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I. Tsuboi et al.
hepatosplenomegaly, pancytopenia, and coagulopathy.9 The clinical entity of HLH is different from that of sepsis or inflammatory response syndrome.15 However, these diseases show a common immunopathological state referred to as a cytokine storm.9,15
Senescence-accelerated mice, senescence-prone (SAMP) show early onset of a decline in immune function such as decreased natural killer-cell and T-cell activities and are susceptible to infection.16-20 Thus, we investigated whether repeated lipopolysaccharide (LPS) treatment induces hyperinflammation by deterioration of the function of the immune system resulting in sHLH in the senescence- prone substrain, SAMP1/TA-1 mice.
Methods
Mice
SAMP1/TA-1 mice were bred and maintained in an experimen- tal facility at Nihon University School of Medicine.16,17 SAMR1/Ta Slc mice were purchased from Japan SLC Inc. (Hamamatsu, Japan). Eight- to 12-week old SAMR1 and SAMP1/TA-1 male mice were used.16,17 All protocols involving laboratory mice were reviewed and approved by the Nihon University Animal Care and Use Committee (experiment codes AP15M033 and AP16M054). The approved experimental protocol was performed humanely in strict accordance with Nihon University rules concerning animal care and use.
Progenitor cell colony assay
Myeloid progenitor (CFU-GM) cells were assayed using MethoCult M3231 (Stem Cell Technologies Inc., Vancouver, Canada) supplemented with 10 ng/mL recombinant murine gran- ulocyte-macrophage colony-stimulating factor. B lymphoid-prog- enitor (CFU-preB) cells were assayed using MethoCult M3630. Erythroid progenitor (BFU-E) cells were assayed using MethoCult M3334 supplemented with 1 ng/mL recombinant murine inter- leukin (IL)-3. Megakaryocytic progenitors (CFU-Mk) were assayed using MegaCult-C supplemented with 50 ng/mL recombinant human thrombopoietin, 10 ng/mL recombinant murine IL-3, 20 ng/mL recombinant human IL-6, and 50 ng/mL recombinant murine IL-11. Cells were cultured in a humidified incubator at 37°C and 5% CO2. CFU-GM, CFU-preB, and CFU-Mk cells were counted 7 days after plating the cells. BFU-E cells were counted 10 days after plating the cells.
Gene expression assay
The levels of gene expression of cytokines were determined with real-time polymerase chain reaction (PCR) using the Applied Biosystems 7500 Fast Sequence Detection System. Briefly, total RNA from splenic and liver cells was isolated using ISOGEN reagent (Nippongene Corp., Toyama, Japan). mRNA was reverse transcribed using Superscript III (Life Technologies, Carlsbad, CA, USA) and Oligo-dT (Promega Corp., Madison, WI, USA). The levels of gene expression were determined with real- time PCR using TaqManTM Universal Fast PCR master mix (Applied Biosystems, Foster City, CA, USA) and specific primers. Specific primers and probes for murine IL-1β, IL-6, IL-10, tumor
necrosis factor (TNF)-α, interferon (IFN)-g, Cxcl9, Cxcl10, and GAPDH genes were purchased from Applied Biosystems as described elsewhere.23,24
Clinical laboratory tests and enzyme-linked immunosorbent assays
Plasma fibrinogen levels were measured by ACL ELITE PRO (Instrumentation Laboratory, Bedford, MA, USA). Serum ferritin levels were evaluated with an enzyme-linked immunosorbent assay kit (Abcam pic, Cambridge, UK).
Flow cytometry analysis for peritoneal macrophage polarization in SAMR1 and SAMP1/TA-1 mice
Peritoneal cells (2 x 105) were labeled using fluorescein isothio- cyanate-conjugated rat anti-mouse CD11b monoclonal antibody and phycoerythrin-conjugated rat anti-mouse inducible nitric oxide synthase (iNOS) monoclonal antibody, or phycoerythrin- conjugated rat anti-mouse CD11b monoclonal antibody and fluo- rescein isothiocyanate-conjugated rat anti-mouse CD206 mono- clonal antibody. Labeled cells were analyzed by flow cytometry (Cytomics FC500, Beckman Coulter, Brea, CA, USA) for direct detection of CD11b+/iNOS+ M1 macrophages and CD11b+/CD206+ M2 macrophages.
Statistical analysis
Data are expressed as the mean ± standard deviation (SD). Data sets were compared using the two-tailed unpaired Student t test and two-way analysis of variance. Differences were considered statistically significant at P<0.05.
Results
Repeated lipopolysaccharide treatment induced pancy- topenia in SAMP1/TA-1 mice but not in SAMR1 mice
The numbers of peripheral white blood cells, red blood cells, and platelets were evaluated in SAMR1 and SAMP1/TA-1 mice after repeated LPS treatment (Figure 1A). The number of white blood cells in LPS-treated SAMR1 mice was slightly decreased until day 21 after the first LPS treatment compared with that of the non- treated control group (day 0). In contrast, the number of white blood cells in LPS-treated SAMP1/TA-1 mice was significantly decreased to 17.5% that of the non-treated control group by day 21 (SAMP1/TA-1; day 0 vs. day 7; P<0.05, day 0 vs. day 14; P<0.005, day 0 vs. day 21; P<0.005). The number of red blood cells in LPS-treated SAMR1 mice remained unchanged compared with that in the non-treated control group. In contrast, the number of red blood cells in LPS-treated SAMP1/TA-1 mice was significantly decreased to 44.7% that in the non-treated control group by day 21 (SAMP1/TA-1; day 0 vs. day 7; P<0.005, day 0 vs. day 14; P<0.005, day 0 vs. day 21; P<0.005). The numbers of platelets in LPS-treated SAMR1 mice on days 7, 14, and 21 were higher than those in the non-treated control group. In contrast, the number of platelets in LPS-treated SAMP1/TA-1 mice was significantly decreased to 9.9% that in the non- treated control group by day 21 (SAMP1/TA-1; day 0 vs. day 7; P<0.005, day 0 vs. day 14; P<0.005, day 0 vs. day 21; P<0.005).
Hemophagocytosis in hematopoietic tissues of lipopolysaccharide-treated SAMP1/TA-1 mice
Hemophagocytosis was observed in SAMP1/TA-1 mice
Lipopolysaccharide treatment
Mice were injected intravenously with a single dose of 25 μg LPS three times at weekly intervals.21,22 The body weight of non- treated SAMR1 and SAMP1/TA-1 mice was 29.6 ± 1.1 g and 31.2 ± 1.3 g, respectively. A control group of SAMR1 and SAMP1/TA- 1 mice was treated with the same volume of pyrogen-free saline.
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haematologica | 2019; 104(10)