A158: CpG‐Induced Macrophage Activation Syndrome Develops Through an Organism Level Resistance to Toll‐like Receptor 9 Tolerance

Background/Purpose: Macrophage activation syndrome (MAS) is a cytokine storm syndrome known to complicate severe inflammatory diseases, and can be seen in patients with systemic rheumatologic conditions. Recent data implicates repeated adjuvant stimulation using the CpG Toll‐like Receptor 9 (TLR9) agonist as a model of the feed forward inflammatory cycle seen in cytokine storm syndromes. These results contradict the well known phenomenon of TLR tolerance, whereby repeated doses of TLR agonists result in diminishing inflammatory responses to additional stimuli. In contrast, CpG‐induced MAS results in a dose‐dependent increase in systemic inflammation in a process dependent on TLR9, Interferon‐γ (IFNγ), and Interleukin (IL)‐12. Methods: C57BL/6 and Yet40 IL‐12 reporter mice were treated with 0–4 doses of CpG before harvesting tissues to measure parameters of MAS. Analysis of ex vivo and in vitro cellular TLR9 tolerance was performed on whole splenocytes, bone marrow‐derived dendritic cells (BMDC) and bone marrow‐derived macrophages (BMMP). IL‐12 production was measured from supernatants by ELISA and intracellular IL‐12 was assessed by flow cytometry. Results: Our data recapitulate the well described phenomenon of TLR9 tolerance whereby repeated stimulation with CpG leads to reduced cytokine production using in vitro cultures of whole splenocytes, BMDC and BMMP. Interestingly, IFNγ does not alter the responsiveness of cells to a TLR9 tolerizing stimulus, as the addition of exogenous IFNγ does not rescue TLR9 tolerance in vitro . In contrast, repeated injections of CpG in whole animals results in a dose‐dependent increase in cytokine production, cellular tissue infiltration, cellular responsiveness to CpG, and immunopathology. This dose responsive systemic inflammation correlates with the continued responsiveness of splenic, liver, and bone marrow leukocytes to CpG after in vivo systemic CpG stimuli. Conclusion: Despite demonstrating TLR9 tolerance in multiple cell types in vitro , we show that cells remain responsive to CpG stimulation ex vivo even in mice previously treated with systemic CpG. Without the negative regulatory mechanisms of TLR tolerance, repeated injections of CpG lead to a feed forward systemic inflammatory response resulting in systemic immunopathology. These observations elucidate a novel mechanism whereby persistent adjuvant is sufficient to drive the uncontrolled inflammatory responses seen in MAS because of in vivo failure of TLR9 tolerance.