Senescence-accelerated mice (SAMP1/TA-1) treated repeatedly with lipopolysaccharide develop a condition that resembles hemophagocytic lymphohistiocytosis

Abstract

Hemophagocytic lymphohistiocytosis is a life-threatening systemic hyperinflammatory disorder with primary and secondary forms. Primary hemophagocytic lymphohistiocytosis is associated with inherited defects in various genes that affect the immunological cytolytic pathway. Secondary hemophagocytic lymphohistiocytosis is not inherited, but complicates various medical conditions including infections, autoinflammatory/autoimmune diseases, and malignancies. When senescence-accelerated mice (SAMP1/TA-1) with latent deterioration of immunological function and senescence-resistant control mice (SAMR1) were treated repeatedly with lipopolysaccharide, SAMP1/TA-1 mice displayed the clinicopathological features of hemophagocytic lymphohistiocytosis such as hepatosplenomegaly, pancytopenia, hypofibrinogenemia, hyperferritinemia, and hemophagocytosis. SAMR1 mice showed no features of hemophagocytic lymphohistiocytosis. Lipopolysaccharide induced upregulation of proinflammatory cytokines such as interleukin-1β, interleukin-6, tumor necrosis factor-α, and interferon-γ, and interferon-γ-inducible chemokines such as c-x-c motif chemokine ligands 9 and 10 in the liver and spleen in both SAMP1/TA-1 and SAMR1 mice. However, upregulation of proinflammatory cytokines and interferon-γ-inducible chemokines in the liver persisted for longer in SAMP1/TA-1 mice than in SAMR1 mice. In addition, the magnitude of upregulation of interferon-γ in the liver and spleen after lipopolysaccharide treatment was greater in SAMP1/TA-1 mice than in SAMR1 mice. Furthermore, lipopolysaccharide treatment led to a prolonged increase in the proportion of peritoneal M1 macrophages and simultaneously to a decrease in the proportion of M2 macrophages in SAMP1/TA-1 mice compared with SAMR1 mice. Lipopolysaccharide appeared to induce a hyperinflammatory reaction and prolonged inflammation in SAMP1/TA-1 mice, resulting in features of secondary hemophagocytic lymphohistiocytosis. Thus, SAMP1/TA-1 mice represent a useful mouse model to investigate the pathogenesis of bacterial infection-associated secondary hemophagocytic lymphohistiocytosis.

Figure 1.

Repeated lipopolysaccharide treatment induced pancytopenia and hemophagocytosis in SAMP/TA-1 mice. (A) Numerical changes in white blood cell (WBC), red blood cell (RBC), and platelet counts in SAMR1 and SAMP1/TA-1 mice after treatment with lipopolysaccharide (LPS). Numerical changes in the counts of WBC (a), RBC (b), and platelets (c) in SAMR1 and SAMP1/TA-1 mice after repeated LPS treatment are shown. The samples of peripheral blood cells were obtained from non-treated control mice (day 0) and mice 7, 14, and 21 days after the first treatment with 25 μg LPS. Each bar represents the mean ± standard deviation obtained from three mice. (B) Hemophagocytosis in hematopoietic tissues of SAMP1/TA-1 mice after LPS treatment. A peripheral blood smear (a) and touch preparations of bone marrow (b) and spleen (c) were made on day 7 after the first treatment with 25 μg LPS. Cells were stained with Wright-Giemsa, and hemophagocytosis was evaluated.

Figure 2.

Repeated lipopolysaccharide treatment induced hepatosplenomegaly in SAMP/TA-1 mice. (A) Changes in the ratio of liver and spleen weight to total body weight (BW) in SAMR1 and SAMP1/TA-1 mice after treatment with lipopolysaccharide (LPS). Changes in the ratio of liver (a) and spleen weight (b) to total BW in SAMR1 and SAMP1/TA-1 mice after repeated LPS treatment are shown. The samples of spleen and liver obtained from non-treated control mice (day 0) and mice 7, 14, and 21 days after the first treatment with 25 μg LPS were weighed and expressed as a ratio of liver and spleen weight to total BW. Each bar represents the mean ± standard deviation obtained from three mice. (B) Photograph of spleen and liver specimens from SAMP1/TA-1 mice 21 days after the first treatment with saline or 25 μg LPS.

Figure 3.

