Prevention of Hemophagocytosis by TNF-α and IL-6 Blockade in TLR9- and Nod1- Ligand Induced Hemophagocytic Syndrome.

Hemophagocytic syndrome (HPS, also referred as hemophagocytic lymphohistiocytosis; HLH) is a life-threatening condition caused by hyperinflammatory response. It can be classified into familial and acquired forms. Familial HPS is a disorder inherited in an autosomal recessive or X-linked manner. Several loci and genes have been implicated in the familial HPS, including perforin, or cytotoxic granule exocytosis genes. While, acquired (secondary) HPS is a sporadic syndrome occurring in association with systemic infection (virus, bacteria, protozoa, and fungi), underlying malignancy, or immunodeficient disorder. The central pathophysiologic abnormality in HPS is cytokine dysfunction, resulting in uncontrolled accumulation of activated T-lymphocytes and activated histiocytes (macrophages) in many organs. Several cytokines including interferon-γ, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, IL-12, and soluble IL-2 receptor (sCD25), found at extremely high levels in the plasma of HPS patients. Treatment of hemophagocytic lymphohistiocytosis is based on epipodophyllotoxin derivatives, etoposide, corticosteroids, cyclosporin A, antithymocyte globulin (ATG), and, in selected patients, intrathecal methotrexate. Recently, anti-TNF-α therapy has been reported to prove patients with refractory HPS, however, there is not enough evidence. In this study, we established murine HPS model by intravenous injection of CpG oligodeoxynucleotide (ODN)-1668, a ligand for Toll-like receptor 9 (TLR9) and combination of CpG and iE-DAP, which is a ligand for Nod1, and investigated the role of TNF-α and other cytokine in HPS model.

C57BL/6J mice were injected intravenously with 200 μg of CpG or combination of CpG and 100 μg of iE-DAP. The treated mice were assayed for temperature, spleen weight, and complete blood count. The serum cytokines levels (TNF-α and IL-6) and triglycerides were determined by ELISA and auto-analyzer, resectively. Hemophagocytosis was determined by flow cytometric (FCM) analysis in the peripheral blood and spleen. The spleen was subjected into immunohistochemistry staining and histological examination.

For TNF-α and IL-6 blockade, the mice were treated with TNF receptor (TNFR)-Fc (etanercept) or combination of TNFR-Fc and anti-mouse IL-6 receptor (IL-6R) monoclonal antibody (MR16-1) by intravenous injection.

CpG injected mouse displayed the typical HPS clinical criteria, such as, fever, cytopenia, splenomegaly, hemophagocytosis in the peripheral blood and spleen. Furthermore, elevations of inflammatory cytokines (TNF-α and IL-6) and triglyceride were detected in the serum of these mice. These clinical symptoms were not observed in CpG-injected TLR2^−/−4^−/−9^−/− mice and LPS-injected wild type mice. Interestingly, combination challenge of CpG and iE-DAP led to synergistic induction of hemophagocytosis and enhanced production of TNF-α and IL-6 in the serum, however, single iE-DAP challenge did not induce HPS. FCM analysis revealed that monocyte-derived dendritic cells, which were CD11b, Ly6c, and F4/80, were mobilized and engulfed TER119⁺ erythrocyte in the peripheral blood and spleen after these challenge. Treatment of mice with TNFR-Fc partially prevented, and combination of TNFR-Fc and anti IL-6R mAb markedly suppressed hemophagocytosis in the peripheral blood and spleen.

Conclusions: Based on these results, CpG or CpG + iE-DAP-induced mouse HPS model is valuable to study for cellular and molecular mechanism of HPS development, and potential system to apply preclinical research. Furthermore, prevention of both TNF-α and IL-6 might represent a novel therapy for HPS.

No relevant conflicts of interest to declare.