key: cord-0003909-5u1u3xoy authors: Chen, Lingye; Welty-Wolf, Karen E.; Kraft, Bryan D. title: Nonhuman primate species as models of human bacterial sepsis date: 2019-01-14 journal: Lab Anim (NY) DOI: 10.1038/s41684-018-0217-2 sha: 4041dbdf50df73b5c4dd4f9d11f74c7335c33564 doc_id: 3909 cord_uid: 5u1u3xoy Sepsis involves a disordered host response to systemic infection leading to high morbidity and mortality. Despite intense research, targeted sepsis therapies beyond antibiotics have remained elusive. The cornerstone of sepsis research is the development of animal models to mimic human bacterial infections and test novel pharmacologic targets. Nonhuman primates (NHPs) have served as an attractive, but expensive, animal to model human bacterial infections due to their nearly identical cardiopulmonary anatomy and physiology, as well as host response to infection. Several NHP species have provided substantial insight into sepsis-mediated inflammation, endothelial dysfunction, acute lung injury, and multi-organ failure. The use of NHPs has usually focused on translating therapies from early preclinical models to human clinical trials. However, despite successful sepsis interventions in NHP models, there are still no FDA-approved sepsis therapies. This review highlights major NHP models of bacterial sepsis and their relevance to clinical medicine. S epsis is a common clinical syndrome arising from a dysregulated host response to systemic infection leading to lifethreatening end-organ damage 1 . While the incidence of sepsis varies 2 , the substantial burden of critical illness reflects an aging and susceptible population with escalating comorbid conditions 3, 4 . With the exception of early antibiotics and fluids [5] [6] [7] , targeted new therapies for sepsis have remained elusive 8 . However, through research, our understanding of sepsis has evolved from being seen solely as an exuberant host immune response 9 to the intersection of pro-and anti-inflammatory, endothelial, coagulation, metabolic, endocrine, and neural pathways 10, 11 . The foundation of such research has been the use of small animals in studies to unravel mechanisms of sepsis pathophysiology and develop novel therapeutics 12, 13 . This review briefly presents the common nonhuman primate (NHP) models of bacterial sepsis and summarizes the contributions to our understanding of sepsis pathophysiology and drug discovery, and how they might impact on future targeted research. An ideal animal model of sepsis must balance biological feasibility and practical considerations. The animal species should recapitulate the same hemodynamic changes to infection seen in humans, such as hypotension, low systemic vascular resistance (SVR), and a compensatory increase in cardiac output (CO) following fluid resuscitation [14] [15] [16] . Animals should also demonstrate similar signs and symptoms of infection, such as decreased activity or oral intake, change in body temperature, and change in white blood cell count 12 . The model should include provision of antibiotics 17, 18 , a standard of care treatment for patients 1 that may alter host cytokine responses 19 and significantly improves survival 20 . The species should also activate similar molecular pathways, such as cytokine release in response to circulating pathogen-associated molecular patterns (PAMPs) 11 , and should lead to development of multiple organ dysfunction (MOD) 21, 22 . Practically, the model must be economically reasonable, easy to support, and large enough for the collection of physiological data. The earliest and most widely used animal species for sepsis studies has been rats and mice. Murines are attractive because they are inexpensive, easy to breed, versatile, and genetically modifiable 8, 12, 13 . The rat model of endotoxemic sepsis with fluid resuscitation relies on intravenous lipopolysaccharide (LPS) to mimic the physiological responses of Gram-negative sepsis in humans. Unlike human sepsis, which is a protracted response to live pathogens, LPS induces a rapid, transient inflammatory surge that causes mainly vascular injury 13 . The rat cecal-ligation and puncture (CLP) technique was developed to mimic human abdominal sepsis from live polymicrobial bacteria 12, 23, 24 . Though this model has been widely considered the gold standard for rodent studies, it is limited by variability in technique (e.g. number of punctures, size of puncture) 23 and pathogen (e.g. polymicrobial) 24 . As an alternative rat model, the monomicrobial Staphylococcus aureus impregnated clot model of peritoneal sepsis was developed to simulate post-operative abdominal wound infections 25 . Another model using intravenous injection of live bacteria simulates the bacteremia seen in up to 69% of human septic shock 24 , but does so by artificially infusing a large inoculum of bacteria into the bloodstream rather than by slow release from an established nidus of infection 12 . Rodent models suffer from important technical limitations. Their small size makes invasive hemodynamic monitoring difficult, and their small total blood volume (approximately 2 ml in mice and 20 ml in rats) limits serial phlebotomy 23 . Mice are also more resistant to LPS and bacterial pathogens than are humans, requiring 10 4 -to 10 5 -fold higher doses to achieve shock 8, 13, 24 . Once shock develops, the hemodynamic and cytokine profiles of small rodents differ greatly from those of humans 8, 24 , though the inconsistent use of fluid resuscitation in these studies may have affected the results. The sizable technical and biological challenges ultimately led to the use of larger mammals such as rabbits, sheep, dogs, and pigs, whose sizes permitted invasive and serial procedures 12, 23 . Many large animals also exhibit sepsis physiology that is similar to humans. For instance, pigs are prone to sepsis-induced capillary leak and development of pulmonary edema, and sheep display similar hyperdynamic responses (i.e. elevated heart rate, cardiac output, and oxygen delivery with depressed systemic vascular resistance) to endotoxin Nonhuman primate species as models of human bacterial sepsis Lingye Chen*, Karen E. Welty-Wolf and Bryan D. Kraft Sepsis involves a disordered host response to systemic infection