Changes in liver histology in SAMP1/TA-1 mice after lipopolysaccharide treatment. (A) Livers obtained from SAMP1/TA-1 mice 21 days after the first treatment with saline (a, c) or 25 μg lipopolysaccharide (LPS) (b, d) were sectioned and stained with hematoxylin & eosin (HE). (B) Changes in high-power liver histology in SAMP1/TA-1 mice after LPS treatment. Congestion (a) and microthrombi (b) were observed in the livers of LPS-treated SAMP1/TA-1 mice.

Figure 4.

Changes in splenic histology in SAMP1/TA-1 mice after lipopolysaccharide treatment. (A-D) Spleens obtained from SAMP1/TA-1 mice 21 days after the first treatment with saline (A, C) or 25 μg lipopolysaccharide (LPS) (B, D) were sectioned and stained with hematoxylin & eosin (HE) (A, B) or Berlin blue to label trivalent iron (Fe) (C, D).

Figure 5.

Changes in plasma fibrinogen levels and serum ferritin levels in SAMP1/TA-1 mice after lipopolysaccharide treatment. (A) Plasma fibrinogen levels were measured with ACL ELITE PRO in plasma obtained from SAMP1/TA-1 mice 21 days after the first injection of saline or 25 μg lipopolysaccharide (LPS). (B) Serum ferritin levels were evaluated with an enzyme-linked immunosorbent assay kit in serum obtained from SAMP1/TA-1 mice 7 days after the first treatment with saline or 25 μg LPS. Each bar represents the mean ± standard deviation obtained from three mice. *P<0.05 vs. saline-treated control.

Figure 6.

Numerical changes in hematopoietic progenitor cells in the bone marrow from SAMR1 and SAMP1/TA-1 mice after lipopolysaccharide treatment. (A-D) The numbers of hematopoietic progenitor cells in the femoral bone marrow of SAMR1 and SAMP1/TA-1 mice after repeated treatment with lipopolysaccharide (LPS) are shown: myeloid progenitors, CFU-GM (A); B lymphoid progenitors, CFU-preB (B); erythroid progenitors, BFU-E (C); and megakaryocytic progenitors, CFU-Mk (D). The samples of femoral bone marrow cells were obtained from non-treated control mice (day 0) and mice 7, 14, and 21 days after the first treatment with 25 μg LPS. Each bar represents the mean ± standard deviation obtained from three mice. *P<0.05, ^†P<0.005 vs. non-treated control.

Figure 7.

Changes in the levels of gene expression of cytokines and chemokines in the liver and spleen in SAMR1 and SAMP1/TA-1 mice after the first lipopolysaccharide treatment. (A, B) Changes in levels of gene expression of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ; anti-inflammatory cytokines such as IL-10; and IFN-γ-inducible chemokines such as Cxcl9 and Cxcl10 in the liver (A) and spleen (B) of SAMR1 and SAMP1/TA-1 mice after the first treatment with lipopolysaccharide (LPS). The expression levels of pro-inflammatory cytokines such as IL-1β (a), IL-6 (b), TNF-α (c), and IFN-γ (d); anti-inflammatory cytokines such as IL-10 (e); and IFN-γ-inducible chemokines such as Cxcl9 (f) and Cxcl10 (g) were evaluated in the liver (A) and spleen (B) of SAMR1 and SAMP1/TA-1 mice 1, 3, and 6 h and 1, 2, 3, 5, and 7 days after the first treatment with 25 μg LPS. Each value shown for SAMR1 (closed circles) and SAMP1/TA-1 (open circles) mice after LPS treatment is relative to the level in non-treated control SAMR1 mice. Each bar represents the mean ± standard deviation obtained from three mice.

Figure 8.

The proportions of M1 and M2 peritoneal macrophages in SAMR1 and SAMP1/TA-1 mice after lipopolysaccharide treatment. (A; B) The changes in the proportions of M1 cells (CD11b⁺/iNOS⁺ cells) (A) and M2 cells (CD11b⁺/CD206⁺ cells) (B) in SAMR1 and SAMP1/TA-1 mice after the first treatment with 25 μg lipopolysaccharide (LPS) were evaluated. The samples of peritoneal macrophages were obtained from non-treated control mice (day 0) and mice 2 and 5 days after the first treatment with 25 μg LPS.