Volume 36, Issue 5, Pages 807-820 (May 2012) Nitric Oxide Increases Susceptibility of Toll-like Receptor-Activated Macrophages to Spreading Listeria monocytogenes  Caroline Cole, Stacey Thomas, Holly Filak, Peter M. Henson, Laurel L. Lenz  Immunity  Volume 36, Issue 5, Pages 807-820 (May 2012) DOI: 10.1016/j.immuni.2012.03.011 Copyright © 2012 Elsevier Inc. Terms and Conditions

Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Analysis of Lm Spread by Flow Cytometry To measure spread, 2.5 × 105 unlabeled recipients were mixed with 2.5 × 104 CellTrace Far Red+ donors that were mock, ΔActA, or WT GFP-Lm infected. 15–18 hr after mixing, cells were collected and analyzed by flow cytometry. Bars indicate standard errors. (A) Flow cytometry identification of CellTrace Far Red+ donors and unlabeled recipients. In the top panel, donors that were ΔActA or WT GFP-Lm infected, but not mock infected, were GFP-Lm+. In the bottom panel, only recipients incubated with WT GFP-Lm-infected donors were GFP-Lm+. Treatment with cytochalasin B (CB) blocked actin polymerization and prevented spread from WT donors into recipients. (B) Quantification of spread into recipients that were mixed with mock, ΔActA, or WT infected donors alone (untreated) or given CB, n = 5. (C and D) Spread into recipients was dependent on (C) the donor to recipient ratio, n = 5, and (D) the time of incubation, n = 3. Histograms are representative of all experiments. Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 TLR Stimulation of Recipients Enhances Spread of Lm (A and B) Flow cytometry analysis for WT GFP-Lm+ recipients 15–18 hr after mixing 2.5 × 105 recipients with 2.5 × 104 WT GFP-Lm-infected donors. (A) Recipients were treated overnight with various concentrations of TLR ligands (LPS at 0.4, 4, and 40 ng/ml, Pam3CSK4 at 10, 100, and 1,000 ng/ml, poly(I:C) (PIC) at 40, 200, and 1,000 ng/ml, and CpG(−) and CpG(+) at 5 and 20 nm), n = 4. TLR stimulation enhanced spread into recipients compared to untreated (Control). (B) Recipients were treated overnight with lipoteichoic acid (LTA), peptidoglycan (PGN), or heat-killed (HK) Lm (MOI = 1), washed, and incubated with infected donors, n = 3. (C) To confirm the enhancement of spread by TLR ligands measured by flow cytometry, confluent monolayers of macrophages were infected at an MOI of 0.01, given LPS, washed, and overlayed with agar. 48 hr later, plaque size was measured from images of plates that had been counterstained with neutral red dye. The average size of plaques was increased by LPS treatment of macrophages, n = 3. (D) Macrophages from C57BL/6, Myd88−/−, or Ticam1−/− mice were used as recipients. Recipients were treated as in (A) with LPS (40 ng/ml), Pam3CSK4 (1,000 ng/ml), or PIC (1,000 ng/ml), washed, and incubated with infected donors, n = 3. (E) Time course analyses showed that LPS enhancement of spread occurred after 9 hr of coincubation, n = 3. (F–H) 2.5 × 104 mock, ΔActA-, or WT GFP-Lm-infected donors were added to 2.5 × 105 Control (−) or LPS-treated recipients, incubated for 15–18 hr, and collected for flow cytomtery. (F) LPS enhanced spread from WT but not ΔActA-infected donors, n = 7. (G and H) LPS also increased then numbers of bacteria in recipients as assessed by (G) the mean fluorescence intensity (MFI) for GFP in recipient cells, n = 6, and (H) the total Lm CFU in the culture, n = 4. Bars indicate standard errors. Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 TLR-Induced NO Enhances Lm Spread Flow cytometry analysis for WT GFP-Lm+ recipients and measurement of nitrite in the supernatant 15–18 hr after mixing 2.5 × 105 recipients with 2.5 × 104 WT GFP-Lm-infected donors. Bars indicate standard errors. (A) Spread of WT GFP-Lm from C57BL/6 (n = 4) or NOS2-deficient (Nos2−/−, n = 3) donors into Nos2−/− recipients was reduced compared to C57BL/6 recipients. Furthermore, the LPS enhancement did not occur in the absence of NOS2. (B) Nitrite concentrations in the supernatants, n = 3. (C and D) The NOS2 inhibitors (Lnil or 1400W) prevented the LPS enhancement of (C) spread (n = 4) and (D) nitrite production (n = 4) from C57BL/6 donors into C57BL/6 recipients. The NOS2 inhibitors had no effect on the spread or nitrite production from C57BL/6 donors into Nos2−/− recipients. (E and F) SNAP, a NO donor, increased (E) spread into Nos2−/− recipients from C57BL/6 donors (n = 4) and (F) the nitrite concentrations, n = 3. (G) Correlation between the percentage of GFP-Lm+ C57BL/6 recipients and the supernatant nitrite, n = 20. Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 NO Is Required for Enhanced Lm Spread but Not Primary Infection (A and B) Microscopy for ΔActA (top) or WT (bottom) GFP-Lm CD45.1− recipients 15 hr after plating 5 × 105 recipients with 2.5 × 104 WT GFP-Lm-infected CD45.1+ donors on coverslips and treating with gentamicin alone (untreated), LPS, LPS+1400W, or SNAP. Pictures of CD45.1+ donors (red) were analyzed for the number of unlabeled recipients that were infected with WT GFP-Lm (green) or uninfected. (A) Representative images of infection foci seen after each treatment. (B) The number of WT GFP-Lm+ recipient per donor (n = 41–47 per treatment) was calculated for each image and the total for each treatment was graphed. Microscopy confirms the flow cytometry results indicating that LPS and SNAP enhance spread and the requirement for NO in enhanced spread, n = 2. (C) The number of uninfected recipient per donor (n = 41–47 per treatment) was calculated for each image and the total for each treatment was graphed to verify that treatments did not affect viability or total cell numbers, n = 2. (D) The role of LPS was tested in primary Lm infection by plating macrophages on coverslips overnight alone or with LPS. Cells were infected the next day at an MOI of 1, washed at 1 hr, and treated with gentamicin to prevent growth of bacteria in the media. The number of colony forming units (CFU) was determined by plating lysates from coverslips collected at 1.5, 3, 6, and 9 hpi. For primary infection, LPS pretreatment suppressed the growth of Lm compared to untreated. Nos2−/− mice showed comparable infection to WT cells, n = 3. Bars indicate standard errors. Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 NO-Dependent Delay in Phagolysosome Fusion Increases Lm Escape from Secondary Vacuoles, thereby Enhancing Spread In Vitro and In Vivo (A and B) Flow cytometry analysis for WT GFP-Lm+ recipients and measurement of nitrite in the supernatant 15–18 hr after mixing 2.5 × 105 recipients with 2.5 × 104 WT GFP-Lm-infected donors. (A) Inhibiting phagosomal acidification with Concanamycin (Con), Bafilomycin (Baf), or ammonium chloride (AC) did not change untreated or LPS-enhanced secondary infection but did prevent the inhibition of spread by LPS+1400W, n = 6. (B) Inhibiting phagosomal acidification did not impact nitrite production, n = 6. (C–G) Flow cytometry analysis was performed to determine the rate of digestion of beads phagocytosed by PKH-membrane-labeled macrophages. Synchronized uptake of latex beads (mimic free Lm for primary infection) or carboxylated beads (mimic pseudopod-contained Lm for secondary infection) by PKH-membrane-labeled macrophages that were untreated, pretreated overnight with LPS or LPS+1400W, or given SNAP 30 min prior to starting the assay. Lysates were collected and stained for the early endosomal marker Rab-5 or the lysosomal marker Lamp-1 at 0, 30, 60, or 90 min after phagocytosis. (C) Flow cytometry plots depicting how beads were identified by forward and side scatter. Phagocytosed beads were identified by their staining for macrophage PKH-membrane dye. (D and E) Colocalization of phagocytosed beads with the early endosomal marker Rab-5. (D) Mean fluorescence intensity (MFI) for Rab-5 staining on phagocytosed PKH+ latex beads that mimic primary infection was not affected by LPS, but was reduced by SNAP treatment, n = 3. (E) The MFI for Rab-5 on carboxylated beads that mimic secondary infection was increased by LPS or SNAP treatment that induce NO, n = 3. (F and G) Colocalization of phagocytoses beads with the lysosomal marker Lamp-1. (F) MFI for Lamp-1 on phagocytosed PKH+ latex beads (primary infection) was not affected by NO, n = 4. (G) NO induced by LPS or SNAP reduced the colocalization of phagocytosed carboxylated beads (secondary infection) with the lysosome as measured by the MFI for Lamp-1, n = 4. Bars indicate standard errors. Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 NO Increases Cytosolic Lm in Secondary Cells To measure WT GFP-Lm secondary infection, 2.5 × 105 unlabeled recipients were mixed with 2.5 × 104 CellTrace Far Red+ donors that were WT GFP-Lm infected and plated on coverslips with no treatment, LPS, LPS+1400W, or SNAP. 9 or 18 hr after mixing, coverslips were collected, fixed and stained for actin, and mounted to slides. Images were obtained from deidentified slides and the percentage of Lm that were colocalized with actin in recipient cells was determined. Bars indicate standard errors. (A) Representative images of GFP-Lm (green) colocalization with actin (red) in the cytoplasm of CellTrace Far Red+ recipient (blue) macrophages that were untreated or given LPS, LPS+1400W, or SNAP. Arrows in the enlarged picture indicate colocalization. (B) Graph of the analysis of images described in (A). The proportion of GFP-Lm colocalizing with actin was increased by LPS and SNAP compared to untreated or LPS+1400W at 18 hr postmixing with infected donors, n = 3. Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 NO Contributes to Enhanced Bacterial Spread In Vivo C57BL/6 mice were treated with saline or 1400W and infected with either WT or ΔActA Lm. Mice were harvested 48 hpi. (A) CFU in the liver of mice shows that there is a decrease in WT CFU when mice were treated with 1400W. (B) CFU in the spleen. (C) The ratio of WT to ΔActA CFU was calculated as a measure of Lm spread for the liver and spleen of saline or 1400W-treated mice. Enhanced spread was seen in the liver compared to the spleen. Treatment with 1400W significantly reduced Lm spread in the liver. Data points represent each mouse, means ± SEM from all mice. n = 5 mice per experiment, performed twice. Immunity 2012 36, 807-820DOI: (10.1016/j.immuni.2012.03.011) Copyright © 2012 Elsevier Inc. Terms and Conditions