leading to high morbidity and mortality. Despite intense research, targeted sepsis therapies beyond antibiotics have remained elusive. The cornerstone of sepsis research is the development of animal models to mimic human bacterial infections and test novel pharmacologic targets. Nonhuman primates (NHPs) have served as an attractive, but expensive, animal to model human bacterial infections due to their nearly identical cardiopulmonary anatomy and physiology, as well as host response to infection. Several NHP species have provided substantial insight into sepsis-mediated inflammation, endothelial dysfunction, acute lung injury, and multi-organ failure. The use of NHPs has usually focused on translating therapies from early preclinical models to human clinical trials. However, despite successful sepsis interventions in NHP models, there are still no FDA-approved sepsis therapies. This review highlights major NHP models of bacterial sepsis and their relevance to clinical medicine. as humans 23 . Nevertheless, notable physiological differences still exist between these large mammals and humans: Sheep and other ruminants have significantly more pulmonary intravascular macrophages compared to primates, which alters their susceptibility to sepsis-induced pneumonia and acute lung injury 26, 27 . Pigs demonstrate a profound rise in pulmonary vascular resistance in response to bacterial sepsis which can be fatal and experiment-limiting 23 . And dog models of septic shock are confounded by significant intestinal mucosal congestion and hemorrhage 23 . To circumvent the limitations of rodent and large mammal models, researchers developed nonhuman primate (NHP) models of bacterial sepsis 20, 28, 29 . Baboons, macaques, and chimpanzees have historically been the most popular primates studied, though as of 2013 the National Institutes of Health has limited its funding support to monkeys, as great apes are widely agreed to be too sentient to ethically justify their use. Originating from a common phylogenetic ancestor, humans and monkeys have nearly identical anatomy and physiology. For example, the baboon and human tracheobronchial trees share a similar dichotomous branching pattern with a bronchiolar transition zone between the small airways and distal alveolar regions 30 . Moreover, the alveolar region of baboons and humans contain a prominent alveolar interstitial compartment 31 . These features are less prominent or less uniform in rodents, which may partly explain differing patterns of pneumonia-induced acute lung injury (ALI) compared with humans [30] [31] [32] . Humans and baboons also display almost identical hemodynamic and cytokine responses to intravenous endotoxin and live bacteria 29, [33] [34] [35] . Following infusion of Gram-negative bacteria (E. coli), baboons develop tachycardia, increased cardiac output, decreased SVR, and hypotension similar to human sepsis 29 . Furthermore, the use of NHPs carries the same advantages seen in other large animals. For instance, the NHPs' large size allows for invasive monitoring, tissue collection, and serial phlebotomy 23 . Their use also satisfies the U.S. Food and Drug Administration (FDA) drug development requirement that investigational new drug (IND) applications include data from at least one non-rodent species 23 . As such, the NHP models are considered quite suitable for pre-clinical drug development studies. Endotoxemia model. Endotoxin plays a central role in the development of Gram-negative sepsis. Located on the outer cell membrane of Gram-negative bacteria, LPS is a pathogen-associated molecular pattern (PAMP) recognized by the innate immune pathogen recognition receptor, toll-like receptor 4 (TLR4), and activates pro-inflammatory and coagulation cascades 36 . Healthy human volunteers given intravenous LPS (2-4 ng/kg) developed tachycardia, decreased SVR, and depressed left ventricular function 34 . These hemodynamic changes are associated with the release of pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α ) and interleukin-6 (IL-6), and activation of coagulation and fibrinolysis pathways 37 (Fig. 1a, b) , similar to the responses of patients in septic shock. NHPs are more resistant to endotoxin than humans. The baboon (Papio ursinus or cynocephalus) requires 0.1 mg/kg 33 and the rhesus macaque (Macaca mulatta) 0.1 mcg/kg 38 to achieve a hyperdynamic state. One exception appears in older studies of chimpanzees (Pan troglodytes) which, like humans, develop a mild and reversible inflammatory state at 4 ng/kg 39, 40 . Nonetheless, NHP endotoxemia models are frequently employed to model Gram-negative sepsis without live pathogen inoculation, and they generally corroborate the hemodynamic and cytokine profiles seen in human sepsis 33 . Pharmacologic inhibitors are commonly used to test mechanism of immune pathways, and monoclonal antibodies have been used in NHP sepsis experiments to inactivate a variety of pro-inflammatory and coagulation mediators 38, 40, 41 . Chimpanzees given monoclonal anti-TNF-α antibodies (15 mg/kg) immediately following LPS injection had decreased downstream production of IL-6 and CXCL8 40 , and within the lungs, had increased fibrinolysis 42 compared to LPS-only controls. Baboons and rhesus monkeys treated with anti-TNF-α antibodies had lower serum TNF-α levels, attenuated coagulation responses, and less severe organ dysfunction than control monkeys 38, 43 . In the rhesus, treatment with anti-TNF-α antibodies significantly improved survival 38 . These findings suggest that TNF-α is not only a catalyst for other downstream inflammatory mediators, but also for sepsis-induced fibrinolysis and organ dysfunction (Fig. 1a) . Anti-TNF-α Ab Like TNF-α , IL-6 is also an early phase mediator (released within one hour) of LPS exposure 33, 37 . In chimpanzees with mild endotoxemia, anti-IL-6 antibodies attenuate thrombin-antithrombin III (TAT) complex activity in both serum 44 and lungs 42 , suggesting its role in the activation of sepsis-induced coagulation. Similarly, pretreatment with monoclonal antibody to tissue factor decreased TAT complex activity and coagulation response, but had no effect on cytokine release or fibrinolysis 41 (Fig. 1a) . IL-6 also influences the development of acute lung