Figure 1.
Intravascular adenovirus administration leads to the release of Ly-6G+7/4+ polymorphonuclear leukocytes from bone marrow into the blood and their retention in the spleen.
Dot-plot of the forward and side light scatters of leukocytes in the bone marrow (A), blood (C), and spleen (E) and their analysis for expression of Ly-6G and 7/4 cell surface marker using flow cytometry. The representative dot-plots, obtained from five mice are shown. The population of cells that stained positive for both markers is depicted on the right panel with an oval. Quantification of Ly-6G+7/4+ cells in the bone marrow (B) (the absolute numbers shown are for one femur bone), blood (D) and spleen (F) of mice injected with saline (Mock group) or Ad vector (HAdv5 group). The data was collected from three independent experiments, N = 5. * - P<0.05; ** - P<0.01.
Figure 2.
Kinetics of Ly-6G+7/4+ leukocyte recruitment and retention in the spleen in response to intravascular administration of adenovirus.
Accumulation of Ly-6G+7/4+ cells in the spleen over time after injection of mice with escalating doses of Ad, shown as virus particles per mouse. Data obtained from three independent experiments are shown. Mock mice were injected with phosphate-buffered saline (PBS) only. Splenocytes were harvested, stained with antibodies for Ly-6G and 7/4 cellular markers and analyzed by flow cytometry. N = 4. * - P<0.05; ** - P<0.01. Error bars represent standard deviation of a mean.
Figure 3.
Specific Ly-6G+ leukocyte retention in the splenic marginal zone occurs in response to Ad, but not AMD3100.
(A) Distribution of Ly-6G+ cells (green, FITC-labeled anti-Ly-6G antibody) on sections of spleen harvested from mice that were injected with either Ad or saline (Mock panels) 2 hours after treatment. The splenic marginal zone (MZ), located between the germinal centers (GC) and red pulp (RP) anatomical compartments, is outlined with dotted lines and was defined based on section staining with DAPI (blue). MZ macrophages trap Ad particles from the blood (Ad-Hexon antibody staining, red). Representative panels are shown. N = 5. Sections at three depth levels were obtained from each mouse. (B) Quantitative representation of PMN distribution on sections of the spleen 2 hours after virus challenge. Ly-6G+ cell (PMN) distribution was analyzed on at least 4 consecutive sections of the spleen, cut at three depth levels from each individual mouse. The pictures of spleen sections with average PMN cell densities were taken and up to 200 total PMN cells were counted and assigned to MZ or RP compartments based on their actual localization. For each experimental condition, the data were combined and averaged from three to 5 individual mice. * - P<0.05. Mock – mice injected with saline. (C) Distribution of Ly-6G+ cells (green, FITC-labeled anti-Ly-6G antibody) on sections of spleen harvested from mice that were injected with small molecular drug AMD3100 2 hours after treatment. The splenic marginal zone (MZ), located between the germinal centers (GC) and red pulp (RP) anatomical compartments, is outlined with dotted lines and was defined based on section staining with DAPI (blue). Representative panels are shown. N = 5. Sections at three depth levels were obtained from each mouse. (D) Quantitative representation of PMN distribution on sections of the spleen 2 hours after the mouse was challenged with small drug AMD3100. The cellular localization was assessed and analyzed as described in (B). n.s. – not statistically significant, compared to corresponding control group. RP – red pulp; MZ – marginal zone.
Figure 4.
Localization of Ly-6G+ cells to the splenic MZ occurs in response to pro-inflammatory stimuli.
