Background/Purpose: Cytokine release is the hallmark feature in mice given repeated injections of the TLR9 agonist, CpG-containing oligodeoxynucleotides (CpG-ODN), resulting in pathology resembling the human disease, MAS. However, an anti-IL-10 Receptor (R) monoclonal antibody (mAb) must be co-administered with CpG-ODN to induce more severe human MAS symptoms including hemophagocytosis (herein referred to as fulminant-MAS). Use of an anti-mouse IFNγ mAb, XMG1.2, has demonstrated the dependence of disease manifestation on this cytokine. This study exploits an in vivo principle that secreted IFNγ, in the presence of XMG1.2, produces a complex that has a relatively long-half-life and consequently allows cytokine accumulation in the serum which can be quantitated (Finkelman, 1999).  An accumulation of complex, from body tissues into the serum, thus, is a reflection of the total IFNγ production of the cytokine in the body. Our in vivo results characterize and correlate the production of IFNγ to the clinical and laboratory parameters in murine models of MAS and fulminant-MAS.

Methods: C57BL/6 mice received i.p. injections of CpG-ODN on days 0, 2, 4, 7 and 9. Neutralizing IL-10R, mAb 1B1.3A at 200µg/mouse (days 0, 2, 4 & 6), and anti-mouse IFNg, mAb XMG1.2 at 100 mg/kg (days 1, 3 & 6) were administered i.v. Q-PCR was used to analyze inflammatory gene expression. Luminex multiplex technology was used to detect serum cytokines. Blood parameters were measured using a haematological counter. Serum concentrations of the cytokine-drug complex were determined by an ELISA method developed for purpose.

Results: TLR9 agonism resulted in a multi-phasic production of IFNγ evidenced by a spike in serum cytokine levels following each CpG-ODN injection. Therapeutic blockade of IFNγ by XMG1.2 reduced body weight loss, splenomegaly, normalized white blood cell counts, significantly reversed the decrease in other laboratory parameters (e.g. platelets, haemoglobin and red blood cells) and controlled hyperferritinemia. IFNγ measured from CpG-ODN mice co-treated with XMG1.2, which accumulated as a complex in the serum, reached steady state levels of 233 ng/ml.  This represented a 200-fold increase over serum IFNγ levels measured in mice treated with CpG-ODN alone (1 ng/ml). Expression of IFNγ induced inflammatory genes demonstrated that spleen and liver are major sites of IFNγ production. Interestingly, although the fulminant-MAS model presented a more severe pathology, in vivo production of IFNγ production was determined to be equivalent in the two models.  Nonetheless, blockade of IFNγ in mice with fulminant-MAS also improved key disease features including body weight loss, splenomegaly, anemia, hyper-cytokinemia, lymphopenia. Notably, XMG1.2 treatment also decreased the platelet loss in mice with fulminant MAS. Ongoing experiments are focused on determining how the blockade of IFNγ influences the consumptive anemia of inflammation observed in these mice.

Conclusion: The substantial production of IFNγ in tissues is intimately associated with the clinical and laboratory features in the CpG-induced model of MAS. These data support the potential therapeutic strategy for IFNγ neutralization in patients afflicted with this severe form of MAS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/correlating-the-in-vivo-production-of-ifn%ce%b3-to-disease-parameters-in-tlr9-induced-macrophage-activation-syndrome-mas-in-mice/