Discrete and conserved inflammatory signatures drive thrombosis in different organs after Salmonella infection

Abstract

Inflammation-induced thrombosis is a common consequence of bacterial infections, such as those caused by Salmonella Typhimurium (STm). The presentation of multi-organ thrombosis post-infection that develops and resolves with organ-specific kinetics raises significant challenges for its therapeutic control. Here, we identify specific inflammatory events driving thrombosis in the spleens and livers of STm-infected mice. IFN-γ or platelet expression of C-type lectin-like receptor CLEC-2, key drivers of thrombosis in liver, are dispensable for thrombosis in the spleen. Platelets, monocytes, and neutrophils are identified as core constituents of thrombi in both organs. Depleting either neutrophils or monocytic cells abrogates thrombus formation. Neutrophils and monocytes secrete TNF and blocking TNF diminishes both thrombosis and inflammation, which correlates with reduced endothelial expression of E-selectin and leukocyte infiltration. Moreover, inhibiting tissue factor and P-selectin glycoprotein ligand-1 pathways impairs thrombosis in both spleen and liver. Therefore, we identify organ-specific, and shared mechanisms driving thrombosis within a single infection. This may inform on tailoring treatments towards infection-induced inflammation, and single- or multi-organ thrombosis, based on the clinical need.

Fig. 1. Interferon (IFN)-γ and C-type lectin (CLEC)-2 are dispensable for thrombosis in the spleen after STm infection.

A Representative immunohistochemistry of spleen sections from WT and IFN-γ-deficient mice (ifng^−/−) infected with STm for one day. Brown = Fibrin, Blue=CD41. B Percentage of section area (left) and number of thrombi per section (right) in spleens from (A); each point represents data from a single mouse, generated from combining results from two independent experiments for a total of n = 7 WT mice and n = 10 IFN-γ-deficient mice. C Representative immunohistochemistry of liver sections from WT and IFN-γ-deficient mice infected with STm for 7 days. Brown= Fibrin, Blue=CD41. D Percentage of section area in livers from (C); each point represents data from a single mouse, generated from combining results from two independent experiments for a total of n = 8 WT mice and n = 8 IFN-γ-deficient mice. E Representative immunohistochemistry of spleen sections from WT and PF4^CreCLEC-2^fl/fl mice infected with STm for one day. Brown = Fibrin, Blue = CD41. F Percentage of section area in spleens from (E); each point represents data from a single mouse, the graph was generated from combining three independent experiments for a total of n = 22 WT mice and n = 12 PF4^CreCLEC-2^fl/fl mice. G Representative immunohistochemistry of spleen sections from WT and PF4^CreCLEC-2^fl/fl mice infected with STm for 7 days. (H) Percentage of section area in spleens from (G); data presented is representative from three independent experiments each with n = 4 mice per group and a single point represents data from a single mouse. Median values shown as horizontal lines on each graph. Error bars depict the 75^th–25^th interquartile range (IQR). Each dot represents an independent mouse. Statistical analyses were performed using the two-tailed Mann–Whitney test. Source data are provided as a Source Data file. ns non-significant, T thrombus, V vessel, RP red pulp, WP white pulp.

Fig. 2. STm infection drives the accumulation of neutrophils and Ly6C⁺ monocytic cells and these cells can be found in thrombi.

