Blood Systems Response to Sepsis

November 9, 2010

Purpose

The Division of Blood Diseases and Resources of the NHLBI convened a Workshop on “Blood Systems Response to Sepsis” on June 15-16, 2010 that assembled an interdisciplinary group of experts to review state of the science advances in blood systems- based investigations and identify scientific priorities for new studies in the field of sepsis with special emphasis on its mechanisms, treatment, and prevention. Consistent with the NHLBI Strategic Plan, this initiative meets three key goals, namely, Goal 1: to increase understanding of the molecular and physiological basis of health and disease and use that understanding to improve diagnosis, treatment, and prevention; Goal 2: To improve understanding of the clinical mechanisms of disease and thereby enable better prevention, diagnosis, and treatment; and Goal 3: to translate research into practice for the benefit of personal and public health (http://apps.nhlbi.nih.gov/strategicplan/).

Background

Sepsis poses a growing public health problem in the United States and globally. In the US, as pointed out in an Opening Statement by Dr. Susan Shurin, Acting Director of NHLBI, it is estimated that 900,000 patients will be diagnosed with sepsis this year, with an estimated mortality rate of 30%. These patients include infants, critically ill, immunocompromised hematopoietic stem cell transplant recipients, and other critically ill patients, as well as patients undergoing elective surgery, diabetics, and a growing elderly population. In the US, the estimated cost of ICU care for patients with sepsis exceeds $20 billion annually. Despite the worldwide “Surviving Sepsis Campaign” that was launched in 2001 to curtail sepsis’ devastating consequences, the mechanism of sepsis progression to irreversible multi-organ failure and death remains an enigma. Due to diversity of microbial agents that cause sepsis, its pathogenesis as well as laboratory and clinical hallmarks are not uniformly established. For example, one of the most significant hallmarks of sepsis is hypotension in the context of febrile response to infection since a delay in its early control correlates with strikingly higher mortality.

Summary of Discussion

Sepsis evolves from local or generalized invasion of the body by pathogenic microbes. Their genetic prowess outpaces development of new anti-microbial therapeutics. Blood systems that respond to microbial virulence factors encompass innate and adaptive immune cells, platelets, and the plasma proteins that represent antibody, complement, coagulation, and fibrinolysis networks. These blood systems are enclosed by the enormous surface of the microvascular endothelium. The blood/tissue barrier formed by endothelium and adjoining structures is vital to maintaining function of key organs during sepsis. The vasculature is profoundly altered by the mix of microbial virulence factors and proinflammatory mediators released from activated blood cells that gain access to surrounding tissue by crossing the leaky endothelial boundary. Severe endothelial dysfunction results from loss of the homeostatic function of the microvascular endothelium and contributes to hypoxic injury to the lungs, heart, brain, kidneys, liver, skin and other organs. Acute Respiratory Distress Syndrome (ARDS) and Disseminated Intravascular Coagulation (DIC) are two major complications of sepsis. The prevailing sepsis paradigm has been accepted as a systemic inflammatory response syndrome, also known as cytokine storm, due to infection. Our current understanding of sepsis expands “the cytokine storm paradigm” to the unifying concept of severe endothelial dysfunction syndrome in response to intravascular or extravascular microbial agents that cause multi-organ failure. In the late stages of sepsis, it appears that a hypofunctional state of the innate and adaptive immune systems also contributes to lethal outcomes. Thus, new strategies are urgently needed to support vascular integrity/barrier function, implement endothelial cytoprotection mechanisms, and limit detrimental hemostatic and inflammatory processes. These new strategies complement the arsenal of anti-microbial agents and supportive respiratory care in order to reduce the heavy toll of sepsis.

Genetic predisposition to sepsis in different age groups is incompletely understood and awaits robust studies evolving from rapid advances in genomic technologies. Application of high-throughput DNA sequencing technologies, such as deep transcriptome, exome, and whole genome sequencing, are now within the reach of individual investigators. This technology surge provides a long-awaited opportunity to analyze sepsis susceptibility genes as potential biomarkers and therapeutic targets. Predisposition to sepsis may be related to defects in innate and adaptive immunity cellular and humoral components, including complement and antibody system proteins. Moreover, proteins that contribute to coagulation, fibrinolysis, and platelet function, different phenotypes of microvascular endothelium in organ-specific vascular beds, and transcriptional and posttranscriptional regulation await in-depth analysis in the context of sepsis. Emerging knowledge of microRNAs (miRs) in heart and vascular remodeling is being followed by evidence for regulatory functions in innate immunity. Moreover, the potential roles of chromatin modifications and of cytotoxicity of histones released from apoptotic and necrotic cells open new vistas for studies of predisposition to sepsis and its progression to a fatal outcome.

