CIRP increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TLR4-IL-2 signaling in the late phase of sepsis

T helper (Th) cell imbalances have been associated with the pathophysiology of sepsis, including the Th1/Th2 and Th17/T regulatory cells (Treg) paradigms. Cold-inducible RNA-binding protein (CIRP), a novel damage-associated molecular pattern (DAMP) was reported that could induce T cell activation, and skew CD4+ T cells towards a Th1 profile. However, the effect and underlying mechanisms of CIRP on Th17/Treg differentiation in sepsis still remains unknown. A prospective exploratory study including patients with sepsis was conducted. Blood samples were collected from patients on days 0, 3 and 7 on admission. The serum CIRP and peripheral blood Treg/Th17 percentage was determined by ELISA and flow cytometry. CD4+ T cells from the spleen and lymph nodes of mice with experimental sepsis were collected after treatment with normal saline (NS), recombinant murine CIRP (rmCIRP) and C23 (an antagonist for CIRP-TLR4) at late stage of sepsis. RNA-seq was conducted to reveal the pivotal molecular mechanism of CIRP on Treg/Th17 differentiation. Naïve CD4+ T cell was isolated from the Tlr4 null and wildtype mice in the presence or absence rmCIRP and C23 to confirmed above findings. A total of 19 patients with sepsis finally completed the study. Serum CIRP levels remained high in the majority of patients up to 1 week after admittance was closely associated with high Treg/Th17 ratio of peripheral blood and poor outcome. A univariate logistic analysis demonstrated that higher CIRP concentration at Day 7 is an independent risk factor for Treg/Th17 ratio increasing. CIRP promotes Treg development and suppresses Th17 differentiation was found both in vivo and in vitro. Pretreated with C23 not only alleviated the majority of negative effect of CIRP on Th17 differentiation, but also inhibited Treg differentiation, to some extent. Tlr4 deficiency could abolish almost all downstream effects of rmCIRP. Furthermore, IL-2 is proved a key downstream molecules of the effect CIRP, which also could amplify the activated CD4+ T lymphocytes. Persistent high circulating CIRP level may lead to Treg/Th17 ratio elevated through TLR4 and subsequent active IL-2 signaling which contribute to immunosuppression during late phases of sepsis.