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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM082852-04
Application #
8308620
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Dunsmore, Sarah
Project Start
2009-08-03
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
4
Fiscal Year
2012
Total Cost
$311,819
Indirect Cost
$105,998
Name
University of Pittsburgh
Department
Surgery
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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
Collage, Richard D; Howell, Gina M; Zhang, Xianghong et al. (2013) Calcium supplementation during sepsis exacerbates organ failure and mortality via calcium/calmodulin-dependent protein kinase kinase signaling. Crit Care Med 41:e352-60
Gomes, Marco Tulio R; Campos, Priscila C; Oliveira, Fernanda S et al. (2013) Critical role of ASC inflammasomes and bacterial type IV secretion system in caspase-1 activation and host innate resistance to Brucella abortus infection. J Immunol 190:3629-38
Guo, Lanping; Stripay, Jennifer L; Zhang, Xianghong et al. (2013) CaMKI* regulates AMP kinase-dependent, TORC-1-independent autophagy during lipopolysaccharide-induced acute lung neutrophilic inflammation. J Immunol 190:3620-8
Howell, Gina M; Gomez, Hernando; Collage, Richard D et al. (2013) Augmenting autophagy to treat acute kidney injury during endotoxemia in mice. PLoS One 8:e69520
Zhang, Xianghong; Guo, Lanping; Collage, Richard D et al. (2011) Calcium/calmodulin-dependent protein kinase (CaMK) Ialpha mediates the macrophage inflammatory response to sepsis. J Leukoc Biol 90:249-61