The coagulation and fibrinolysis systems are critical components of the host response to microbial pathogens. These include Escherichia coli, Pseudomonas aeruginosa, pathogenic Staphylococci, Streptococci Group A and Group B, and Candida albicans, among others. Intravascular or extravascular microbial pathogens frequently express virulence factors that are specifically designed to engage the components of the coagulation and fibrinolysis systems in their vertebrate hosts. For example, Staphylococcus aureus, including the growing threat of methicillin-resistant strains (MRSA), has evolved a particularly impressive repertoire of factors that mediate bacteria-hemostatic factor interactions to control fibrin deposition and dissolution. Known and suspected virulence factors include the bacterial fibrinogen receptor, clumping factor A (ClfA), the bacterial prothrombin activators, coagulase and von Willebrand factor binding protein, and the bacterial plasminogen activator, staphylokinase. These virulence factors contribute to ~156,000 invasive staphylococcal infections annually, with a 50% survival rate. The estimated annual cost of hospital-and community-acquired staphylococcal infections in the US is $16 billion. Similarly, Group A streptococci (GAS) have adapted a key virulence factor, streptokinase, to human plasminogen, contributing to devastating and often deadly invasive infections. GAS is estimated to account for over 700 million human infections and over 500 thousand deaths per year world-wide. Taken together, these findings lend support to the working hypothesis that coagulation and fibrinolysis factors, in general, and fibrinogen and plasminogen, in particular, are likely to be important determinants of bacterial virulence/host defense. Evolutionary pressures may account for the wide distribution of the normal physiologic levels for many coagulation and fibrinolysis factors, including von Willebrand factor and fibrinogen, which span ranges of 3- to 5-fold in the normal population. This variability also accounts for significant differences in thrombosis and bleeding risk. These findings also suggest the potential for developing novel therapeutic agents targeted at coagulation/fibrinolysis-related virulence factors produced by the sepsis-causing pathogens.

The known refractoriness of small laboratory animals and non-human primates to sepsis-causing pathogens awaits a comparative genetic analysis to draw lessons from divergent evolutionary guideposts. These studies will help to understand not only genetic basis of sepsis susceptibility but also molecular mechanism of host-pathogen interaction. There is also a perceived need to identify and characterize specific microbial virulence factors that perturb the coagulation and fibrinolysis systems, the function of platelets and other blood cells, and/or endothelial physiology. Furthermore, we need to understand the mechanisms of microbial entry and spread, evasion of innate immunity, and strategies that these microbial virulence factors employ to cause microvascular damage and ultimately failure of key organs, especially the heart and lung. This knowledge deficit can be best addressed by interdisciplinary teams working to develop new animal models of sepsis that can reveal the molecular, cellular and end-organ pathophysiologic changes ensuing during this syndrome. Advanced animal models of sepsis would also stimulate and enable comprehensive genomic analyses of the genetic requirements of pathogens to cause and sustain this syndrome, thereby resulting in new knowledge and catalyzing novel translational research. Other critical knowledge gaps are a lack of understanding of the environmental cues that lead microbes to activate the expression of specific virulence genes prior to and during sepsis progression and the complex regulatory networks that control them. Studies of how microbial virulence factors interact with host molecules will unravel key mechanisms that lead to sepsis initiation and progression and offer great promise to promote the discovery of new, innovative therapies for sepsis. The primary purpose of such investigation is not the identification of new antibiotics. Rather, comprehensive analyses of the key host factors and pathogen genes contributing to and sustaining sepsis will identify and validate targets for the development of new classes of anti-sepsis therapeutics.

