Rocha, E. I. S.; Samo, K.; Miller-Jensen, K.

Cytokine dysregulation during microbial infections can dangerously increase the severity of associated diseases. Non-additive responses to simultaneous activation of toll-like receptor (TLR) signaling pathways are one potential contributor. Here we explored mechanisms underlying the TLR2/MyD88 and TLR3/TRIF-mediated responses in macrophages for canonical pro-inflammatory and antiviral targets including TNF, IL-6, IL-12p40, IFN-{beta} and CXCL10. We found that all targets exhibited characteristic levels of synergistic cytokine activation that varied with TLR-ligand concentration and exposure time. These trends were conserved when the TLR3-ligand polyI:C (PIC) was combined with either of the TLR2-mediated pathogens S. aureus or L. pneumophila. Using pharmacological inhibitors, genetic knockouts, and recombinant cytokines, we explored how TLR2-TLR3-induced paracrine signaling via TNF and IFN-{beta} affected synergy. While TNF synergy is paracrine-independent, IFN-{beta} contributes to synergistic activation of IL-6 and IL-12p40. When IFN-{beta} was directly combined with P3C or S. aureus infection, low concentrations produced modest synergy for both, while high concentrations increased IL-6 but antagonized IL-12p40. Thus, multiple mechanisms regulate TLR2/TLR3-mediated synergistic cytokine activation in macrophages, including type I interferon modulation of TRIF-independent pathogen stimulation that increases inflammatory signaling. These findings have important implications for therapeutic modification of cytokine-driven inflammation, including cytokine storms and the development of vaccine adjuvants.