William P. Lafuse, Larry S. Schlesinger  Cell Reports  Volume 21, Issue 1, Pages 126-140 (October 2017) DOI: 10.1016/j.celrep.2017.09.034 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 CTL Expression by Human Macrophages (A–E) Day 5 PBMCs were immunostained for (A) MR/CD206, (B) Dectin-1, (C) Mincle, (D) Dectin-2, or (E) DC-SIGN/CD209. hMDMs were gated by forward and side scatter, and surface expression on non-permeabilized hMDMs was determined by flow cytometry. Flow cytometry data were analyzed using FlowJo software. (F) The specific mean fluorescence intensity (MFI) (mean ± SEM, n = 3) was calculated by subtracting the MFI for the respective isotype control Abs (MFIs for isotype Abs were as follows: CD206, 363; Dectin-1, 190; Mincle, 314; Dectin-2, 72.1; DC-SIGN, 190). (G and H) CTL expression in non-permeabilized MDMs was examined by confocal microscopy (G) and qRT-PCR (H). The data shown in (H) are from a representative experiment (mean ± SD) of 3 independent experiments using 3 different donors. Scale bar, 10 μm. Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 MR-Interacting Proteins in Human Macrophages upon Activation (A and B) hMDMs were incubated with M.tb (MOI 20:1) by synchronized phagocytosis (A) or ManLAM (5 μg/mL) (B). Cells were lysed at the indicated times, and cell lysates were analyzed by WB using a phosphor-tyrosine Ab (4G10) and re-probed with anti-MR Ab. The WBs shown are from representative experiments (n = 3, using 3 different donors). The graphs at the bottom show cumulative data of band intensities of phosphorylated MR (normalized to total MR) from the 3 donors (mean ± SEM). (C) hMDMs were incubated with M.tb (MOI 20:1), and after the indicated times (min), cells were lysed and immunoprecipitated with phosphor-tyrosine Ab (4G10) followed by immunoblot with anti-MR Ab. The arrow shows the presence of MR in the immunoprecipitate (n = 2). (D) Synthetic peptides corresponding to 28 amino acids of the MR cytoplasmic tail with (MRCTP, peptide 2) or without (MRCT, peptide 1) phosphorylation of the tyrosine residue at the 18th position were N-terminally tagged with biotin. (E) hMDM lysates were mixed with MR peptides, pulled down with streptavidin beads, and separated by gradient SDS-PAGE. The protein bands were visualized with Gel Code Blue. The boxes depict the locations where the lanes were cut and analyzed by mass spectrometry. (F) The identified proteins were compared between the MRCT and MRCTP groups. (G) Selected proteins associated with human MR cytoplasmic peptides. (H) hMDM lysates were mixed with MR peptides and pulled down with streptavidin beads, and binding of Grb2 (molecular weight [MW], 25.3 kDa), SHP-1 (MW, 67.9 kDa), and FcRγ-chain (MW, 9.7 kDa) was determined by WB (n = 3). See also Figure S1. Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 FcRγ-Chain Is Required for MR Cell Surface Expression on Human Macrophages (A) hMDM lysates were incubated with anti FcRγ-chain Ab or isotype control IgG Ab and immunoprecipitated, and proteins were resolved by gradient SDS-PAGE and then immunoblotted with human MR Ab followed by anti-FcRγ-chain Ab. The data shown are representative of 3 experiments. (B) Scramble control or FcRγ-chain siRNA-transfected hMDM monolayers were fixed with/without permeabilization and stained with anti-MR Ab or isotype control Ab. Representative images (n = 2) are shown (top). Scale bar, 10 μm. MR surface expression and total MR levels were determined by MFI (bottom). (C) Transfected hMDMs were incubated with mCherry-M.tb (MOI 10:1) and fixed, and M.tb association was examined by fluorescence microscopy. Approximately 100 macrophages were counted for each experiment. (D and E) Control or MR siRNA-transfected hMDMs were incubated with (D) mCherry-M.tb (MOI 10:1) for 2 hr or (E) with mannose BSA beads (MOI 20:1) for 1 hr. Cells were fixed, and M.tb association or percent bead uptake (uptake index) was determined by counting 100 and 150 cells, respectively. The graphs show cumulative data (mean ± SEM) from two independent donors. ∗p < 0.05, ∗∗p < 0.005. MR knockdown was confirmed in each experiment by WB using MR Ab followed by actin Ab. See also Figures S2C and S2G. Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Grb2 Interacts with the MR and Initiates Rac-1/Cdc42 Activation in hMDMs (A) Grb2 binding motif in the human MR. (B) hMDMs were infected with M.tb (MOI 20:1) for the indicated times or left uninfected (R). The cells were lysed, and cell lysates were immunoprecipitated with Grb2 or isotype control IgG Ab; proteins were visualized by WB using anti-MR Ab and re-probed with Grb2 Ab. The arrows indicate the MR (top) and Grb2 (bottom). (C and D) hMDMs were incubated with mannose BSA beads (C and D) or BSA beads (D) (MOI 20:1) for the indicated times. The amount of Rac-1 or Cdc42 bound to PAK (indicative of active GTP-Rac-1/Cdc42) was determined by WB (n = 2). (E) Grb2 siRNA- or control-transfected hMDMs were incubated with mannose BSA beads (MOI 20:1) for the indicated times, and Rac-1 activity was determined as described above. The WB shown is representative of 2 independent experiments using 2 different donors. The graph (bottom) shows cumulative data from n = 2 (mean ± SEM; ∗p < 0.05; ∗∗p < 0.005). (F) Bead uptake in transfected cells was assessed at 2 hr by dividing the number of cells that took up beads by total cells counted in each test group (250 total cells). Shown are cumulative data from 2 experiments (mean ± SEM). ∗∗p < 0.005. (G) Transfected hMDMs were incubated with M.tb-Lux (MOI 5:1), and luciferase activity was determined. Shown are cumulative data from 2 experiments (mean ± SEM). ∗∗p < 0.005. RLU, relative luciferase units. See also Figures S2 and S3. Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 MR Regulates the Recruitment and Activation of SHP-1 in hMDMs (A) hMDMs underwent synchronized phagocytosis with mCherry-M.tb (MOI 20:1) for the indicated times, and SHP-1 phosphorylation was determined using phospho-specific SHP-1 Ab followed by total SHP-1 Ab. Shown are a representative blot (top) and band intensities of cumulative data from n = 3 (bottom, mean ± SEM). (B) hMDMs were infected with mCherry-M.tb (MOI 10:1) and stained with SHP-1 Ab (which recognizes total SHP-1) or rabbit isotype control Ab. Nuclei were stained with DAPI. (C) Twenty M.tb-containing phagosomes were counted for SHP-1 co-localization and calculated as percent SHP-1 co-localization (mean ± SD). (D) Control or MR siRNA-transfected hMDMs were infected with mCherry-M.tb (MOI 10:1) and stained with SHP-1 Ab. (E) Percent SHP-1 co-localization with M.tb was determined for the experiment shown in (D) (mean ± SD). (F) Phosphatase activity of SHP-1 in M.tb-infected hMDMs was determined following synchronized phagocytosis with mCherry-M.tb (MOI 20:1) (mean ± SD). (G) Transfected hMDMs were incubated with mCherry-M.tb (MOI 10:1). The cells were lysed, and SHP-1 phosphatase activity was determined (mean ± SD). (B–G) show representative of 3 independent experiments. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005. Scale bars, 10 μm (isotype) and 20 μm (SHP-1 labeling). See also Figures S2 and S4. Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 MR Regulates P-L Fusion through SHP-1 in hMDMs (A) hMDMs were pre-treated with the SHP-1 inhibitor SSG (10 μg/mL) or DMSO for 1 hr and infected with mCherry-M.tb (MOI 10:1). Scale bars, 15 μm (left) and 20 μm (right). White arrows show bacteria not co-localized with LAMP-1. (B) Percent P-L fusion was determined by localization of LAMP-1 with mCherry-M.tb. (C) Day 5 PBMCs were transfected with control siRNA or SHP-1 siRNA, and after 48 hr of incubation, the transfected hMDMs were infected with mCherry-M.tb (MOI 5:1), fixed, permeabilized, and stained with LAMP-1 Ab. White arrows show bacteria not co-localized with LAMP-1. Scale bar, 25 μm. (D) Percent LAMP-1 co-localization with mCherry M.tb. (E and F) Vehicle control or SHP-1 inhibitor-treated hMDMs (E) or control or SHP-1 siRNA-transfected hMDMs (F) were infected with M.tb-Lux (MOI 5:1), and M.tb growth was determined by luciferase assay. Shown are representative images and graphs of 3 independent experiments. (B) and (D)–(F) shown mean ± SD. ∗p < 0.05, ∗∗p < 0.005. See also Figure S2. Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 SHP-1 Regulates Class III PI3K Activity in Human Macrophages (A) SHP-1 binding motif on hVps15 and hVps34. (B) hMDMs underwent synchronized phagocytosis with mCherry-M.tb (MOI 20:1) for the indicated times, and cell lysates were immunoprecipitated with SHP-1 or control IgG Ab, followed by WB with Vps15 (top), Vps34 (center), and SHP-1 Abs (bottom). The WBs shown are representative of 2 independent experiments. (C) hMDMs underwent synchronized phagocytosis with mCherry-M.tb (MOI 20:1) for the indicated times. Cell lysates were immunoprecipitated with anti-hVps34 or isotype IgG control Ab, and the immunoprecipitates were used to determine PI3K activity. (D) Transfected hMDMs were infected with M.tb (MOI 20:1), and PI3K activity was determined. (E and F) hMDMs were transfected with p40PX-EGFP, and after 16 hr, cells were pre-treated with the SHP-1 inhibitor SSG (10 μg/mL) or DMSO for 1 hr and then incubated with mannose BSA beads (E) or mCherry-M.tb (MOI 10:1) (F). (G and H) Percent co-localization of p40PX-EGFP with beads (G) or M.tb (H) was determined from the images acquired using confocal microscopy (∼20 images, representing ∼150 cells). Red arrows signify co-localization. White arrows signify no co-localization. Scale bars, 25 μm. The graphs shown in (C), (D), (G), and (H) are representative of 2 independent experiments (mean ± SD). ∗p < 0.05, ∗∗p < 0.005. See also Figure S2. Cell Reports 2017 21, 126-140DOI: (10.1016/j.celrep.2017.09.034) Copyright © 2017 The Author(s) Terms and Conditions