Julie A. Stortz

Microbial recognition and danger signals in sepsis and trauma

Early host recognition of microbial invasion or damaged host tissues provides an effective warning system by which protective immune and inflammatory processes are initiated. Host tissues responsible for continuous sampling of their local environment employ cell surface and cytosolic pattern recognition receptors (PRRs) that provide redundant and overlapping identification of both microbial and host alarmins. Microbial products containing pathogen-associated molecular patterns (PAMPs), as well as damage-associated molecular patterns (DAMPs) serve as principle ligands for recognition by these PRRs. It is this interaction which plays both an essential survival role in response to infection and injury, as well as the pathologic role in tissue and organ injury associated with severe sepsis and trauma. Elucidating the interaction between ligands and their respective PRRs can provide both a better understanding of the host response, as well as a rational basis for therapeutic intervention. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Murine Models of Sepsis and Trauma: Can We Bridge the Gap?

Sepsis and trauma are both leading causes of death in the United States and represent major public health challenges. Murine models have largely been used in sepsis and trauma research to better understand the pathophysiological changes that occur after an insult and to develop potential life-saving therapeutic agents. Mice are favorable subjects for this type of research given the variety of readily available strains including inbred, outbred, and transgenic strains. In addition, they are relatively easy to maintain and have a high fecundity. However, pharmacological therapies demonstrating promise in preclinical mouse models of sepsis and trauma often fail to demonstrate similar efficacy in human clinical trials, prompting considerable criticism surrounding the capacity of murine models to recapitulate complex human diseases like sepsis and traumatic injury. Fundamental differences between the two species include, but are not limited to, the divergence of the transcriptomic response, the mismatch of temporal response patterns, differences in both innate and adaptive immunity, and heterogeneity within the human population in comparison to the homogeneity of highly inbred mouse strains. Given the ongoing controversy, this narrative review aims to not only highlight the historical importance of the mouse as an animal research model but also highlight the current benefits and limitations of the model as it pertains to sepsis and trauma. Lastly, this review will propose future directions that may promote further use of the model.

Immunological Defects in Neonatal Sepsis and Potential Therapeutic Approaches

Despite advances in critical care medicine, neonatal sepsis remains a major cause of morbidity and mortality worldwide, with the greatest risk affecting very low birth weight, preterm neonates. The presentation of neonatal sepsis varies markedly from its presentation in adults, and there is no clear consensus definition of neonatal sepsis. Previous work has demonstrated that when neonates become septic, death can occur rapidly over a matter of hours or days and is generally associated with inflammation, organ injury, and respiratory failure. Studies of the transcriptomic response by neonates to infection and sepsis have led to unique insights into the early proinflammatory and host protective responses to sepsis. Paradoxically, this early inflammatory response in neonates, although lethal, is clearly less robust relative to children and adults. Similarly, the expression of genes involved in host protective immunity, particularly neutrophil function, is also markedly deficient. As a result, neonates have both a diminished inflammatory and protective immune response to infection which may explain their increased risk to infection, and their reduced ability to clear infections. Such studies imply that novel approaches unique to the neonate will be required for the development of both diagnostics and therapeutics in this high at-risk population.

Innate Immunity in the Persistent Inflammation, Immunosuppression, and Catabolism Syndrome and Its Implications for Therapy

Clinical and technological advances promoting early hemorrhage control and physiologic resuscitation as well as early diagnosis and optimal treatment of sepsis have significantly decreased in-hospital mortality for many critically ill patient populations. However, a substantial proportion of severe trauma and sepsis survivors will develop protracted organ dysfunction termed chronic critical illness (CCI), defined as ≥14 days requiring intensive care unit (ICU) resources with ongoing organ dysfunction. A subset of CCI patients will develop the persistent inflammation, immunosuppression, and catabolism syndrome (PICS), and these individuals are predisposed to a poor quality of life and indolent death. We propose that CCI and PICS after trauma or sepsis are the result of an inappropriate bone marrow response characterized by the generation of dysfunctional myeloid populations at the expense of lympho- and erythropoiesis. This review describes similarities among CCI/PICS phenotypes in sepsis, cancer, and aging and reviews the role of aberrant myelopoiesis in the pathophysiology of CCI and PICS. In addition, we characterize pathogen recognition, the interface between innate and adaptive immune systems, and therapeutic approaches including immune modulators, gut microbiota support, and nutritional and exercise therapy. Finally, we discuss the future of diagnostic and prognostic approaches guided by machine and deep-learning models trained and validated on big data to identify patients for whom these approaches will yield the greatest benefits. A deeper understanding of the pathophysiology of CCI and PICS and continued investigation into novel therapies harbor the potential to improve the current dismal long-term outcomes for critically ill post-injury and post-infection patients.