injury (ALI). In endotoxemic chimpanzees given anti-IL-6 antibody, procoagulant proteins were significantly reduced in bronchoalveolar lavage fluid (BALF) compared with untreated animals 42 . These studies also found that pro-coagulant proteins were elevated in the BALF without evidence of vascular permeability, suggesting that coagulation in ALI is a local response 42 . E. coli bacteremia model. Despite its ability to duplicate the initial hours of Gram-negative sepsis, the LPS model nonetheless cannot replace a live bacterial infection model. E. coli bacteremia in the baboon (Papio cynocephalus) is one of the earliest and most versatile models of NHP sepsis and has greatly expanded the understanding of sepsis-induced inflammation, coagulation, and organ injury [45] [46] [47] . A frequent complication of sepsis, disseminated intravascular coagulation (DIC) is a syndrome of dysregulated microvascular coagulation that can lead to MOD 45, 47, 48 . In the baboon with E. coli bacteremia, the degree of coagulopathy varies directly with the dose of pathogen. The three most common dosing models, lethal dose 100 (LD 100 ), LD 50 , and LD 10 , corresponding to a predicted mortality of 100%, 50%, and 10%, respectively 48 , have been used to elucidate mechanisms of sepsis-induced DIC and cellular injury, and to test novel sepsis therapies. The LD 100 model (10 10 CFU/kg E. coli) produces a highly virulent phenotype wherein all animals die within 24 to 32 hours 48, 49 . Administration of intravenous E. coli triggers the release of TNF-α , IL-6, and CXCL8 48 , activation of endothelial cells, and microvascular migration of leukocytes 50, 51 . Activated endothelial cells also express tissue factor (TF), a stimulus for intravascular and extravascular fibrin deposition 45, 47, 48 . Figures 1a,b demonstrate the TF-dependent coagulation pathways culminating in fibrin clot formation. Moreover, fibrinolysis is simultaneously triggered and inhibited by upregulation of tissue plasminogen activator (tPA), TAT complexes, and plasminogen activator inhibitor (PAI) 45, 46 . These competing processes result in unchecked and widespread microvascular clot formation and MOD 47 . NHP studies of coagulation cascade inhibitors further support DIC as a contributor to MOD (Fig. 1b, Table 1 ). Compared with control animals, baboons that received recombinant tissue factor-pathway inhibitor (TFPI) shortly after LD 100 infusion had an attenuated coagulation response, significantly reduced organ injury, and 100% survival at 7 days 52 . Likewise, LD 100 E. coli animals given monoclonal antibodies against tissue factor 53 or inactivated factor VIIa 54 had attenuated coagulopathy and lethality. Moreover, treatment of E. coli-infected baboons with recombinant human antithrombin III (ATIII) reduced inflammation, coagulation, fibrinolysis, and mortality 55 . Multi-organ dysfunction. The LD 100 model demonstrated that inflammation and coagulation interconnect through tissue factor, and that derangement of coagulation pathways culminates in DIC and lethal organ failure. However, due to its high lethality, the LD 100 model was unable to define the cellular injury and repair that occurs in sublethal sepsis, prompting development of LD 10 and LD 50 models. The baboon LD 50 model (10 9 CFU/kg E. coli) produced hypotension and progressive MOD that was fatal in 50% of animals. The baboon LD 10 model (10 7 -10 8 CFU/kg E. coli) produced mild hypotension and non-progressive organ dysfunction with spontaneous resolution. Unlike the LD 100 model where animals expired rapidly, these sub-lethal models survived long enough to demonstrate that sepsis is a multistage process driven by distinct mechanisms 48 . Although these experiments preceded the current standard of fluid resuscitation in early sepsis 6 , and ischemia-reperfusion is recognized and prevented clinically, these experiments show the natural course of under-resuscitated sepsis. The initial stage is a TNF-αand tissue factor-dependent and PAMP-mediated intravascular innate immune activation leading to microvascular DIC and organ ischemia. The second stage is the extravascular (organ) sequelae of ischemia-reperfusion injury 48 , with accumulation of free radicals and further release of inflammatory mediators even after pathogen clearance 56 . Thus, whereas the LD 100 animals expired from intravascular events (hemodynamic collapse and DIC) before reperfusion could develop, the LD 50 animals expired from extravascular tissue injury 48 . This host-mediated, pathogen-independent injury is due to widespread complement activation 48, 56, 57 . Specifically, serum C5b9 levels were significantly elevated in LD 50 non-survivors, comparable to that of LD 100 animals, but not in LD 50 survivors 48 . These findings provide a deeper understanding of the mechanisms of organ injury and rationale for modern clinical practice including early aggressive fluid resuscitation. The role of complement activation in lethal MOD was demonstrated in greater detail by NHP complement inhibitor studies ( Table 1) . Administration of compstatin, a C3 convertase inhibitor, to LD 50 baboons inhibited complement activation, protected against organ injury, and attenuated sepsis-mediated coagulopathy 58 . Concurrent administration of E. coli with a C5 inhibitor similarly decreased the inflammatory and coagulation responses, leading to organ protection and improved survival 59 . Acute respiratory distress syndrome. Because of their similarities to human respiratory anatomy 32 and physiology during Gram-negative sepsis 60 , baboon models are central to the study of sepsis-induced acute respiratory distress syndrome (ARDS), an ALI arising from systemic inflammatory responses 61 . ARDS is characterized by bilateral pulmonary infiltrates, reduced lung compliance, and impaired gas exchange, and in severe cases can lead to respiratory failure, MOD, and death [62] [63] [64] . The histopathologic hallmark is protein-rich alveolar edema and fibrin deposition due to breakdown of the alveolar-capillary barrier 61, 63 . Compared with healthy controls, bronchoalveolar lavage fluid (BALF) from patients with ARDS demonstrates high factor X, extrinsic pathway inhibitor, and anti-plasmin activity, suggesting that a