The complex biological processes of sepsis are poorly understood, which has hindered the development of sepsis-specific therapies, reflected by a mortality rate that has remained unchanged for 25 years. Impaired calcium (Ca2+) handling has been cited as the mediator of aberrant inflammation underlying the cell death and organ dysfunction of sepsis. Surprisingly, little is known of how this prevalent (88% of severely septic patients) pathophysiologic condition develops nor the signaling pathways and cellular responses regulated by these altered Ca2+ signals. Recently, we identified the calcium/calmodulin-dependent protein kinases (CaMK), a family of serine/threonine kinases responsive to intracellular Ca2+ concentration [Ca2+], mediate Ca2+-dependent signaling in the MF. We now recognize that CaMK, specifically CaMKI and IV, are operant in vivo and regulate key mediators of septic inflammation implicated in organ dysfunction and death. This proposal will focus upon the mechanisms by which CaMKI and IV mediate LPS induced Ca2+ signaling in MF. We propose that following LPS stimulation of the TLR4 pathway in MF, a ryanodine receptor (RyR)-gated cytosolic Ca2+ transient activates CaMKI and CaMKIV. CaMKI and CaMKIV, in turn, regulate the release of HMGB1, TNFa, and IL-10, key mediators of septic inflammation. CaMKI and CaMKIV assume distinct roles in mediating this inflammation, which we will define.
In Aim 1 we will utilize high-speed, depth resolved optical mapping of Ca2+ transients and genetically engineered mice lacking key components of TLR4 and CaMK signaling pathways to characterize the mechanisms of LPS-induced Ca2+ signaling and CaMK activation in MF.
Aim 2 will determine the mechanisms of CaMKI- and CaMKIV-dependent regulation of HMGB1 release from MF, as HMGB1 has been causally associated with septic mortality.
In Aim 3 we show that these mechanisms are operant in an in vivo CLP model of surgical sepsis. We will show, using in vivo CaMK RNAi and mice deficient in the expression of CaMKIV, that CaMKI and CaMKIV regulate the inflammatory response during sepsis, and we will define their roles in organ dysfunction and death. The combined studies may provide key insights linking Ca2+ signaling in MF to the dysregulated Ca2+ handling underlying the inflammation and organ dysfunction of sepsis.

Public Health Relevance

This project will determine TLR4-dependent calcium and calcium/calmodulin-dependent protein kinase (CaMK) signaling in macrophages and establish perturbations in these transduction systems as a biological mechanism underlying the inflammation and organ dysfunction of sepsis. An understanding of these mechanisms will increase our understanding of inflammation, prove useful in the design of novel forms of immunomodulatory therapy, and provide insight into the potential detriment of current practices of calcium supplementation in critical illness.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Dunsmore, Sarah
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University of Pittsburgh
Schools of Medicine
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Lewis, Anthony J; Griepentrog, John E; Zhang, Xianghong et al. (2018) Prompt Administration of Antibiotics and Fluids in the Treatment of Sepsis: A Murine Trial. Crit Care Med 46:e426-e434
Lewis, Anthony J; Zhang, Xianghong; Griepentrog, John E et al. (2018) Blue Light Enhances Bacterial Clearance and Reduces Organ Injury During Sepsis. Crit Care Med 46:e779-e787
Lewis, Anthony J; Lee, Janet S; Rosengart, Matthew R (2018) Translational Sepsis Research: Spanning the Divide. Crit Care Med 46:1497-1505
Zhang, Xianghong; Yuan, Du; Sun, Qian et al. (2017) Calcium/calmodulin-dependent protein kinase regulates the PINK1/Parkin and DJ-1 pathways of mitophagy during sepsis. FASEB J 31:4382-4395
Lewis, Anthony; Zuckerbraun, Brian; Griepentrog, John et al. (2017) Reducing Animal Use with a Biotelemetry-Enhanced Murine Model of Sepsis. Sci Rep 7:6622
Lewis, Anthony J; Yuan, Du; Zhang, Xianghong et al. (2016) Use of Biotelemetry to Define Physiology-Based Deterioration Thresholds in a Murine Cecal Ligation and Puncture Model of Sepsis. Crit Care Med 44:e420-31
Lewis, Anthony J; Billiar, Timothy R; Rosengart, Matthew R (2016) Biology and Metabolism of Sepsis: Innate Immunity, Bioenergetics, and Autophagy. Surg Infect (Larchmt) 17:286-93
Yuan, Du; Collage, Richard D; Huang, Hai et al. (2016) Blue light reduces organ injury from ischemia and reperfusion. Proc Natl Acad Sci U S A 113:5239-44
Zhang, Xianghong; Howell, Gina M; Guo, Lanping et al. (2014) CaMKIV-dependent preservation of mTOR expression is required for autophagy during lipopolysaccharide-induced inflammation and acute kidney injury. J Immunol 193:2405-15
Zhao, Y; Xiong, Z; Lechner, E J et al. (2014) Thrombospondin-1 triggers macrophage IL-10 production and promotes resolution of experimental lung injury. Mucosal Immunol 7:440-8

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