The Postinjury Inflammatory State and the Bone Marrow Response to Anemia

Rationale: The pathophysiology of persistent injury‐associated anemia is incompletely understood, and human data are sparse. Objectives: To characterize persistent injury‐associated anemia among critically ill trauma patients with the hypothesis that severe trauma would be associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, and decreased erythropoiesis. Methods: A translational prospective observational cohort study comparing severely injured, blunt trauma patients who had operative fixation of a hip or femur fracture (n = 17) with elective hip repair patients (n = 22). Bone marrow and plasma obtained at the index operation were assessed for circulating catecholamines, systemic inflammation, erythropoietin, iron trafficking pathways, and erythroid progenitor growth. Bone marrow was also obtained from healthy donors from a commercial source (n = 8). Measurements and Main Results: During admission, trauma patients had a median of 625 ml operative blood loss and 5 units of red blood cell transfusions, and Hb decreased from 10.5 to 9.3 g/dl. Compared with hip repair, trauma patients had higher median plasma norepinephrine (21.9 vs. 8.9 ng/ml) and hepcidin (56.3 vs. 12.2 ng/ml) concentrations (both P < 0.05). Bone marrow erythropoietin and erythropoietin receptor expression were significantly increased among patients undergoing hip repair (23% and 14% increases, respectively; both P < 0.05), but not in trauma patients (3% and 5% increases, respectively), compared with healthy control subjects. Trauma patients had lower bone marrow transferrin receptor expression than did hip repair patients (57% decrease; P < 0.05). Erythroid progenitor growth was decreased in trauma patients (39.0 colonies per plate; P < 0.05) compared with those with hip repair (57.0 colonies per plate; P < 0.05 compared with healthy control subjects) and healthy control subjects (66.5 colonies per plate). Conclusions: Severe blunt trauma was associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, erythroid progenitor growth suppression, and persistent injury‐associated anemia. Clinical trial registered with www.clinicaltrials.gov (NCT 02577731).

Sepsis is associated with reduced spontaneous neutrophil migration velocity in human adults

Sepsis is a common and deadly complication among trauma and surgical patients. Neutrophils must mobilize to the site of infection to initiate an immediate immune response. To quantify the velocity of spontaneous migrating blood neutrophils, we utilized novel microfluidic approaches on whole blood samples from septic and healthy individuals. A prospective study at a level 1 trauma and tertiary care center was performed with peripheral blood samples collected at <12 hours, 4 days, and/or 14 days relative to study initiation. Blood samples were also collected from healthy subjects. Ex vivo spontaneous neutrophil migration was measured on 2 μl of whole blood using microfluidic devices and time-lapse imaging. For each sample, individual neutrophils were tracked to calculate mean instantaneous velocity. Forty blood samples were collected from 33 patients with sepsis, and 15 blood samples were collected from age- and gender-matched healthy, control subjects. Average age was 61 years for septic patients with a male predominance (67%). Overall, average spontaneous neutrophil migration velocity in septic samples was 16.9 μm/min, significantly lower than controls samples at 21.1 μm/min (p = 0.0135). Neutrophil velocity was reduced the greatest at <12 hours after sepsis (14.5 μm/min). Regression analysis demonstrated a significant, positive correlation between neutrophil velocity and days after sepsis (p = 0.0059). There was no significant association between neutrophil velocity and age, gender, APACHE II score, SOFA score, sepsis severity, total white blood cell count, or percentage of neutrophils. Circulating levels of the cytokines IL-6, IL-8, IL-10, MCP-1, IP-10, and TNF were additionally measured using bead-based multiplex assay and found to peak at <12 hours and be significantly increased in patients with sepsis at all three time points (<12 hours, 4 days, and 14 days after sepsis) compared to healthy subjects. In conclusion, these findings may demonstrate an impaired ability of neutrophils to respond to sites of infection during the proinflammatory phase of sepsis.

Chronic Critical Illness and the Persistent Inflammation, Immunosuppression, and Catabolism Syndrome

Dysregulated host immune responses to infection often occur, leading to sepsis, multiple organ failure, and death. Some patients rapidly recover from sepsis, but many develop chronic critical illness (CCI), a debilitating condition that impacts functional outcomes and long-term survival. The “Persistent Inflammation, Immunosuppression, and Catabolism Syndrome” (PICS) has been postulated as the underlying pathophysiology of CCI. We propose that PICS is initiated by an early genomic and cytokine storm in response to microbial invasion during the early phase of sepsis. However, once source control, antimicrobial coverage, and supportive therapies have been initiated, we propose that the persistent inflammation in patients developing CCI is a result of ongoing endogenous alarmin release from damaged organs and loss of muscle mass. This ongoing alarmin and danger-associated molecular pattern signaling causes chronic inflammation and a shift in bone marrow stem cell production toward myeloid cells, contributing to chronic anemia and lymphopenia. We propose that therapeutic interventions must target the chronic organ injury and lean tissue wasting that contribute to the release of endogenous alarmins and the expansion and deposition of myeloid progenitors that are responsible for the propagation and persistence of CCI.

