Neutralization of IFNgamma defeats haemophagocytosis in LCMV-infected perforin- and Rab27a-deficient mice

Abstract

Hereditary haemophagocytic lymphohistiocytosis (HLH) is a fatal inflammatory disease and treatments currently may lead to serious side effects. There is a pressing need for effective, less toxic treatments for this disease. Previous reports have suggested that interferon gamma (IFNgamma) has a role in the pathogenesis of HLH. Here, we report that blocking IFNgamma had a therapeutic effect in two different murine models of human hereditary HLH (perforin-deficient and Rab27a-deficient mice, both infected with lymphocytic choriomeningitis virus). Therapeutic administration of an anti-IFNgamma antibody induced recovery from haemophagocytosis in both genetic models, as evidenced by increased survival in perforin-deficient mice and correction of blood cytopenia, moderation of body temperature changes, decreased cytokinaemia, restoration of splenic architecture and reduced haemophagocytosis in the liver of both murine models. Involvement of the central nervous system in Rab27a-deficient mice was prevented by anti-IFNgamma therapy. Hepatic T-cell infiltrates and virus persisted, with no detectable harm during the time course of these studies. These data strongly suggest that neutralization of IFNgamma could be used in humans to safely alleviate the clinical manifestations of haemophagocytosis.

Figure 1. Anti-IFNγ treatment improves survival and clinical as well as haematological recovery

In the panels on the left-hand side, data from pfp−/− mice are shown that were injected with LCMV (100 pfu) on day 0 and treated either with anti-IFNγ or with control antibodies from day 8 to day 20 (five injections); in the panels on the right-hand side, data from Rab27a−/− mice are shown that were injected with LCMV (500 pfu) on day 0 and treated either with anti-IFNγ or with control antibody from day 13 to day 22 (four injections); open squares indicate control mice (wt and Rab27a+/−), black triangles mice treated with anti-IFNγ (pfp−/− and Rab27a−/−) and grey dots mice treated with control antibody (pfp−/− and Rab27a−/−). A—C. (A) Survival, (B) body weight and (C) body temperature of pfp−/− mice. D—F. (D) Survival, (E) body weight and (F) body temperature of Rab27a−/− mice. G. Blood haemoglobin level and thrombocyte and neutrophilic granulocyte counts in pfp−/− mice. H. Blood haemoglobin level and thrombocyte and neutrophilic granulocyte counts in Rab27a−/− mice. The dashed lines in (G) and (H) correspond to normal values in C57BL/6J wt mice given in the book by Metcalf (2005). Blood counts are representative for two independent experiments; * p < 0.05; **p < 0.005, ***p < 0.001.

Figure 2. Histopathological features of bone marrow, spleen and brain before and after anti-IFNγ treatment

A, B. Bone marrow sections of the right femur from (A) wt and pfp−/− mice and (B) Rab27a+/− and Rab27a−/− mice at different times after LCMV-injection, without any antibody treatment, after treatment with control or with anti-IFNγ antibody; treatment schedules were the same as indicated for Fig 1; 25× objective lens. C. Spleen sections from wt and pfp−/− mice at different times after LCMV-injection, without any antibody treatment or after treatment with anti-IFNγ antibody (r = red pulp, m = marginal zone and w = white pulp). D. Spleen sections from a Rab27a+/− and Rab27a−/− mice at different times after LCMV-injection, without any antibody treatment, after treatment with control or with anti-IFNγ antibody (r = red pulp, m = marginal zone and w = white pulp); 10× objective lens. E. Left panels: Brain sections showing parenchyma with discrete lymphocytic infiltrates in a Rab27a+/− mouse; Middle panels: a diffuse lymphocytic infiltrate in the subcortical region (red arrowheads), meningitis (arrow), a perivascular cuff (asterix) and an intraparenchymal lymphocytic cluster (white arrowhead) in a Rab27a−/− mouse after control antibody treatment; Right panels: discrete lymphocytic infiltrates in a Rab27a−/− mouse after anti-IFNγ treatment, all analysed on day 25 after LCMV injection; Upper panels: 25× objective lens, lower panels: 100× objective lens. Representative histological sections stained with haematoxylin and eosin are shown.