A Percentage of tissue area occupied by thrombi in the spleen (0 h n = 4, 4 h n = 4; 8 h n = 3, 18 h n = 3) and liver (0 days n = 4, 1 day n = 4, 7 days n = 3) after STm infection across the timepoints indicated. Each point represents data from a single mouse. B The percentage thrombus area staining positive for Ly6G (orange) or Ly6C (green), with each point representing individual thrombi from spleens (left, 1 day post-infection) or livers (right; 7 days post-infection). Each dot represents a different thrombus analyzed from n = 4 mice per group. C Representative images of a spleen thrombus from day 1 (upper row) and a liver thrombus from day 7 post-infection (bottom row). Sections are stained for CD41 (red), Ly6G (blue), Ly6C (green), and fibrin (gray). n = 4 mice per group. D Flow cytometric analysis of the frequency of neutrophils and Ly6C⁺ monocytes from live CD45⁺ cells in spleens (0 h n = 4, 4 h n = 4; 8 h n = 3; 18 h n = 4) and livers (0 days n = 4, 1 day n = 5, 7 days n = 4) from mice infected with STm. Representative data from two experiments, each point represents data from a single mouse. E Representative fields of view (FOV) of spleen (Upper panel) and liver (bottom panel) obtained by intra-vital microscopy in non-infected mice (NI) and STm infected mice, 1 day (spleen) or 7 days (liver) post-infection. Cells were stained with CD49b (red), Ly6G (blue) and F4/80 (white). Data are representative of two independent experiments each with NI n = 3 and STm n = 4 mice per group. Yellow arrows indicate the presence of platelets, neutrophils and F4/80⁺ aggregates. F Representative immunohistochemistry of liver sections from non-immunized (NI) or mice infected with STm for the indicated times. The graph shows the quantification of Ly6G⁺ areas per section generated from F. Each dot represents a different field of view assessed from different time points. Non-immunized (NI) n = 4, 4 h n = 4; 8 h n = 3, 18 h n = 3, 7 days n = 4 mice. The data are expressed as dot plots, with horizontal lines depicting the medians. Error bars depict the 75^th–25^th interquartile range (IQR). Statistical analyses were performed using the two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons tests. Source data are provided as a Source Data file. ns non-significant, T thrombus, V vessel.

Fig. 3. Neutrophils and monocytic cells are required to drive thrombosis in the spleen and liver after STm infection.

A Total numbers of CD11b⁺Gr1^hi cells in isotype control treated (IC) or anti-Ly6G treated mice in the spleen (1 day post-infection) or liver (day 7 post-infection). B Numbers of neutrophils per mm³ in the peripheral blood in isotype control (IC) or anti-Ly6G treated mice that were infected with STm for 1 day (left) or 7 days (right). Representative data from two independent experiments with n = 4 mice per group. C Representative immunohistochemistry sections from spleens (top row; day 1 post-infection) and livers (bottom row; day 7 post-infection) from isotype control (IC) or anti-Ly6G treated mice (Fibrin= brown, CD41=blue). D Quantification of the area occupied by thrombi in spleens and livers from mice represented in (C). Data presented are from two independent experiments combined for a total of n = 8 mice per group. E Representative immunohistochemistry of spleens (day 1 post-infection) and livers (day 7 post-infection) from mice treated with PBS liposomes or clodronate liposomes (Fibrin= brown, CD41=blue). F Quantification of the area occupied by thrombi in the spleens and livers of mice represented in (E). Data presented are from two independent experiments combined for a total of n = 8 mice per group. The data are expressed as dot plots, with each point representing data from a single mouse, and horizontal lines depict the medians. Error bars depict the 75^th–25^th interquartile range (IQR). Statistical analyses were performed using the two-tailed Mann–Whitney test. Source data are provided as a Source Data file. V Vessel, T Thrombus.

Fig. 4. Blocking TNF prevents thrombosis after STm infection.

A Total numbers of TNF-producing neutrophils (CD11b⁺Ly6G⁺) and Ly6C⁺ monocytes (CD11b⁺ Ly6C⁺) determined by flow cytometry in the spleens (NI n = 4, STm n = 5) and livers (NI n = 4, STm n = 4) of mice infected for 1 or 7 days respectively. Representative data shown from at least two independent experiments. B Representative images of spleen sections from mice infected for 1 day stained for TNF (green), CD31 (gray) and F4/80 (blue); n = 4 mice per group. C Representative images of liver sections from mice infected for 7 days stained for TNF (green), DAPI (gray) and F4/80 (blue); n = 4 mice per group. In (B) and (C) the right-hand image corresponds to a higher magnification of the area within the dotted white square and the yellow arrows indicate sites of positive TNF staining. D Representative immunohistochemistry of spleen and liver sections from mice infected for 1 or 7 days, respectively, and treated with either isotype control antibody (IC) or an anti-TNF blocking antibody (α-TNF) (Fibrin=blue, CD41=brown). E Quantification of the area occupied by thrombi in spleen and liver from (D). Data presented are from two independent experiments combined. Spleen IC n = 7, spleen α-TNF n = 10, liver IC n = 7, liver α-TNF n = 8. Each point represents data from an individual mouse and horizontal lines depict the medians. Error bars depict the 75^th–25^th interquartile range (IQR). Statistical analyses were performed using the two-tailed Mann-Whitney test. Source data are provided as a Source Data file. V Vessel, T Thrombus.