NHLBI-supported research provided the basis for the development of recombinant human activated protein C (APC) for sepsis treatment. Through an NHLBI-sponsored RFA that funded “Plasma Proteins with Potential Therapeutic Usefulness", the discovery of the hereditary human deficiencies of protein C that cause fatal neonatal purpura fulminans was subsequently translated into the only successful phase III trial for mortality reduction in adult severe sepsis (PROWESS trial) that led to rapid FDA approval of APC. In the face of the failures of antithrombin or tissue factor pathway inhibitor to reduce death in severe sepsis, the efficacy of recombinant APC (XIGRIS from Lilly) raises the possibility that APC's life-saving actions are not primarily anticoagulant in nature but rather include APC's anti-apoptotic and anti-inflammatory activities.

In summary, sepsis poses a serious challenge and a growing public health problem in the US and worldwide. However, recent advances in mammalian and microbial genetics, innate immunity and the cell biology of blood and vascular cells provide an unprecedented opportunity to launch new interdisciplinary studies focused on the fundamental role of blood systems in sepsis pathogenesis, the identification of predisposing factors, and the development of new targets for anti-sepsis therapy and prevention.

Research Opportunities and Priorities

HARNESSING GENOMIC APPROACHES TO ESTABLISH LANDMARKS OF SEPSIS SUSCEPTIBILITY, PROGRESSION AND OUTCOME

The dynamic interplay between host blood components and sepsis-causing microbes is a key driver of evolutionary variation in the genomes of both host and pathogen. These genetic variations are in turn key determinants of host disease susceptibility, progression and outcome. Although recent advances in genomic technology have contributed dramatically to our understanding of human and microbial population genomics, the interaction between polymorphisms in host blood components and pathogen remain largely unexplored. Further investigation of these interactions is likely to lead to enhanced understanding of sepsis pathogenesis and new opportunities for novel approaches to diagnosis, prevention, and therapy. Areas of research emphasis that would address this unmet need include:

• Application of state-of-the art genomic technologies to investigate the role of genetic variation in host blood components and genomes of sepsis-causing microbes in the pathogenesis of sepsis and multi-organ damage.
• Leveraging existing patient and pathogen biobanks and developing new, well-curated resources to address susceptibility and clinical outcomes in specific subsets of sepsis-prone patients. Examples include identification of host and pathogen genetic determinants contributing to the coagulation/fibrinolysis networks and/or vascular system dysfunction associated with Disseminated Intravascular Coagulation (DIC).
• Advancing the characterization of the dynamic host and pathogen interaction in sepsis initiation and progression, including gene-, protein- or metabolite-specific expression patterns.
• Identifying novel, biologically significant polymorphic genetic variations in host blood system components in bacterial and/or fungal infections.
• Exploiting the power of genetic variation in blood system components as potential biomarkers for sepsis diagnosis, prognosis and choice of personalized therapy.

EXPLORING HOW SEPSIS LEADS TO ABERRANT HEMOSTASIS, MICROVASCULAR THROMBOSIS, AND VASCULAR PATHOBIOLOGY

Although the blood/tissue barrier formed by the endothelium and adjoining structures is vital to maintaining the function of all major organs, our current knowledge of the mechanisms that lead to endothelial alterations during sepsis is severely limited. For example, the mechanisms responsible for the extremely rapid microvascular injury induced by the mix of microbial virulence factors and proinflammatory mediators released from activated blood cells in sepsis -associated purpura fulminans, is inadequately understood. Addressing these and other unmet needs within the fields of hemostasis, thrombosis and vascular biology as related to sepsis, should not only advance the understanding of the cardinal mechanisms of cardiovascular collapse but also pave the way for innovative therapies. Areas of research emphasis that would catalyze advances in our knowledge far beyond where the field is currently include:

• Better understanding of structural and functional biology of blood and vascular cells (endothelium, smooth muscle, pericytes) as well as coagulation and fibrinolysis networks in terms of their responses to microbial virulence factors and proinflammatory mediators and the role of regulatory mechanisms such as microRNAs in sepsis. These studies could be conducted as clinical investigations or in vivo animal models.
• Better understanding of the role of innate immunity mediated by its cellular effectors, phagocytes, as well as platelet and endothelial cell activation involving innate immunity receptors (Toll-like Receptors)-initiated pathways. The mechanism of Neutrophil Extracellular Traps (NETs) formation and function in sepsis also awaits elucidation.
• Development of new or refined sepsis models that can be perturbed at multiple levels of experimental specificity (e.g. genetics of the host and microbe, facile blood sampling and analysis, imaging, gene expression profiling, etc.) and that can be tested for multiple sepsis-causing pathogens coupled to the analysis of novel therapeutics and vaccines.
• Better understanding of mechanisms for organ-specific vascular damage, involving heart, lung, brain, kidneys, and other sepsis- affected organs. These investigations can be complemented by development and application of novel methods for studies of vascular integrity and vascular injury in animal models of sepsis, and innovative approaches to cytoprotection of the vasculature.

