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Macrophage Depletion Mitigates Platelet Aggregate Formation in Splenic Marginal Zone and Alleviates LPS-Associated Thrombocytopenia in Rats

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Macrophage Depletion Mitigates Platelet Aggregate Formation in Splenic Marginal Zone and Alleviates LPS-Associated Thrombocytopenia in Rats

Ying Li et al. Front Med (Lausanne).

Abstract

Sepsis is often accompanied with thrombocytopenia partly due to platelet sequestration in the lung and liver. The spleen can store up to one-third of circulating platelets and can also significantly affect platelet transfusion outcomes by accumulating platelets. However, in sepsis, it is not clear whether there are platelet changes in the spleen which could contribute to sepsis-associated thrombocytopenia and also influence platelet transfusion outcomes. By using confocal microscopy, we examined endogenous rat platelets and infused human platelets in the spleen of severe combined immune deficient Rag2 KO rats which were injected intraperitoneally with lipopolysaccharide (LPS). LPS-injected Rag2 KO rats developed sepsis as indicated by increased TNFa, IL-6, IL-1b, and IL-10 levels and thrombocytopenia. Large platelet aggregates were observed in the spleen with majority located in the marginal zone and closely associated with CD169+ macrophages. Depletion of macrophages by clodrosome resulted in reduction of LPS-induced cytokine generation and alleviated LPS-induced thrombocytopenia. Macrophage depletion also remarkedly diminished large platelet aggregate formation in the splenic marginal zone but had less effect on those in red pulp. Infusion of human platelets into LPS-injected rats failed to raise platelet counts in the peripheral blood. In LPS-injected rat spleen, human platelets interacted with aggregated rat platelets in the marginal zone. In contrast, human platelets infused into control rats were located outside of splenic marginal zone. This study provides morphological evidence of platelet aggregates in the splenic marginal zone in sepsis which can interact with infused platelets and thus can contribute to platelet infusion refractoriness in sepsis. It indicates that macrophages play an important role in LPS-associated thrombocytopenia. It also suggests that CD169+ macrophages support platelet aggregate formation in the splenic marginal zone.

Keywords: CD169; infusion; macrophages; platelets; sepsis; spleen; thrombocytopenia.

Figures

Figure 1
Figure 1
LPS treatment induces septic response in Rag2 KO rats. Intraperitoneal injection of LPS dramatically increased TNFa (A), IL-6 (B), IL-1b (C), and IL-10 (D) levels in the circulation and caused significant platelet count decrease (E) in Rag2 KO rat 4 h after LPS administration. Three to four animals were analyzed in each group. *P < 0.05; ***P < 0.001; ****P < 0.0001. Students t-test.
Figure 2
Figure 2
Platelets aggregate in the marginal zone in LPS-injected Rag2 KO rat spleen. (A) Platelet aggregates consist of activated platelets. Upper panels show, in a PBS-injected rat spleen, both anti-CD42d (red, resting platelets) and anti-rat platelet (clone BR4, green, resting and activated platelets) antibodies recognize platelets (white arrows) and megakaryocytes (white arrowheads). Lower panels show, in a LPS-injected rat spleen, the anti-rat platelet antibody clone BR4 recognizes large platelet aggregates in the marginal zone which are largely CD42d negative. Yellow arrowheads indicate the presence of a few weak CD42d+ resting platelets in the aggregates. Nuclei were stained with TOPRO3 (blue). Scale bars, 10 μm. (B,C) Platelet aggregates in the marginal zone are closely associated with CD169+ macrophages in LPS-injected Rag2 KO rat spleen (B) and LPS-injected wild type rats (C). Rat spleens were stained for rat platelet (Rt-PLT, red) and CD169 (green) expression 4 h after intraperitoneal injection of LPS. Left panels show representative pictures from PBS-injected rat spleens and right panels from LPS-injected rat spleens. White arrowheads indicate large platelet aggregates formed in the marginal zone associated with CD169+ macrophages. Yellow arrowheads indicate large platelet aggregates in the red pulp. White arrows indicate single rat platelets. Yellow arrows indicate megakaryocytes. Three to four animals were analyzed in each group. Nuclei were stained with TOPRO3 (blue). Scale bars, 20 μm.
Figure 3
Figure 3
Macrophage depletion by clodrosome diminishes platelet aggregation in splenic marginal zone in LPS-injected rats. (A) Clodrosome injection depleted macrophages in the spleen. Note the dramatic decreases in macrophage marker CD68 and CD169 expression. Scale bars, 100 μm. (B) Encapsome injection had little effects on LPS-induced platelet aggregation in splenic marginal zone (arrows) and in red pulp (arrowheads) compared to PBS+LPS-injected rats (left and middle panels, respectively). Right panel shows that clodrosome injection diminished large platelet aggregates in the marginal zone but had less effects on large aggregates formation in red pulp (arrowheads) in LPS-injected rat spleen. Dashed line indicates the marginal zone. Three to four animals were analyzed in each group. Scale bars, 50 μm.
Figure 4
Figure 4
Macrophage depletion mitigates LPS-induced cytokine generation and thrombocytopenia in Rag2 KO rats. Clodrosome and encapsome injection alone did not affect LPS-induced cytokine generation and thrombocytopenia in rats. Compared to encapsome + LPS-injected rats, clodrosome injection significantly alleviated TNFa (A), IL-1b (B), IL-6 (C), and IL-10 (D) generation in LPS-injected rats. (E) Clodrosome injection mitigated platelet count decreases in LPS-treated rats. Three to four animals were analyzed in each group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA with Tukey's multiple comparisons test.
Figure 5
Figure 5
Infused human platelets interact with rat platelet aggregates in the splenic marginal zone in LPS-treated rats. (A) Left panel shows infused human platelets (human CD41+, green) and rat platelets (red, BR4 clone) are located outside of splenic marginal zone indicated by CD169+ macrophages (blue) in a PBS-injected rat. White arrows indicate single human platelets and yellow arrows indicate single rat platelets. Right panel shows infused human platelets interacted with aggregated rat platelet in a LPS-treated Rag2 KO rat spleen. Scale bars, left panel, 10 μm; right panel, 20 μm. (B) Zoomed-in view of boxed region in (A) right panel. Arrowheads indicate the colocalization (yellow/orange) of human platelets and rat platelets. Arrows indicate single human platelets. Scale bars, 10 μm. (C) Infusion of human platelets into LPS-injected rats did not increase, but even decreased, the peripheral blood platelet counts. Three to four animals were analyzed in each group. *P < 0.05; ****P < 0.0001. One-way ANOVA with Tukey's multiple comparisons test.

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