(A) Mouse cytokine array panel showing differences in the proinflammatory cytokines and chemokines in spleen of wild type mice injected with saline (Mock), Ad5 (HAdv5), Ad5/35S, and Ad-ts1 viruses 1 hour after virus injection, determined by Proteome Profiler antibody array. Representative blots from four independent experiments are shown. Pos-C are dots that show the manufacturer's internal positive control samples on the membrane. Mock – the spleen protein sample of a mouse injected with saline. (B) The amounts of proinflammatory cytokines and chemokines in the spleens of mice 1 hour after Ad injection. HU – histogram units. Graphs show means ± s.d. n = 4. Mock – negative control mice injected with saline. (C) Kinetics of Ly-6G+7/4+ leukocyte accumulation in the spleen after injection of mice with 1010 virus particles of Ad5 (HAdv5), Ad5/35S and Ad-ts1 viruses. The data obtained from three independent experiments is shown. Mock group of mice was injected with phosphate-buffered saline (PBS) only. Splenocytes were harvested, stained with antibodies for Ly-6G and 7/4 cellular markers and analyzed by flow cytometry. N = 4. ** - P<0.01. Error bars represent standard deviation of a mean. (D) Distribution of Ly-6G+ leukocytes on sections of the spleens harvested from mice injected 1010 virus particles of Ad (HAdv5), Ad5/35S, and Ad-ts1. Mock – mice were administered with saline. The MZ is outlined by the dotted lines. Spleen sections were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. Representative spleen sections for each experimental setting are shown. N = 5. Scale bar is 75 µm (E) Quantitative representation of Ly-6G+ (PMN) distribution on sections of the spleens of mice administered with 1010 virus particles of indicated viruses 2 hours after virus challenge. The quantification of Ly-6G+ cell distribution was done as described in Figure 3B. N = 5. RP – red pulp; MZ – marginal zone. * - P<0.05, compared to corresponding Mock groups. Inverted triangle – P<0.05, compared to Ly-6G+ cell distribution observed for corresponding compartments in mice administered with HAdv5. Error bars represent standard deviation of a mean.
Figure 5.
The recruitment of Ly-6G+7/4+ leukocytes to the splenic marginal zone partially depends on functional IL-1α-IL-1RI signaling, but not on IL-1β.
(A) Splenocytes were harvested from WT, Il1a−/−, Il1b−/−, Il1a/b−/−, and Il1r1−/− mice injected with Ad 2 hours after the virus challenge. Mock – WT mice were injected with saline. N = 5. * - P<0.05. n.s. – not statistically significant, between indicated experimental groups. Error bars represent standard deviation of a mean. (B) Distribution of Ly-6G+ leukocytes on sections of the spleens harvested from WT, Il1a−/−, and Il1a/b−/− mice injected with Ad virus (HAdv5). Mock – mice were injected with saline. The MZ is outlined by the dotted lines. Spleen sections were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. Representative spleen sections for each experimental setting are shown. N = 5. (C) Quantitative representation of Ly-6G+ (PMN) distribution on sections of the spleens of mice injected with Ad 2 hours after the virus administration. The quantification of Ly-6G+ cell distribution was done as described in Figure 3B. N = 5. RP – red pulp; MZ – marginal zone. * - P<0.05, compared to corresponding mock groups. Inverted triangle – P<0.05, compared to Ly-6G+ cell distribution observed for corresponding compartments in WT mice.
Figure 6.
The retention of Ly-6G+ cells in the splenic marginal zone requires CXCR2, but not CXCR1, signaling.
(A) Splenocytes were harvested from WT, Cxcr1−/−, and Cxcr2−/− mice injected with Ad 2 hours after the virus challenge. Mock – WT mice were injected with saline. N = 5. * - P<0.05. n.s. – not statistically significant, between indicated experimental groups. Error bars represent standard deviation of a mean. (B) Distribution of Ly-6G+ leukocytes on sections of the spleens harvested from Cxcr1−/− and Cxcr2−/− mice injected with Ad virus (HAdv5). Spleen sections were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. Representative spleen sections for each experimental setting are shown. N = 5. Scale bar is 150 µm. (C) Quantitative representation of Ly-6G+ (PMN) distribution on sections of the spleens of indicated gene deficient mice injected with Ad 2 hours after the virus administration. The quantification of Ly-6G+ cell distribution was done as described in Figure 3B. N = 5. RP – red pulp; MZ – marginal zone. * - P<0.05, compared to corresponding mock groups. Inverted triangle – P<0.05, compared to Ly-6G+ cell distribution observed for corresponding compartments in WT mice.
Figure 7.
MARCO+ marginal zone cells are eliminated from the spleen after sequestering Ad from the blood.