CHARACTERIZING EVOLVING PATTERNS OF PATHOGEN-HOST INTERACTION IN SEPSIS

There is a significant knowledge gap in understanding the mechanisms by which bacterial proteins engage host factors in innate immunity including phagocytes, complement, coagulation and fibrinolysis networks, and enable pathogen virulence and vascular injury. Areas of research emphasis that would address this unmet need include:

• Evaluating the dynamic response of the host following development of sepsis, with specific focus on discovering key "turning points" at which intervention could alter the course of disease.
• Biochemical/biophysical, structural, and physiological studies of the unique interplay between microbes and their corresponding host factors to characterize already identified targets for the development of new therapeutics.
• Analysis of host factors in humans and/or animal models of sepsis uniquely required for the pathogenesis of this syndrome in the context of an infection with any one or multiple clinically relevant microbes. The main goal of these studies is the identification and validation of targets for the development of new classes of anti-sepsis therapies.

SEEKING AND EVALUATING NOVEL THERAPIES FOR SEPSIS

There is a dearth of successful novel therapeutics for sepsis in spite of extensive efforts to target the innate immune response and coagulation pathways. New efforts are urgently needed to translate emerging concepts into novel therapeutics. Areas of research that would address this unmet need include:

• Exploration of the therapeutic potential of small molecule and novel recombinant protein derivatives that counteract deleterious effects of procoagulant, proinflammatory, and proapoptotic pathways activated at the blood and tissue interface.
• Pharmacological and chemical-genomic studies for molecules that impact the outcome of sepsis using either animals or in vitro models simulating sepsis process and investigation of their use in sepsis caused by various pathogens.
• Development of small molecules, antibodies, vaccines and novel recombinant proteins targeting bacterial factors known to perturb host regulatory mechanisms involved in maintaining hemostasis and vascular integrity.
• Development of novel cytoprotective agents encompassing second generation APC and other therapeutics that emerge from mechanistic insights into the ability of APC and of the protein C system to reduce mortality in animal sepsis models.

Publication Plans: NHLBI website, publication in scientific journals encompassing research fronts covered by this Report.

Suggested Funding Mechanisms for Research, Training, and Career Development:
The Workshop participants enthusiastically endorsed the interdisciplinary team approach to address unmet needs in research areas related to blood systems response to sepsis-causing pathogens. They recommend the development of an RFA to support investigator -initiated R01 grants and R01 style collaborative grants, linking 2-4 investigators with complementary expertise to address key research areas of sepsis genetic predisposition, microbial pathobiology, mechanisms of vascular injury, and new therapies. A new cadre of investigators to participate in these interdisciplinary efforts needs to be trained and nurtured through innovative training and career development programs.

Additional Comments from Consultants

Comments from Kenneth Bauer, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston

The document provides an excellent summary of the current state of the field and touches on much of the important research done in this area. I concur that collaborative research projects, particularly those between investigators with a focus on virulence factors of pathogens and vascular biologists, would be productive in moving the field forward.

Comments from Shaun Coughlin, University of California, San Francisco

The document is very good, and the focus on acquiring a better basic understanding of sepsis at the molecular, cell biological and tissue levels is appropriate. Additionally, I do worry that this field will continue to be stuck between the lab and the clinical trial for want of a path to validating targets and a means of developing meaningful information before endpoint trials, and that this has and will continue to diminish overall interest in the basic science. Fixing this is a long-term proposition. I would invest in:

1. Better characterization of human sepsis at the molecular and physiological levels, with the latter including serial measurements of vascular permeability and perfusion as well as organ metabolism using modern imaging techniques and methods for hunting for biomarkers
2. A parallel detailed characterization of the same endpoints in animal models, with an attempt to identify a set of models that most identify human sepsis.
3. A focus on identifying measurements in the above that predict outcome.

The notion is that such a program might both generate new targets and paths to target validation. Most of these thoughts are explicit or implied in the document.

