A. C. Bolognese1,2, A. Sharma3, W. Yang1,2,3, J. Nicastro1, G. F. Coppa1, P. Wang1,2,3 1Hofstra North Shore-LIJ School Of Medicine,Department Of Surgery,Manhasset, NY, USA 2Elmezzi Graduate School Of Molecular Medicine,Manhasset, NY, USA 3The Feinstein Institute For Medical Research,Center For Translational Research,Manhasset, NY, USA
Introduction: Cold-inducible RNA-binding protein (CIRP) was recently discovered as a proinflammatory mediator in sepsis that stimulates cytokine release from macrophages. In light of the immune dysregulation seen in sepsis, the effect of CIRP on the innate and adaptive immune response of T cells is an area of interest that has not yet been characterized. Here, we hypothesized that extracellular CIRP activates splenic T cells via a toll-like receptor 4 (TLR4)-dependent mechanism and promotes a proinflammatory Th1 response.
Methods: C57BL/6 wild type (WT) and TLR4 knockout (TLR4KO) mice received intravenous (IV) injection of recombinant murine CIRP (rmCIRP, 5 mg/kg BW) or PBS (vehicle). At 20 h after injection, splenocytes were isolated, labeled, and analyzed by flow cytometry. The early T cell activation marker CD25 was used to identify activated T cells. CD4 T cells isolated from the spleens of WT and TLR4KO mice were incubated on anti-CD3/anti-CD28-coated plates with or without rmCIRP (1 µg/ml) for 20 h, followed by flow cytometry or PCR array analysis.
Results: After rmCIRP injection in WT mice, the CD25+ CD4 T cell population was increased 4.1-fold compared to PBS; however, there was no significant change in the number of CD25+ CD4 T cells in TLR4KO mice (Table). Furthermore, incubation of WT CD4 T cells with rmCIRP and CD3/CD28 co-stimulation increased the population of CD25+ CD4 T cells by 11% compared to PBS control. In contrast, CD4 T cells isolated from TLR4KO mice showed a decreased percentage of CD25+ CD4 T cells with rmCIRP incubation (Table). Additionally, an 84-gene Th1 & Th2 response PCR array (QIAGEN) showed significant upregulation of seven genes in CD4 T cells treated with rmCIRP compared to PBS control (n=3, P < 0.05). Of these, four were Th1-related genes; interferon-γ, interleukin 12 receptor beta 2, T-bet and colony stimulating factor 2 were increased 7.1-, 5.3-, 3.7- and 2.3-fold, respectively. Two CD4 T cell markers, interleukin 6 and suppressor of cytokine signaling 3, were increased 3.9- and 2.9-fold, respectively. Lastly, the Th2 cytokine interleukin 13 was increased 3.7-fold. No genes were downregulated.
Conclusion: Our findings demonstrate that CIRP activates T cells via a TLR4-dependent mechanism. We have further shown that CIRP promotes a proinflammatory Th1 response profile in activated CD4 T cells. These findings demonstrate that CIRP plays an important role in T cell dysregulation in sepsis.