Figure 3. Neutralization of IFNγ and decrease of macrophage derived cytokine levels with anti-IFNγ treatment

A, B. IFNγ serum levels in (A) pfp−/− and (B) Rab27a−/− mice at various times after LCMV injection, as determined by ELISA. LCMV injections and antibody treatments in all experiments were performed as indicated in Fig 1. Open squares indicate serum levels in wt (or Rab27a+/−) mice; grey dots, pfp−/− (or Rab27a−/−) mice treated with control antibody; black triangles, pfp−/− (or Rab27a−/−) mice treated with anti-IFNγ antibody. Values represent mean ± standard error of the mean (SEM) (n ≥ 3 per group) and are representative for two independent experiments. C. Serum levels of immune complexes of rat IgG and mouse IFNγ in pfp−/− mice. Serum levels of immune complexes were measured on days 0, 11 and 14 by ELISA with goat anti-rat IgG as capture antibody and goat anti-mouse IFNγ as revealing antibody. Dilution buffer was used as a negative control and rat anti-mouse IFNγ that was pre-incubated with mouse IFNγ was used as a positive control. Values represent mean+SD of duplicate measurements (n = 3 per group). D. IFNγ serum levels of pfp−/− mice on day 8 (i.e. before anti-IFNγ) and day 14 (i.e. during anti-IFNγ treatment) as determined by ELISA. E. Serum-induced IGTP transcript levels in RAW264.7 cells by the same serum samples as in (D), measured by quantitative RT-PCR. The data show the fold difference in the IGTP-transcript compared to stimulation with cell culture medium alone. Values represent mean ± SEM (n = 5 per group from two independent experiments). F, G. Serum levels of TNF-α, GM-CSF, IL-12p70, CCL5 and IL-17 in (F) pfp−/− mice on day 12 (or day 36) and in (G) Rab27a−/− mice on day 18 after LCMV-injection treated with control or anti-IFNγ antibody, measured by Multiplex cytokine assay. Values represent mean +SD (n ≥ 3 per group) and are representative of two independent experiments. White bars indicate measurements in wt (or Rab27a+/−) mice, grey bars pfp−/− (or Rab27a−/−) mice treated with control antibody and black bars pfp−/− (or Rab27a−/−) mice treated with anti-IFNγ antibody. *p < 0.05, **p < 0.005, ***p < 0.001.

Figure 4. Reduced macrophage activation after anti-IFNγ treatment

A, B. Liver sections stained with anti-macrophage antibody F4/80 in (A) wt and pfp−/− mice and in (B) Rab27a+/− and Rab27a−/− mice at different times after LCMV-injection, without any antibody treatment, after treatment with control or with anti-IFNγ antibody; treatment schedules were the same as indicated for Fig 1; arrows indicate the corresponding cells at different magnifications; arrowheads indicate: H = nucleus of hepatocyte, M = nucleus of macrophage, p = phagocytosed cell, M + P indicating haemophagocytosis; 25× and 100× objective lens as indicated. C, F. Serum triglyceride levels in (C) pfp−/− and (E) Rab 27a−/− mice. D, F. Serum ferritin levels in (D) pfp−/− and (F) Rab 27a−/− mice. Mean + SD of measurements (n ≥ 4 per group). ^#p > 0.05, *p < 0.05, ***p < 0.001.

Figure 5. Persistence of virus and lymphocytes in the liver of anti-IFNγ-treated mice

A, B. Viral load in livers and spleens of (A) pfp−/− and (B) Rab27a−/− mice as measured by quantitative PCR, expressed as number of LCMV-copies per mill β-actin-copies. Measurements in (A) were performed on day 0, 8 and 27, in (B) on day 0, 13 and 25 after LCMV-injection. Striped bars indicate measurements in LCMV-infected mice before any antibody treatment, black bars mice treated with anti-IFNγ antibody and grey bars Rab27a−/−mice treated with control antibody. Values represent mean + SD from two independent experiments. C. Infiltration in the portal tract of liver sections stained with antibodies to LCMV (left panel), CD3 (middle panel) and Granzyme B (right panel) from a pfp−/− mouse that was treated with anti-IFNγ antibody from day 8 to day 20 and analysed on day 27 after LCMV injection; 25× objective lens. Images are also representative for immunohistochemical stainings in Rab27a−/− under anti-IFNγ as well as under control antibody treatment. D, E. Aspartate aminotransferase (ASAT) levels in the serum of (D) pfp−/− and (E) Rab27a−/− mice before and after LCMV-injection, receiving anti-IFNγ or control antibody treatment. Values represent mean + SD (n = 4 per group); LCMV injections and antibody treatments in all experiments were performed as indicated in Fig 1. ^#p > 0.05, *p < 0.05, ***p < 0.001.