hypercoagulable environment develops within the lungs 65 . The LD 100 intravenous E. coli baboon model was further optimized to enhance ALI by the addition of a pre-inoculation bacterial priming step. Baboons pretreated with 10 9 CFU/kg heat-killed E. coli (65°C water bath for 30 minutes) prior to inoculation with 9 x 10 10 CFU/kg live E. coli developed endotoxin tolerance with less shock and renal injury than unprimed animals 66 . However, the tolerance did not extend to the lungs, as priming worsened alveolar inflammation and fibroblast proliferation compared to unprimed animals 66 . These findings suggest that prior activation of the systemic inflammatory system leads to more severe lung injury in subsequent infections. Using this primed LD 100 E. coli model, the same tissue factordependent pathways involved in systemic endothelial activation were found to also mediate pulmonary microvascular injury (alveolar fibrin deposition) and contribute to ARDS 18, 67, 68 . Additionally, tissue factor is not only expressed on endothelial cells but also on pulmonary macrophages and alveolar epithelium, suggesting that these extravascular cells also regulate the procoagulant environment of the lung seen in ALI 67, 69 . Further dysregulation comes from LaB aNimaL | VOL 48 | FEBRUARY 2019 | 57-65 | www.nature.com/laban decreased expression of tissue factor pathway inhibitor (TFPI), a major checkpoint of the coagulation pathway 68 (Fig. 1b) . Baboons treated with site-inactivated factor VIIa 18 or with factor X inhibitor 70 to block tissue factor binding had reduced lung injury and improved renal function. These experiments support the causative relationship between coagulation and MOD. The lung pathology from patients at autopsy or from patients undergoing open lung biopsies show that ARDS is an acute inflammatory process followed by either resolution or fibrosis 63 , but less is known about long term ARDS sequelae 61 . Keshari et al. used the LD 50 E. coli model to characterize the evolution of ARDS-associated fibro-inflammatory changes for up to 27 months post-inoculation 71 . Lung histopathology within the first 24 to 48 hours after inoculation demonstrated an acute exudative process with increased alveolar-capillary permeability and intra-alveolar hemorrhage, fibrin, and edema. By day 7, a restorative phase had developed with proliferation of type 2 epithelial cells. In baboons surviving to six to 27 months, lung tissue universally showed fibroblast proliferation, collagen deposition, and significantly increased macrophage accumulation compared to controls, suggesting the long-term sequelae of ARDS is fibrosis and chronic inflammation 71 . Treatment of LD 50 baboons with the complement inhibitor compstatin, however, decreased expression of fibrosis mediators, such as transforming growth factor-beta (TGF-β ) and connective tissue growth factor (CTGF), myofibroblast accumulation, and collagen deposition 72 . In addition to the pathophysiology of sepsis, NHPs are also used to model organ-specific disease. The most common source of sepsis in adults is pneumonia followed by abdominal and urinary tract infections 21 . While no NHP model for urinary tract infections has been reported, abdominal sepsis has been modeled in baboons via intraperitoneal implantation of E. coli-laden fibrin clot at a dose of 10 11 CFU 73 . This model produced coagulopathy, MOD, and a 42% mortality rate. However, the interpretation was limited because the animals did not receive antibiotics or surgical source control, while both are standard-of-care treatments for patients with peritonitis. Necrotizing fasciitis has also been modeled in baboons by intramuscular injection with Streptococcus pyogenes (group A Streptococcus) 74 . The animals develop a multistage, suppurative, soft tissue infection similar to that of humans and characterized by abscess formation, violaceous skin discoloration, and bullae formation. Histopathology showed early intense neutrophilic influx at the site of infection followed by lymphoplasmacytic infiltration. Nonsurvivors, however, had a paucity of neutrophilic infiltration and a peripheral blood leukopenia, suggesting death may be linked to secondary immune paralysis. Neither of these surgical sepsis models has gained widespread use. Pneumonia. The most extensively studied organ system is the lungs. The use of NHP to model pneumonia dates back to the 1970s, most commonly using S. pneumoniae, the leading cause of bacterial community-acquired pneumonia (CAP) in adults 75 . An inoculation is performed under direct visualization by bronchoscopic instilment of live bacteria into the airway 17, 76, 77 . Multiple species of NHP are susceptible to S. pneumoniae infection. Squirrel monkeys endotracheally inoculated with either influenza A or low-dose Streptococcus pneumoniae (770 CFU) develop a self-limited illness with cough, fever, and tachypnea much like in humans 76 . However, the two pathogens together produce severe bronchopneumonia, leading to death in three of four animals 76 . The rhesus macaque (Macaca mulatta) demonstrates a dosedependent response to S. pneumoniae ranging from no clinical symptoms at 10 6 CFU to fever, leukocytosis, and respiratory distress at 10 8 -10 9 CFU 77 . A more recent model of S. pneumoniae pneumonia was published in baboons (P. cynocephalus). Animals given 10 6 CFU experienced spontaneous bacterial clearance without signs or symptoms of pneumonia, whereas animals given 10 9 19 . The inflammatory responses are also consistent across primate species. In baboons infected with high dose S. pneumoniae (10 9 CFU), peripheral blood cytokines and chemokines such as IL-6, CCL2, G-CSF, IL-1ra, and IL-10 are significantly elevated by 24 to 48 hours post-inoculation, but return to normal or near normal levels with time (~ 1 week) and antibiotic therapy 17, 19 . Reyes et al. found that antibiotic treatment may reduce cytokine and chemokine concentrations in the lung tissue itself, although differences in experimental duration between the antibiotic-treated and untreated groups (9-14 days vs. 4-9 days, respectively), could also account for this observation 19 . In the BALF of baboons and squirrel monkeys with S. pneumoniae pneumonia, TNF-α , IL-1β , and IL-6 levels are