Editorial: Myeloid‐derived suppressor cells: a new therapeutic target in sepsis patients

This issue of the Journal of Leukocyte Biology headlines with some impressive findings from the El Gazzar laboratory [1]. In their work, “Myeloid cellspecific deletion of Cebpb decreases sepsisinduced immunosuppression in mice,” the authors investigated the role of C/EBPb in the generation, maturation, and differentiation of murine Gr1CD11b MDSCs in both septic and nonseptic mice. Their research approach used sophisticated methodology, including but not limited to the creation of BALB/cJ Cebpb conditional knockout mice, adoptive cell transfer, and transfection of miRNA precursors. Previous work from their laboratory demonstrated that in septic murine hosts, C/EBPb induces miR-21 and miR-181b expression, both of which promote the expansion of MDSCs [2]. The silencing of these miRNAs using antagomirs, which are small, chemically modified oligonucelotides that bind and inactivate specific miRNAs, in turn, blocked the generation of MDSCs and reduced late-sepsis mortality [2]. Likewise, McPeak et al. [1] found in their current study that C/EBPb-deficient septic mice exhibit reduced mortality to sepsis, presumably as a result of decreased MDSC expansion. From a functional standpoint, the MDSCs from these conditional knockout mice were less immunosuppressive and more proinflammatory, producing greater levels of TNF-a and significantly less IL-10. In addition, C/EBPb-deficient bone marrow MDSCs were more likely to differentiate into macrophages and dendritic cells than control MDSCs when cultured in the presence of M-CSF and IL-4. Finally, transfection of miR-21 and miR-181b into Gr1CD11b cells isolated from the bone marrow of C/EBPb conditional knockout mice led to decreased differentiation of these cells compared with controls. Taken together, these findings suggest that C/EBPb plays a critical role in the expansion, differentiation, and functional transition of Gr1CD11b MDSCs during sepsis (Fig. 1). Although MDSCs did not gain popularity in the literature until more recently, they were first discovered in tumor-bearing mice .50 yr ago [3]. Initially, MDSCs were referred to as “natural suppressor” or “null cells,” given their lack of conventional T, B, and NK cell markers. These immature myeloid precursors were present in increased quantities during periods of rapid tumor growth and as their name implies, suppressed cytotoxic T cell activity [4]. This suppressive activity was determined to occur through multiple mechanisms, including the production of arginase, reactive oxygen species, iNOS, and IL-10, as well as sequestration of amino acids, such as cysteine [5, 6]. Since the initial discovery of MDSCs, characterization of these cells has been difficult, as a result of the absence of unique phenotypic markers that distinguish MDSCs from other cells. Common phenotypic markers expressed by MDSCs include Gr1 and CD11b in mice, as well as CD33 and CD11b in humans, which are either myeloid differentiation antigens or transmembrane receptors expressed on myeloid cells [7]. However, these cell surface markers are also present on non-MDSC cell populations. For this reason, some authors propose that proper identification of MDSCs requires not only phenotypic characterization but also functional characterization as well, with sine qua non evidence of T cell suppression [7]. Even with this coupled assessment, classification of MDSCs remains difficult as a result of their morphologic and functional heterogeneity. In addition to challenges relating to the proper identification of these cells, the exact role of MDSCs is unclear. Whereas it is known that the MDSC population expands during numerous pathologic processes, including cancer, traumatic injury, and sepsis, whether these cells are beneficial or detrimental to the host remains uncertain [5]. It becomes an even more complicated question when one considers the variability of the time points at which they are present, the organ system in which they exist, as well as specific aspects of precision medicine (such as gender, age, etc.) that may affect their role in the host. In addition, the pathology that is present in the host may determine whether MDSCs have a net positive or negative effect on host protective immunity. Several authors propose that these cells are maladaptive as a result of their immunosuppressive capacity, which contributes to tumor evasion in cancer and increased incidence of nosocomial infections in septic hosts [8, 9]. However, blocking MDSC expansion with anti-Gr1 antibodies in mice is paradoxically associated with dramatically decreased survival