Excessive complement activation is associated with a wide spectrum of pathologies including malaria, sickle cell disease, autoimmune diseases, trauma injury, and sepsis. In the U.S., sepsis alone is responsible for over 210,000 deaths each year, with associated costs estimated at over $16.7 billion. Our current understanding of sepsis is that complement-mediated host immune responses to infection are responsible for microcirculatory distress and severe organ damage. Red blood cells (RBCs) have critical, non- redundant roles in maintaining a non-inflammatory intravascular environment by capturing complement- opsonized particles through complement receptor 1 (CR1) and delivering them to macrophages in the liver and spleen. We have found that during excessive complement activation the additional engagement of glycophorin A (GPA) by soluble complement fragments significantly inhibits RBC membrane deformability and promotes RBC ATP release. Our new data challenge the classic paradigm of RBCs as singularly non-inflammatory cells, revealing that during excessive complement activation, RBCs cease to maintain a non-inflammatory environment and actively promote a pro-inflammatory intravascular milieu. Mechanisms responsible for changing RBCs into proinflammatory cells have not been known. A better understanding of the """"""""reprogramming"""""""" of RBCs into proinflammatory cells will allow the development of novel, effective therapies for septic patients. The long-term objective of this work is to understand the contribution of RBCs and complement to tissue and organ damage during sepsis. Our overall hypothesis is that during excessive complement activation, engagement of GPA by excess complement fragments reprograms RBCs into pro- inflammatory cells through a critical ATP-dependent autocrine signaling mechanism. The overall objective of our proposal is to determine the mechanisms by which engagement of GPA by complement fragments promote ATP release and inhibit RBC functions. The rational for these studies is that we have revealed a unique and crucial purinergic requirement for the GPA-mediated detrimental effect of complement on RBC functions, which suggests that RBC-generated ATP, through autocrine and paracrine purinergic mechanisms, promotes and maintains an inflammatory intravascular milieu. The innovation of our studies is that we have discovered a novel facet of RBC-complement interaction that may considerably impact the efficacy and design of targeted therapeutic approaches that will significantly lower the morbidity and mortality of sepsis. Our studies will have a significant impact on our understanding of the potential of RBCs and purinergic signaling as novel targets for therapy in pathological situation associated with excessive complement activation, as well as on our basic understanding of RBC biology in normal and pathological situations.

Public Health Relevance

The objectives of this proposal are to determine the effects of complement and purinergic signaling on red blood cell (RBC) functions during sepsis. Our in vitro findings regarding the functional consequences of complement-initiated CR1 and GPA (glycophorin A) signaling will be validated in vivo using RBCs from septic patients. In addition, changes in hemorrheology, due to decreased RBC membrane deformability, will be directly visualized and quantified directly in capillary circulation from septic patients using sidestream darkfield microscopy. Therefore, the results of the study will uniquely enhance understanding of the mechanisms responsible for multiorgan injury following sepsis and septic shock. The studies will have a significant impact on the current understanding of the mechanisms responsible for mortality and morbidity associated with sepsis by addressing the newly recognized pro-inflammatory role of RBCs in sepsis. By uncovering the complement- and purinergic-dependent mechanisms responsible for altering RBCs in sepsis, the studies may allow the development of novel, effective and targeted therapeutic approaches that will intercept the complement and purinergic signaling cascades, and significantly lower morbidity and mortality of sepsis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL096795-04
Application #
8631096
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Qasba, Pankaj
Project Start
2011-05-01
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
4
Fiscal Year
2014
Total Cost
$426,300
Indirect Cost
$181,300
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Andersen, Mikkel S; Lu, Shulin; Lopez, Gregory J et al. (2018) A Novel Implementation of Magnetic Levitation to Quantify Leukocyte Size, Morphology, and Magnetic Properties to Identify Patients with Sepsis. Shock :
Andersen, Mikkel Schou; Howard, Emily; Lu, Shulin et al. (2017) Detection of membrane-bound and soluble antigens by magnetic levitation. Lab Chip 17:3462-3473
Khoory, Joseph; Estanislau, Jessica; Elkhal, Abdallah et al. (2016) Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function. PLoS One 11:e0141206
Danielson, Kirsty M; Estanislau, Jessica; Tigges, John et al. (2016) Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry. PLoS One 11:e0144678
Baday, Murat; Calamak, Semih; Durmus, Naside Gozde et al. (2016) Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living. Small 12:1222-1229
Elkhal, Abdallah; Rodriguez Cetina Biefer, Hector; Heinbokel, Timm et al. (2016) NAD(+) regulates Treg cell fate and promotes allograft survival via a systemic IL-10 production that is CD4(+) CD25(+) Foxp3(+) T cells independent. Sci Rep 6:22325
Gokhin, David S; Nowak, Roberta B; Khoory, Joseph A et al. (2015) Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane. Mol Biol Cell 26:1699-710
Kreimer, Simion; Belov, Arseniy M; Ghiran, Ionita et al. (2015) Mass-spectrometry-based molecular characterization of extracellular vesicles: lipidomics and proteomics. J Proteome Res 14:2367-84
Knowlton, S M; Sencan, I; Aytar, Y et al. (2015) Sickle cell detection using a smartphone. Sci Rep 5:15022
Knowlton, Stephanie; Yu, Chu Hsiang; Jain, Nupur et al. (2015) Smart-Phone Based Magnetic Levitation for Measuring Densities. PLoS One 10:e0134400

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