significantly elevated by 24 to 48 hours 17, 77 and similarly decline during the resolution phase 17 . These plasma and BALF cytokine profiles corroborate observations in patients with pneumonia 78 . Lung histopathology at 168 hours also mirrors human disease and shows ALI with intra-alveolar fibrin, edema, neutrophilic infiltration, and hemorrhage, and in some cases, early organization with collagen deposition 17 . Models for methicillin-resistant Staphylococcus aureus (MRSA) pneumonia have also been developed in the cynomolgus macaque (Macaca fasicularis) 79 and rhesus macaque 80 to study virulence factors. Historically considered a nosocomial pathogen, MRSA is now a recognized cause of severe, necrotizing CAP 75,81 , especially following influenza infection 82, 83 . The MRSA cytotoxin Panton-Valentine leukocidin (PVL) has been implicated in necrotic S. aureus soft tissue infections and CAP 84 , however in macaques inoculated with wildtype MRSA strain USA300 versus PVL deletion-mutant strain, no difference in disease severity was seen 79 . Influenza A with MRSA coinfection also did not potentiate morbidity in this NHP model 85 , suggesting that other host factors must be present to predispose healthy individuals to highly morbid secondary bacterial pneumonia. NHPs have also been used to develop models of Bacillus anthracis pneumonia 86 . Baboons were intravenously infused with B. anthracis at 10 5 -10 9 CFU and then treated with i.v. levofloxacin daily starting at 2 hours post-inoculation. Infected animals displayed a dose-dependent increase in severity of ALI, DIC, systemic inflammation, and capillary leak (vascular permeability and pleural effusions) that closely mirrored the human disease. At the highest doses (≥ 10 8 CFU), mortality was 100% 86 . Similar findings were seen in baboons infused with B. anthracis-derived peptidoglycan, the predominant carbohydrate of the Gram-positive bacterial cell wall, indicating this molecule is a pathogen-associated molecular pattern (PAMP) likely responsible for the inflammation and hypercoagulability seen with clinical anthrax infection 87 . Other studies using rhesus or cynomolgus macaques administered aerosolized B. anthracis spores via nose-or head-only exposure chambers [88] [89] [90] . Animals developed a moribund, disseminated anthrax infection with sepsis and respiratory failure over several days 88, 89 . The inhalational model mimics the likely mechanism of exposure to B. anthracis (airborne dispersion) in a possible terrorist attack and is therefore invaluable to researchers developing novel anthrax therapies for such a public health emergency 90, 91 . The E. coli baboon bacteremia model has been tested for proofof-mechanism studies to test inhibitors of the inflammatory and coagulation pathways 8, 92 . Some of these inhibitors, such as antibodies to the cell surface glycoproteins E-and L-selectin, which mediate leukocyte migration 93 , worsened organ dysfunction and survival and never progressed beyond animal studies 94 . Other inhibitors did improve sepsis survival and/or organ dysfunction, and NHP models have been instrumental in translating these preclinical studies into clinical trials. Table 1 shows a summary of the experimental therapies and their associated NHP models. One of the earliest inhibitor trials targeted TNF-α . Studies from the 1980s showed that LD 100 baboons given monoclonal antibodies against TNF-α had preserved organ function and reduced mortality 95, 96 . Subsequent studies showed the drug attenuated the coagulopathy 97 and reduced mortality in recurrent LD 10 E. coli bacteremia 98 . These positive preclinical studies eventually led to the advancement of afelimomab, a murine monoclonal antibody against TNF-α , into phase III trials 99, 100 . Similarly, recombinant TFPI reduced mortality in LD 100 baboons 52 and moved on to phase II 101 and phase III trials 102 in humans. However, neither afelimomab nor recombinant TFPI improved outcomes in patients and were ultimately abandoned. One mediator-targeted sepsis therapy advanced as far as FDAapproval-activated protein C (APC). APC inhibits factors V and VIII and enhances fibrinolysis, providing negative feedback to the coagulation pathway 103 (Fig. 1b) . Levels of protein C, however, are depleted during septic shock 104 . Administration of APC to LD 100 baboons prevented coagulopathy, hepatic injury, and lethality 49 . Conversely, administration of an antibody to protein C in LD 10 (10 7 -10 8 CFU/kg E. coli) baboons turned sub-lethal sepsis into lethal septic shock 49 . In contrast to the other coagulation inhibitors, APC attenuated the early stage mediators of the inflammatory and coagulation pathways, including TNF-α and fibrinogen 48 . These positive studies eventually led to the phase III clinical trial (PROWESS) of recombinant human APC (drotrecogin alfa) for the treatment of sepsis 105 , which showed a significant reduction in mortality in the treatment group. Drotrecogin alfa was approved by the FDA as a first-in-class drug for the treatment of sepsis. Subsequent phase IV trials however failed to demonstrate benefit 106, 107 , and actually showed increased risk of major bleeding, eventually prompting the removal of drotrecogin alpha from the market. Despite decades of research, there are currently no inflammation-or coagulation-targeted therapies approved for the treatment of sepsis. One reason for the discrepant findings between the animal and human studies is a lack of concordance between experimental design and clinical practice. For example, in baboons the TNF-α inhibitor was given prior to 97 or immediately after 96 E. coli infusion. Similarly, the recombinant TFPI was more efficacious if administered 30 minutes (rather than 4 hours) after E. coli infusion 52 . These animal studies do not reflect the real-world initiation of sepsis treatment, which typically occurs a day or more after onset of infection. The negative human trials also demonstrate the complexity and redundancy of molecular pathways wherein blockade of one mediator is counteracted by activation of another. Another promising potential therapy for sepsis-induced ARDS that has been tested in baboons with S. pneumoniae pneumonia (10 8 -10 9 CFU) is low-dose inhaled carbon