(A) Distribution of Ad particles (stained with anti-Hexon Ab, red) and MACRO+ cells (stained with anti-MARCO Ab, green) on spleen sections 1 hour after intravascular virus injection. Co-localization of virus particles with MARCO+ staining appears as yellow. The anatomic borders of splenic germinal centers are depicted with dotted lines. Scale bar is 50 µm. Representative images are shown. N = 25. (B) Immunohistochemical analysis of MARCO+ marginal zone macrophages on sections of spleen at different times after Ad administration. Spleens of mice injected with the virus were harvested at indicated times and stained with MARCO-specific antibodies. Sections were counter-stained with hematoxylin to visualize splenic anatomical compartments. Mock – spleen sections were prepared from mice injected with saline only. GS – germinal center. RP – red pulp. Representative fields are shown. N = 5. (C) Quantitative representation of MARCO+-specific staining on splenic sections of mice after Ad administration. N = 4 per experimental group per time point. Marker-specific pixels (brown staining) were quantified using MetaMorph software on low-power images of spleen sections with average distribution of MARCO-specific staining collected from three sections and at three depth levels. * - P<0.05. Error bars represent standard deviation of mean. (D–G) Transmission electron microscopy analysis of ultra-thin sections of spleens harvested from saline-injected mice (D) or Ad-injected mice (E) 4 hours after the virus injection. MZ – marginal zone; GC – germinal center. The anatomic borders of germinal centers are depicted with a punctuated line. (F and G) Punctuated rectangle depicts a marginal zone macrophage that contains Ad particles (red arrows on G) and morphologically distorted mitochondria (F and black arrows on G). N – nucleus. Representative images of virus-containing cells in the marginal zone are shown.
Figure 8.
MARCO+ cells are eliminated from the splenic MZ through an IRF3-mediated necrosis-independent pathway.
(A) Quantification of propidium iodide (PI)-positive cells on unprocessed sections of livers and spleens of mice injected with 1010 virus particles of Ad (HAdv5), Ad-ts1, or Ad5/35S viruses 1 hour after the virus challenge. Mice were injected with indicated viruses intravenously and 1 hour later, mice were administered with PI and sacrificed 5 minutes later. Spleens and livers were harvested for histological analyses. Four sections of the spleen at three depth levels were prepared for each mouse and the number of PI-positive cells per low-power view-field was quantified. N = 5. * - P<0.05. n.s. – not significant. Mock – control group of mice injected with saline. (B) Distribution of Ad particles (stained with anti-Hexon Ab, red) and MACRO+ cells (stained with anti-MARCO Ab, green) on spleen sections of Irf3−/− mice 1 hour after intravascular virus injection. Co-localization of virus particles with MARCO+ staining appears as yellow. The anatomic borders of splenic germinal centers are depicted with dotted lines. Scale bar is 50 µm. Representative images are shown. N = 8. (C) Immunohistochemical analysis of MARCO+ marginal zone macrophages on sections of spleens of Irf3−/− mice at different times after Ad administration. Spleens of Irf3−/− mice injected with the virus were harvested at indicated times and stained with MARCO-specific antibodies. Sections were counter-stained with hematoxylin to visualize splenic anatomical compartments. Representative fields are shown. N = 5. Scale bar is 40 µm (D) Quantitative representation of MARCO+-specific staining on splenic sections of WT and Irf3−/− mice after Ad administration. N = 4 per experimental group per time point. Marker-specific pixels (brown staining) were quantified using MetaMorph software on low-power images of spleen sections with average distribution of MARCO-specific staining collected from three sections and at three depth levels. * - P<0.05. Error bars represent standard deviation of mean. (E) Immunohistochemical analysis of MARCO+ marginal zone macrophages on sections of spleens of WT mice at 1 hour and 24 hours after administration of Ad-ts1 virus. Spleens of mice injected with Ad-ts1 virus were harvested at indicated times and stained with MARCO-specific antibodies. Sections were counter-stained with hematoxylin to visualize splenic anatomical compartments. Representative fields are shown. N = 5. Scale bar is 40 µm.
Figure 9.
Complement component C3 is required for recruitment and retention of Ly-6G+ cells in the splenic MZ after Ad injection.