Comments from William P. Faye, University of Missouri, Columbia

Thank you for asking my opinion. I read the document. It is excellent and exciting. I strongly endorse the strategy of multi-disciplinary groups and bundled RO1s as a funding strategy. For these research networks to be successful, it will be essential to include bacteriologists, immunologists, and other related experts along with investigators from the hemostasis and thrombosis field. In addition, it is imperative to develop animal models that better reflect the process of sepsis in humans.

Comments from Steven Holland, National Institute of Allergy and Infectious Diseases, NIH, Bethesda

The NHLBI Working Group on Sepsis lays out fundamental areas that are ripe for investigation along with targeted approaches in the critical areas of host and microbial genetics with an eye toward basic understandings that will help identify novel areas for intervention. The summary is both insightful and panoramic, taking into account the most recent experiences in drug development and their disappointments as well as areas for deeper exploration. Development of well curated specimens and biobanks is likely to be especially important. The suggestions for areas of funding should guide NHLBI in its strategic planning for this critical area.

Comments from Evan Sadler, Washington University, St. Louis

I read the document carefully and have no specific suggestions for changes, but do have a couple of comments:
One of the hardest problems in studying sepsis may be the heterogeneity of subjects available to study. Patients with sepsis are often very complex, difficult to compare. Efforts to improve early identification of study subjects, to reduce heterogeneity of underlying conditions and disease course would be useful.
Exposure of patients to treatments other than antibiotics and fluids may be important to monitor because some such treatments may modulate the immune response or alter host pathogen interactions. In particular, transfusion of any blood products and intravenous gamma globulins may be useful to monitor accurately for later analysis or stratification.

Comments from Ajit Varki, University of California, San Diego

Overall I like the report very much, and have just a few comments and suggestions that may be helpful. While comparisons amongst individual human are emphasized appropriately, the single big omission is the lack of emphasis on the evolutionary comparative approach. Why is it that certain rodents and monkeys are relatively so much more resistant to sepsis than humans? What determines such differences even amongst closely related species and taxa? The answers to this big question may teach us a lot about human sepsis, and what one might do about it.

While high-throughput large scale studies are very popular these days, I suggest that their overall value and yield of useful information per research dollar spent should be frequently compared with more focused molecular/process-oriented research. Included in such an analysis should be the potential negative value of large-scale approaches, which can generate confusing data, sometimes making it hard to see the forest for the numerous trees. In this regard, I applaud the emphasis on investigator-initiated R01s, as apposed to "NIH-initiated research". On the other hand large-scale approaches might well help to sub-classify sepsis into more definable sub-syndromes for further study.

Finally, I suggest that the focus on very well-defined targets be supplemented with continued re-analysis of complex old drugs like heparin, which are highly multifunctional. In a complex and unpredictable syndrome like sepsis it may be more beneficial to weakly hit multiple targets than to punch very hard on a single one.

NHLBI Contacts:

Rita Sarkar, Ph.D., NHLBI, NIH
Email: sarkarr@nhlbi.nih.gov

Working Group Members:

Chair: Jack J. Hawiger, M.D., Ph.D., Vanderbilt University, Nashville, TN

Co-Chair: David Ginsburg, M.D., University of Michigan, Ann Arbor, MI

Members:

Jean-Laurent Casanova, M.D., Ph.D., The Rockefeller University, New York, NY

Jay Degen, Ph. D., University of Cincinnati, Cincinnati, OH

Bruce Furie, M.D., Harvard Medical School, Boston, MA

John H. Griffin, Ph.D., The Scripps Research Institute, La Jolla, CA

James M. Musser, M.D., Ph.D., The Methodist Research Institute, Houston, Texas

Olaf Schneewind, M.D., Ph.D., University of Chicago, Chicago, IL

Denisa D. Wagner, Ph. D., Harvard Medical School, Boston, MA

Aimee Zaas, M.D., MHS, Duke University, Durham, N.C.

Consultants:

Kenneth A. Bauer, M.D., Harvard Medical School, Boston, MA

Shaun Coughlin, M.D., Ph.D., University of California, San Francisco, CA

William (Bill) Fay, M.D., University of Missouri, Columbia, MO

Steven Holland, M.D., NIAID, NIH, Bethesda, MD

J. Evan Sadler,M.D., Ph.D., Washington University, St. Louis, MO

Ajit Varki, M.D., University of California, San Diego, CA