Fig. 5. Blocking TNF induced by STm infection reduces CD62E expression.

A, B Representative immunofluorescence images of spleen (A) and liver (B) sections from mice infected for 1 or 7 days, respectively, and treated with either isotype control antibody (IC) or an anti-TNF blocking antibody (αTNF). Sections were stained for CD62E (yellow) and CD31 (red). In each case, images in the bottom panel are a higher magnification of the areas marked by the dotted white squares. NI non-immunized, IC Isotype control, α-TNF anti-TNF antibody. Cyan arrows indicate areas of positive CD62E staining. C, D The frequency of CD62E⁺ area per field of view (FOV) in spleen (C) and liver (D) from sections represented in (A) and (B) generated from n = 4 mice per group and a minimum of 5 FOV per mouse, with each dot representing a single FOV. Total numbers of CD11b⁺Ly6G⁺ cells (neutrophils) and CD11b⁺Ly6C⁺ cells (Ly6C⁺ monocytes) determined by flow cytometry in spleens (E) and livers (F) of non-immunized (NI), isotype control (IC) or anti-TNF treated mice infected as described for (A) and (B). Representative data are from two independent experiments. Spleen NI n = 8, spleen IC n = 4, spleen anti-TNF n = 5, liver NI n = 8, liver IC n = 4, liver anti-TNF n = 4. The data are expressed as dot plots, where each dot represents an individual mouse. Horizontal lines depict the medians. Error bars depict the 75^th–25^th interquartile range (IQR). Statistical analyses were performed using the two-tailed Mann-Whitney test. V Vessel, T Thrombus.

Fig. 6. Longitudinal expression of TNF and CD62E in the spleen after STm infection.

A Representative images of spleen sections from mice infected with 5 × 10⁵ CFU STm SL3261 for 4 h, 8 h or 18 h. Sections were stained for TNF (yellow), Ly6G (blue) and F4/80 (red). The bottom row shows the area within the dotted white squares at a higher magnification. White arrows show positive TNF staining. B Spleen sections from (A) were stained for CD62E (yellow), CD41 (blue), CD31 (red). The central row shows the area within the dotted white squares at a higher magnification. The bottom row shows CD62E staining only (gray) from the above image. Non-immunized (NI) n = 4, 4 h n = 4; 8 h n = 3, 18 h n = 3 mice. NI Non-immunized, V Vessel, T Thrombus.

Fig. 7. Longitudinal expression of TNF and CD62E in the liver after STm infection.

A Representative images of liver sections from mice infected with 5 × 10⁵ CFU STm SL3261 for 4 h, 8 h, 18 h or 7 days. Sections were stained for TNF (yellow), Ly6G (blue) and F4/80 (red). The bottom row shows the area within the dotted white squares at a higher magnification. Pink arrows show positive TNF staining. B Liver sections from (A) were stained for CD62E (yellow), CD41 (blue) and CD31 (red). The central row shows the area within the dotted white squares at a higher magnification. The bottom row shows CD62E staining only (gray) from the above image. Pink arrows indicate positive staining for CD62E. Non-immunized (NI) n = 4, 4 h n = 4; 8 h n = 3, 18 h n = 3, 7 days n = 4 mice. Representative images from 3-4 mice per group from two independent experiments. NI Non-immunized, V Vessel, T Thrombus.

Fig. 8. STm-induced thrombosis is driven by tissue factor.