(A) Localization of Ly-6G+ cells in the MZ of Il1a/b−/− mice 2 hours after Ad injection in relation to virus-containing MZ cells. Spleen sections of virus-injected Il1a/b−/− mice were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. The regions of the spleen that are selected by rectangles A and B on the left panel are shown on the right panels at higher magnification. Scale bar is 25 µm. Anatomical borders of germinal centers are depicted by the dotted lines. Ly-6G+ cells that are in contact with virus-containing cells in the MZ are indicated by arrows. Representative spleen sections are shown. N = 5. (B) Distribution of Ly-6G+ leukocytes on sections of the spleens harvested from WT and complement component C3-deficient (C3−/−) mice injected with Ad virus. Spleen sections were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. Representative spleen sections for each experimental setting are shown. N = 5. Scale bar is 100 µm. (C) Quantitative representation of Ly-6G+ (PMN) distribution on sections of the spleens of WT and C3−/− mice injected with Ad 2 hours after the virus administration. The quantification of Ly-6G+ cell distribution was done as described in Figure 3B. N = 5. RP – red pulp; MZ – marginal zone. * - P<0.05, compared to corresponding mock groups. Inverted triangle – P<0.05, compared to Ly-6G+ cell distribution observed for corresponding compartments in WT mice. (D) Mouse cytokine array panel showing expression of the proinflammatory cytokines and chemokines in spleens of WT and C3−/− mice injected with saline (Mock) or Ad5 (HAdv5) 1 hour after virus injection, determined by Proteome Profiler antibody array. Representative blots from three independent experiments are shown. Pos-C are dots that show the manufacturer's internal positive control samples on the membrane.
Figure 10.
IL-1α-dependent signaling and complement cooperate in promoting the recruitment of Ly-6G+ leukocytes that eliminate MARCO+ cells from the splenic MZ.
(A) Distribution of Ly-6G+ leukocytes on sections of the spleens harvested from WT and Il1a−/− mice pre-treated with complement activation inhibitor CR2-Crry (50 µg/mouse) 1 hour prior to Ad injection. Spleen sections were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. Representative spleen sections for each experimental setting are shown. N = 5. Scale bar is 50 µm. (B) Distribution of Ly-6G+ leukocytes on sections of the spleens harvested from WT and Il1a−/− mice pre-treated with alternative complement activation pathway inhibitor CR2-fH (50 µg/mouse) 1 hour prior to Ad injection. Spleen sections were simultaneously stained with FITC anti-Ly-6G (green) and anti-Ad-hexon (red) antibodies and pictures were taken using a Leica fluorescent microscope. Representative spleen sections for each experimental setting are shown. N = 5. Scale bar is 50 µm. (C–D) Quantitative representation of Ly-6G+ (PMN) distribution on sections of the spleens of WT and Il1a−/− mice pre-treated with CR2-Crry (C) or CR2-fH (D) complement inhibitors 1 hour prior to Ad administration. The quantification of Ly-6G+ cell distribution was done as described in Figure 3B. N = 5. RP – red pulp; MZ – marginal zone. * - P<0.05, n.s.- not significant compared to corresponding mock (saline-injected) groups. (E) Immunohistochemical analysis of MARCO+ marginal zone macrophages on sections of spleens of mock-injected or Ad-injected (HAdv5) Il1a−/− mice pre-treated with CR2-fH 1 hour prior to Ad administration. The scale bar is 40 µm. Representative pictures are shown. N = 5. (F) Quantitative representation of MARCO+-specific staining on splenic sections of WT and Il1a−/− mice pre-treated with 50 µg of CR2-fH complement inhibitor prior to Ad administration (HAdv5). N = 4 per experimental group per time point. Marker-specific pixels (brown staining) were quantified using MetaMorph software on low-power images of spleen sections with average distribution of MARCO-specific staining collected from three sections and at three depth levels. * - P<0.05. Error bars represent standard deviation of mean.
Figure 11.
The model of distinct molecular mechanisms engaged by tissue residential macrophages in the liver and spleen to limit systemic spread of adenovirus.
After intravascular adenovirus administration, macrophages in the liver and spleen sequester virus particles from the blood. In the liver, tissue residential macrophages, Kupffer cells, undergo cell-autonomous IRF3-dependent necrosis. In the spleen, MARCO+ marginal zone macrophages activate IL-1α-IL-1RI-signaling and complement that recruit PMNs, which eliminate virus-containing cells. In both tissues, the functional consequence of host response to Ad is elimination of virus-containing cells and restriction of systemic virus spread.