monoxide (CO) gas. In mice, low-dose inhaled CO exerts anti-inflammatory and anti-apoptotic effects, reducing severity of sepsis-induced organ injury 108 . In S. pneumoniae-infected baboons, the pharmacokinetics and delivery of inhaled CO were described and preliminary evidence reported for reduction of ALI severity in CO-treated animals compared with historical controls 109 . This was linked to CO-mediated induction of mitochondrial biogenesis in alveolar type 2 cells and generation of specialized proresolving lipid mediators 110, 111 . The pharmacokinetics data generated from this NHP study was used to support an FDA IND application, and a multicenter phase I clinical trial 112 . The macaque anthrax model has also been used to develop novel anthrax therapies. Cynomolgus macaques inoculated with nebulized B. anthracis experienced significantly improved survival after receiving the novel tetracycline class antibiotic TP-271 88 . Survival of inhalational anthrax in this primate species was also significantly improved by administering a post-exposure prophylaxis vaccine, AV7909 89 . The combination of both post-exposure treatments was tested in rhesus macaques where antibiotics (ciprofloxacin x 14 days) LaB aNimaL | VOL 48 | FEBRUARY 2019 | 57-65 | www.nature.com/laban and vaccine therapy (anthrax vaccine adsorbed, AVA) significantly improved post-exposure survival compared to antibiotics alone, likely due to delayed germination of anthrax spores occurring after completion of antibiotics leading to anthrax infection 90 . Additionally, a pre-exposure vaccine study in rhesus macaques using a conjugated anti-capsule vaccine showed robust immune response and improved survival of anthrax infection compared to either component alone 113 . Given the nearly identical pathophysiology amongst primates, it would appear that NHP can readily replace other animal models of sepsis in biomedical research. The reality, however, is that the use of NHPs in research is still subject to limitations 23 . As highly intelligent animals, NHPs require much more stimulation and environmental enrichment than rodents, and must be housed in spacious containment facilities subject to rigorous monitoring regulations 23 . Additionally, the cost of one NHP experiment, when taking into account housing, personnel, and equipment costs is equivalent to that of hundreds of mice 23 . Significant resources and expertise are especially essential for NHP experiments involving intravenous infusions, phlebotomy, or vital sign measurements; these procedures generally require sedation, which when administered to septic animals can worsen cardiopulmonary instability (similar to patients). While such sepsis experiments have been reported for chimpanzees 40 , baboons 70 , and rhesus macaques 114 , they have required provision of ICU-level support (e.g., mechanical ventilation, vasopressors, fluids). An alternative strategy involves inserting and tethering indwelling intravascular catheters or vital sign recording devices to a harness worn by the animal 19, 115 . Such devices can continuously administer fluids or medications, and measure telemetry, heart rate, blood pressure, and body temperature 19, 115 . The implantation procedure itself is still a surgery that requires technical expertise, equipment, sedation, and a recovery period, but obviates the need for and inherent risks of sedation while the animal is septic. There can be device complications, however, such as catheterassociated infections 116 , which could confound sepsis experiments. Cost aside, NHPs require greater skill and expertise to handle than small mammals. They are several times stronger than humans per pound and can carry communicable diseases, such as tuberculosis and herpes B virus 23 . While NHPs share much of the same physiology and cell biology, the clinical course of sepsis in experimental models can nonetheless be different from that of patients. Because animals receive direct inoculation of pathogen, sepsis tends to be quicker onset with either rapid death or resolution. In the clinical arena, however, sepsis can be indolent and develop slowly as the nidus of infection grows. Furthermore, the outcomes of certain studies may be confounded when standard of care treatments are withheld, such as antibiotics or surgery 8 . And the timing of therapies administered to animals (either before or immediately after inoculation) does not reflect clinical practice when patients present several days after the onset of symptoms. NHP models also suffer from the same limitations in experimental design as all other animal models: experiments are tightly controlled and frequently performed on a population of young, typically male animals with no comorbid diseases 8, 24 . Baboons are generally three to eight years of age at the time of experimentation 71,109 , which is equivalent to an adolescent human. These conditions differ greatly from clinical sepsis, which is a notoriously heterogeneous disease affecting older adults of both sexes with multiple comorbidities. Thus the homogeneity that characterizes high quality experimental design becomes one of the biggest hindrances to integrating the findings into the clinical area. Finally, there are ethical considerations that must be considered when discussing NHP sepsis research. The 2006 Weatherall Report commissioned by medical groups in the United Kingdom concluded that the use of NHPs in medical research is justified, provided that 1) there are no alternative means (e.g. lower animals or in vitro work) to answer the biological question; 2) the NHP model is representative of the human disease; 3) there are adequate regulations in place to ensure the welfare of NHPs; and 4) the work significantly advances human health 117 . The report further qualifies that each use of NHPs for medical research should be adjudicated on a case-by-case basis 117 . This ethical justification is predicated on the utilitarian principle that suffering by a small number of NHPs may be acceptable if the research leads to substantial benefits to many humans 118 . Applications of this principle include using NHP studies to prevent a harmful drug from going to clinical trial, or conversely, redirecting resources towards drugs that