A Percentage section area positive for Tissue Factor (TF) in spleens and livers from non-infected controls (NI) or mice infected for 1 day (spleen) or 7 days (liver). Each dot represents a different field of view (FOV) and represents data from a minimum of 10 FOV from each tissue and from a minimum of n = 4 mice for each group. B Representative images of spleen and liver thrombi (from A) stained gray for TF or fibrin (Fib), CD31 (red) and CD41 (blue). C Representative images of spleen and liver thrombi (from A) stained for TF (gray), Ly6G (red) and CD11b (blue). For (B, C) n = 4 spleens and n = 4 livers from four individual mice were assessed from two independent experiments. D Representative images of spleen and liver sections (from A) stained for (gray), α-SMA (red) and CD31 (blue); n = 4 spleens and n = 4 livers from four individual mice were assessed from two independent experiments. E Representative FOV of thrombin activation in the spleen (1 day post-STm infection) and liver (7 days post-STm infection) obtained by intra-vital microscopy. T = 0 min shows the first frame of the recording before the administration of the thrombin probe. T = 5 min shows the frame in the same FOV 5 min after administration of the thrombin probe. Yellow arrows indicate positive thrombin staining. F Quantification of thrombin+ regions 5 min after administration of the thrombin probe in non-infected and infected mice. Data presented are from one experiment with n = 4 mice per group and are representative of at least two experiments for each organ. G Left is a representative image of a splenic thrombus (within the dashed white lines) captured by intra-vital microscopy 24 h after infection, stained for CD49b (red), Ly6G (blue) and F4/80 (gray) next to two images showing thrombin detection (yellow) at t = 0 min and T = 5 min after the administration of the thrombin probe. Images representative of experiments from four infected mice. H Representative sections stained for fibrin (brown) and CD41 (blue) from the spleens (left) and livers (right) of heterozygous mice (expressing human TF and mouse TF, mTF^+/−, hTF^+/−) and mice with low tissue expression (mTF^−/−, hTF^+/+). Data representative of two independent experiments each with n = 4 mice per group. I Quantification of the area occupied by thrombi from G (n = 4 mice per group). Each point represents data from one mouse, horizontal lines depict the medians. Error bars depict the 75^th–25^th interquartile range (IQR). Statistical analyses were performed using the two-tailed Mann-Whitney test. Source data are provided as a Source Data file. T thrombus, V vessel.

Fig. 9. Targeting PSGL-1 prevents thrombosis and modulates the accumulation of neutrophils and monocytes within organs.

A Representative images of thrombi from spleens (top row) and livers (bottom row) stained for CD41 (red), tissue factor (TF, gray), and PSGL-1 (blue) from mice infected with STm for 1 (spleens) or 7 days (livers). n = 4 mice per group, two independent experiments. B Representative images from spleen (top row) and liver (bottom row) sections from mice infected with STm (as in A) and treated either with isotype control (IC) or anti-PSGL1 (α-PSGL1). Sections are stained for fibrin (brown) and CD41 (blue). C Quantification of the area occupied by thrombi in spleens at day 1, or livers at day 7 post-infection (from B), from mice treated with isotype control (IC) or anti-PSGL1(α-PSGL1). Data presented are combined from two independent experiments. Spleen IC n = 8, spleen anti-PSGL1 n = 9, liver IC n = 7, liver anti-PSGL1 n = 9. D, E Frequencies of neutrophils (CD11b⁺Ly6G⁺) or Ly6C⁺ monocytes (CD11b⁺Ly6C⁺) quantified by flow cytometry in the spleens (D) or livers (E) of mice infected as in (C) and treated with isotype control (IC) or anti-PSGL1 (α-PSGL1) antibodies. Representative from two independent experiments with n = 5 mice per group. F Representative images from spleen sections stained for CD31 (green), CD11b (blue), and Ly6G (red) from non-infected mice (NI), isotype control-treated mice (IC) or anti-PSGL-1-treated mice infected for 1 day with STm (n = 4 mice per group). G Representative images from liver sections stained for Ly6G (red), CD11b (blue), and DAPI (gray) from non-infected mice (NI), isotype control-treated mice (IC) or anti-PSGL-1-treated mice infected for 7 days with STm (n = 4 mice per group). In graphs, each point represents the data from one mouse, and horizontal lines show the medians. Error bars depict the 75^th–25^th interquartile range (IQR). Statistical analyses were performed using the two-tailed Mann–Whitney test. Source data are provided as a Source Data file T thrombus, V vessel.