Cold-inducible RNA-binding protein (CIRP), released into the circulation during sepsis, causes lung injury via an as yet unknown mechanism. Since endoplasmic reticulum (ER) stress is associated with acute lung injury (ALI), we hypothesized that CIRP causes ALI via induction of ER stress. To test this hypothesis, we studied the lungs of wild-type (WT) and CIRP knockout (KO) mice at 20 h after induction of sepsis by cecal ligation and puncture (CLP). WT mice had significantly more severe ALI than CIRP KO mice. Lung ER stress markers (BiP, pIRE1α, sXBP1, CHOP, cleaved caspase-12) were increased in septic WT mice, but not in septic CIRP KO mice. Effector pathways downstream from ER stress – apoptosis, NF-κB (p65), proinflammatory cytokines (IL-6, IL-1β), neutrophil chemoattractants (MIP-2, KC), neutrophil infiltration (MPO activity), lipid peroxidation (4-HNE), and nitric oxide (iNOS) – were significantly increased in WT mice, but only mildly elevated in CIRP KO mice. ER stress markers were increased in the lungs of healthy WT mice treated with recombinant murine CIRP, but not in the lungs of TLR4 KO mice. This suggests CIRP directly induces ER stress via TLR4 activation. In summary, CIRP induces lung ER stress and downstream responses to cause sepsis-associated ALI.

Cold-inducible RNA-binding protein (CIRP) is a novel inflammatory mediator that stimulates the release of proinflammatory cytokines from macrophages in sepsis. Given the immune dysregulation that characterizes sepsis, the effect of CIRP on other immune cells is an area of increasing interest that has not yet been studied. In the present study, we hypothesized that extracellular CIRP promotes activation of T lymphocytes in the spleen during sepsis. We observed that mice subjected to sepsis by cecal ligation and puncture showed significantly higher expression of the early activation markers CD69 and CD25 at 20 h on CD4 splenic T cells, and significantly higher CD69 expression on CD8 splenic T cells compared with sham-operated controls. Furthermore, at 20 h after receiving intravenous injection of recombinant murine CIRP (rmCIRP, 5 mg/kg body weight (BW)) or PBS (vehicle), those mice receiving rmCIRP showed significantly increased expression of CD69 and CD25 on both CD4 and CD8 splenic T cells. This effect, however, was not seen in TLR4-deficient mice after rmCIRP injection. In addition, treatment with CIRP predisposed CD4 T cells to a Th1 hyperinflammatory response profile, and influenced CD8 T cells toward a cytotoxic profile. Taken together, our findings indicate that CIRP is a proinflammatory mediator that plays an important role in T-cell dysregulation during sepsis in a TLR4-dependent manner.

Inhibition of receptor-interacting protein kinase 1 by necrostatin-1 decreases systemic and pulmonary inflammation, decreases lung injury, and increases survival in neonatal mice with sepsis. Targeting the necroptosis pathway might represent a new therapeutic strategy for neonatal sepsis.

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Introduction Sepsis remains one of the leading causes of infant death worldwide. It is characterized as uncontrolled inflammatory responses due to proven bacterial infection. Despite improvement in supportive care and the availability of effective antibiotics, no specific therapy targeting the dysregulated inflammatory response is available for neonatal sepsis. Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a secretory glycoprotein abundantly present in human milk. MFG-E8 suppresses the systemic inflammatory responses in adult murine injury models by improving the clearance of dying cells. We hypothesized that exogenous administration of recombinant mouse (rm) MFG-E8 could inhibit the exaggerated inflammatory response and lung injury in a murine model of neonatal sepsis. Methods Neonatal sepsis was induced in 5–7 day old male and female C57BL6 mice using an intraperitoneal injection of cecal slurry (CS). At 1h after sepsis induction, a single dose of 40 μg/kg rmMFG-E8 or vehicle was administered via retro-orbital injection. All neonates were returned to their mothers as a group. At 10 h after CS injection, pups were euthanized and blood and lung tissues were collected. Control mice underwent similar procedure with the exception of CS IP injection. Results Serum LDH, IL-1β and IL-6 were significantly increased 10 h after CS injection. Treatment with rmMFG-E8 decreased these levels by 30%, 56% and 37%, respectively. Lung morphology was significantly compromised in the vehicle group after CS injection whereas the rmMFG-E8 treated groups demonstrated a 48% improvement in the lung injury score. Lung IL-6 and MIP-2 protein levels were significantly reduced with rmMFG-E8 treatment. Lung neutrophil infiltration as observed by Gr-1 staining and, TUNEL positive cells were also significantly reduced with rmMFG-E8 treatment. Conclusion Treatment with rmMFG-E8 attenuated inflammation and lung injury in murine neonatal sepsis. Thus, MFG-E8 could be developed as a possible therapy for neonatal sepsis.