show significant benefits in NHPs 119 . Risks associated with this calculus can be attenuated by minimizing the number of animals used, and be ensuring adequate attention to animal welfare 118 . The former can be addressed by changes to experimental design, such as use of "rolling controls" where the control group changes by one or two animals every experiment, or "before-and-after" studies where an animal serves as its own control. Strategies to address the latter including providing the animals with adequate food, water, space, enrichment, and social interactions 117 . The latter can also be addressed by using humane endpoints, or predetermined changes in animal physiology, rather than experimental endpoints, such as mortality. Furthermore, distinctions between different NHP species are likely relevant. For instance, the use of use great apes (chimpanzees, bonobos, gorillas, orangutans) in medical research was banned in the European Union in 2011 and phased out in the U.S. in 2013 119 . These ethical and policy issues are far from settled, and the justification for using monkeys or lesser apes (siamangs and gibbons) in medical research going forward must be continually reassessed. At the very least, the authors propose that their use should be limited to therapeutic studies (rather than mechanistic ones), which are more likely to directly impact human health. NHP models mimic human sepsis more faithfully than any other species because of anatomic, molecular, and physiological similarities. They have helped to deeply shape our current understanding of sepsis pathophysiology. These models have evolved from discovery of biochemical pathways to drug development. The E. coli bacteremia model has translated dozens of therapies targeting the inflammatory and coagulation pathways from rodent experiments to human clinical trials. However, despite their initial promise in preclinical NHP studies, no pharmacologic therapy has yet demonstrated benefit in humans. Their lack of success may not be due to failure of the animal model itself, but rather to the need to identify crucial targets and pathways in sepsis. Owing to their high cost and intelligence, the use of NHPs in sepsis research today is reserved exclusively for preclinical drug and therapy development, rather than for mechanistic studies in order that novel sepsis treatments can either move to clinical testing or be abandoned before the expense of a clinical trial. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) Benchmarking the incidence and mortality of severe sepsis in the United States Population burden of long-term survivorship after severe sepsis in older Americans Long-term cognitive impairment and functional disability among survivors of severe sepsis Empiric antibiotic treatment reduces mortality in severe sepsis and septic shock from the first hour: results from a guidelinebased performance improvement program Early goal-directed therapy in the treatment of severe sepsis and septic shock A randomized trial of protocol-based care for early septic shock Animal models of sepsis SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference Sepsis: current dogma and new perspectives Host innate immune responses to sepsis Sepsis and septic shock-a review of laboratory models and a proposal Animal models of sepsis: setting the stage Hemodynamic determinants of mortality in human septic shock Serial cardiovascular variables in survivors and nonsurvivors of human septic shock: heart rate as an early predictor of prognosis Sequential cardiorespiratory patterns in septic shock Development of a novel preclinical model of pneumococcal pneumonia in nonhuman primates Blockade of tissue factor: treatment for organ injury in established sepsis A Non-Human Primate Model of Severe Pneumococcal Pneumonia Survival of primates in LD100 septic shock following steroid/antibiotic therapy Severe sepsis and septic shock Multiorgan failure is an adaptive, endocrine-mediated, metabolic response to overwhelming systemic inflammation Animal models of sepsis Experimental models of sepsis and their clinical relevance Mitochondrial biogenesis restores oxidative metabolism during Staphylococcus aureus sepsis Large animal models: baboons for trauma, shock, and sepsis studies Animal models of acute lung injury Survival of primates in lethal septic shock following delayed treatment with steroid Detection of the 'hyperdynamic state' of sepsis in the baboon during lethal E. coli infusion Lower Respiratory Tract Structure of Laboratory Animals and Humans: Dosimetry Implications Morphometric characteristics of cells in the alveolar region of mammalian lungs. The American review of respiratory disease The non-human primate as a model for studying COPD and asthma Lipopolysaccharide dose response in baboons The cardiovascular response of normal humans to the administration of endotoxin Effectiveness of steroid/antibiotic treatment in primates administered LD100 Escherichia coli The immunopathogenesis of sepsis Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways Monoclonal antibody to tumor necrosis factor-alpha prevents lethal endotoxin sepsis in adult rhesus monkeys Pentoxifylline attenuates neutrophil activation in experimental endotoxemia in chimpanzees Differential effects of anti-tumor necrosis factor monoclonal antibodies on systemic inflammatory responses in experimental endotoxemia in chimpanzees Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees Differential effects of anti-cytokine treatment on bronchoalveolar hemostasis in endotoxemic chimpanzees Efficacy of monoclonal antibody against tumor necrosis factor alpha in an endotoxemic baboon model Elimination of interleukin 6 attenuates coagulation activation in experimental endotoxemia in chimpanzees Staging of the pathophysiologic responses of the primate microvasculature to Escherichia coli and endotoxin: examination of the elements of the compensated response and their links to the corresponding uncompensated lethal variants Activation patterns of coagulation and fibrinolysis in baboons following infusion with lethal or sublethal dose of Escherichia coli Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis staging and therapy of severe sepsis in baboon models Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon Comparison of endothelial activation during endotoxic and posttraumatic conditions by serum analysis of soluble E-selectin in nonhuman primates Expression of endothelial leukocyte adhesion molecule-1 in septic but not traumatic/hypovolemic shock in the baboon Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli Activation of the complement system in baboons challenged with live Escherichia coli: correlation with mortality and evidence for a biphasic activation pattern Anti-TNF treatment of baboons with sepsis reduces TNF-alpha, IL-6 and IL-8, but not the degree of complement activation Complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of Escherichia coli sepsis Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of Escherichia coli sepsis VA/Q abnormalities during gram negative sepsis What have anatomic and pathologic studies taught us about acute lung injury and acute respiratory distress syndrome? Clinical review: Early treatment of acute lung injury-paradigm shift toward prevention and treatment prior to respiratory failure The acute respiratory distress syndrome The acute respiratory distress syndrome Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome Bacterial priming increases lung injury in gram-negative sepsis Tissue factor in experimental acute lung injury Sepsis-induced coagulation in the baboon lung is associated with decreased tissue factor pathway inhibitor Coagulation and inflammation in acute lung injury Blockade of tissue factor-factor X binding attenuates sepsis-induced respiratory and renal failure Acute lung injury and fibrosis in a baboon model of Escherichia coli sepsis Complement inhibition decreases early fibrogenic events in the lung of septic baboons Peritonitis in the baboon: a primate model which stimulates human sepsis Staging of the baboon response to group A streptococci administered intramuscularly: a descriptive study of the clinical symptoms and clinical chemical response patterns Community-Acquired Pneumonia Requiring Hospitalization among U Influenza alone and in sequence with pneumonia due to Streptococcus pneumoniae in the squirrel monkey Experimental infection of rhesus macaques with Streptococcus pneumoniae: a possible model for vaccine assessment Compartmentalized cytokine production within the human lung in unilateral pneumonia Lack of a major role of Staphylococcus aureus Panton-Valentine leukocidin in lower respiratory tract infection in nonhuman primates Influenza A and methicillin-resistant Staphylococcus aureus co-infection in rhesus macaques -A model of severe pneumonia Invasive methicillin-resistant Staphylococcus aureus infections in the United States Centers for Disease Control & Prevention. Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza A (H1N1) -United States Staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia Seasonal H3N2 influenza A virus fails to enhance Staphylococcus aureus co-infection in a non-human primate respiratory tract infection model Sepsis and pathophysiology of anthrax in a nonhuman primate model Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Bacillus anthracis Infection in BALB/c Mice and Cynomolgus Macaques Correlation between anthrax lethal toxin neutralizing antibody levels and survival in guinea pigs and nonhuman primates vaccinated with the AV7909 anthrax vaccine candidate Short-course postexposure antibiotic prophylaxis combined with vaccination protects against experimental inhalational anthrax Anthrax as a biological weapon, 2002: updated recommendations for management Such stuff as dreams are made on: mediator-directed therapy in sepsis Antibody to E-and L-selectin does not prevent lung injury or mortality in septic baboons Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia Survival of primates in LD100 septic shock following therapy with antibody to tumor necrosis factor (TNF alpha). Circulatory shock 30 Pretreatment with a 55-kDa tumor necrosis factor receptor-immunoglobulin fusion protein attenuates activation of coagulation, but not of fibrinolysis, during lethal bacteremia in baboons Anti-tumor necrosis factor antibody treatment of recurrent bacteremia in a baboon model Randomized, placebo-controlled trial of the anti-tumor necrosis factor antibody fragment afelimomab in hyperinflammatory response during severe sepsis: The RAMSES Study Efficacy and safety of the monoclonal anti-tumor necrosis factor antibody F(ab')2 fragment afelimomab in patients with severe sepsis and elevated interleukin-6 levels Assessment of the safety of recombinant tissue factor pathway inhibitor in patients with severe sepsis: a multicenter, randomized, placebo-controlled, single-blind, dose escalation study Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme Activated protein C in sepsis Efficacy and safety of recombinant human activated protein C for severe sepsis Drotrecogin alfa (activated) in adults with septic shock Drotrecogin alfa (activated) for adults with severe sepsis and a low risk of death Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway Effects of inhaled CO administration on acute lung injury in baboons with pneumococcal pneumonia Cell-cell interactions and bronchoconstrictor eicosanoid reduction with inhaled carbon monoxide and resolvin D1 The Regulation of Proresolving Lipid Mediator Profiles in Baboon Pneumonia by Inhaled Carbon Monoxide A phase I trial of low-dose inhaled carbon monoxide in sepsis-induced ARDS Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits and monkeys Delivering Prolonged Intensive Care to a Non-human Primate: A High Fidelity Animal Model of Critical Illness Cardiovascular and neuroendocrine responses to baroreceptor denervation in baboons Long-term parenteral nutrition in unrestrained nonhuman primates: an experimental model The use of non-human primates in research The ethical justification for the use of non-human primates in research: the Weatherall report revisited The Ethics of Infection Challenges in Primates The authors thank Dr. Claude A. Piantadosi for his critical review of this manuscript. Reprints and permissions information is available at www.nature.com/reprints.Correspondence should be addressed to L.C.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.