The post-septic peripheral myeloid compartment reveals unexpected diversity in myeloid-derived suppressor cells.

Introduction: Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Methods: Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. Results: We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. Discussion: The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.

Sex differences associate with late microbiome alterations after murine surgical sepsis.

BACKGROUND: Sepsis-induced gut microbiome alterations contribute to sepsis-related morbidity and mortality. Given evidence for improved postsepsis outcomes in females compared with males, we hypothesized that female mice maintain microbiota resilience versus males. METHODS: Mixed-sex C57BL/6 mice underwent cecal ligation and puncture (CLP) with antibiotics, saline resuscitation, and daily chronic stress and were compared with naive (nonsepsis/no antibiotics) controls. For this work, the results of young (3-5 months) and old (18-22 months) adult mice were analyzed by sex, independent and dependent of age. Mice were sacrificed at days 7 and 14, and 16S rRNA gene sequencing was performed on fecal bacterial DNA. α and ß diversity were determined by Shannon index and Bray-Curtis with principal coordinate analysis, respectively. False discovery rate (FDR) correction was implemented to account for potential housing effect. RESULTS: In control mice, there was no difference in α or ß diversity between male and female mice (FDR, 0.76 and 0.99, respectively). However, male mice that underwent CLP with daily chronic stress had a decrease in microbiota α diversity at 7 days post-CLP (Shannon FDR, 0.005), which was sustained at 14 days post-CLP (Shannon FDR, 0.001), compared with baseline. In addition, male mice maintained differences in ß diversity even at day 14 compared with controls (FDR, <0.0001). In contrast, female mice had a decreased microbiota α diversity (Shannon FDR, 0.03) and ß diversity (FDR, 0.02) 7 days post-CLP but recovered their α and ß diversity by post-CLP day 14 (Shannon FDR, 0.5, and FDR, 0.02, respectively). Further analysis of females revealed that only young female mice were not different (ß diversity) post-CLP day 14 to controls. CONCLUSION: Although sepsis-induced perturbations of the intestinal microbiota occur initially in both male and female C57BL/6 mice, females demonstrate different microbiota by day 14. This may be seen primarily in younger females. This difference in recovery may play a role in outcome differences between sexes after sepsis.

A Novel Single Cell RNA-seq Analysis of Non-Myeloid Circulating Cells in Late Sepsis.

Background: With the successful implementation of the Surviving Sepsis Campaign guidelines, post-sepsis in-hospital mortality to sepsis continues to decrease. Those who acutely survive surgical sepsis will either rapidly recover or develop a chronic critical illness (CCI). CCI is associated with adverse long-term outcomes and 1-year mortality. Although the pathobiology of CCI remains undefined, emerging evidence suggests a post-sepsis state of pathologic myeloid activation, inducing suboptimal lymphopoiesis and erythropoiesis, as well as downstream leukocyte dysfunction. Our goal was to use single-cell RNA sequencing (scRNA-seq) to perform a detailed transcriptomic analysis of lymphoid-derived leukocytes to better understand the pathology of late sepsis. Methods: A mixture of whole blood myeloid-enriched and Ficoll-enriched peripheral blood mononuclear cells from four late septic patients (post-sepsis day 14-21) and five healthy subjects underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). Results: We identified unique transcriptomic patterns for multiple circulating immune cell subtypes, including B- and CD4+, CD8+, activated CD4+ and activated CD8+ T-lymphocytes, as well as natural killer (NK), NKT, and plasmacytoid dendritic cells in late sepsis patients. Analysis demonstrated that the circulating lymphoid cells maintained a transcriptome reflecting immunosuppression and low-grade inflammation. We also identified transcriptomic differences between patients with bacterial versus fungal sepsis, such as greater expression of cytotoxic genes among CD8+ T-lymphocytes in late bacterial sepsis. Conclusion: Circulating non-myeloid cells display a unique transcriptomic pattern late after sepsis. Non-myeloid leukocytes in particular reveal a host endotype of inflammation, immunosuppression, and dysfunction, suggesting a role for precision medicine-guided immunomodulatory therapy.

Identification of unique microRNA expression patterns in bone marrow hematopoietic stem and progenitor cells after hemorrhagic shock and multiple injuries in young and old adult mice.

BACKGROUND: After severe trauma, the older host experiences more dysfunctional hematopoiesis of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs), and dysfunctional differentiation of circulating myeloid cells into effective innate immune cells. Our main objective was to compare BM HSPC microRNA (miR) responses of old and young mice in a clinically relevant model of severe trauma and shock. METHODS: C57BL/6 adult male mice aged 8 to 12 weeks (young) and 18 to 24 months (old) underwent multiple injuries and hemorrhagic shock (polytrauma [PT]) that engenders the equivalent of major trauma (Injury Severity Score, >15). Pseudomonas pneumonia (PNA) was induced in some young and old adult mice 24 hours after PT. MicroRNA expression patterns were determined from lineage-negative enriched BM HSPCs isolated from PT and PT-PNA mice at 24 and 48 hours postinjury, respectively. Genome-wide expression and pathway analyses were also performed on bronchoalveolar lavage (BAL) leukocytes from both mouse cohorts. RESULTS: MicroRNA expression significantly differed among all experimental conditions (p < 0.05), except for old-naive versus old-injured (PT or PT-PNA) mice, suggesting an inability of old mice to mount a robust early miR response to severe shock and injury. In addition, young adult mice had significantly more leukocytes obtained from their BAL, and there were greater numbers of polymorphonuclear cells compared with old mice (59.8% vs. 2.2%, p = 0.0069). Despite increased gene expression changes, BAL leukocytes from old mice demonstrated a more dysfunctional transcriptomic response to PT-PNA than young adult murine BAL leukocytes, as reflected in predicted upstream functional pathway analysis. CONCLUSION: The miR expression pattern in BM HSPCs after PT (+/-PNA) is dissimilar in old versus young adult mice. In the acute postinjury phase, old adult mice are unable to mount a robust miR HSPC response. Hematopoietic stem and progenitor cell miR expression in old PT mice reflects a diminished functional status and a blunted capacity for terminal differentiation of myeloid cells.

Septic Stability? Gut Microbiota in Young Adult Mice Maintains Overall Stability After Sepsis Compared to Old Adult Mice.

BACKGROUND: Older adults have worse outcomes after sepsis than young adults. Additionally, alterations of the gut microbiota have been demonstrated to contribute to sepsis-related mortality. We sought to determine if there were alterations in the gut microbiota with a novel sepsis model in old adult mice, which enter a state of persistent inflammation, immunosuppression, and catabolism (PICS), as compared with young adult mice, which recover with the sepsis model. METHODS: Mixed sex old (∼20 mo) and young (∼4 mo) C57Bl/6J mice underwent cecal ligation and puncture with daily chronic stress (CLP+DCS) and were compared with naive age-matched controls. Mice were sacrificed at CLP+DCS day 7 and feces collected for bacterial DNA isolation. The V3-V4 hypervariable region was amplified, 16S rRNA gene sequencing performed, and cohorts compared. α-Diversity was assessed using Chao1 and Shannon indices using rarefied counts, and ß-diversity was assessed using Bray-Curtis dissimilarity. RESULTS: Naive old adult mice had significantly different α and ß-diversity compared with naive adult young adult mice. After CLP+DCS, there was a significant shift in the α and ß-diversity (FDR = 0.03 for both) of old adult mice (naive vs. CLP+DCS). However, no significant shift was displayed in the microbiota of young mice that underwent CLP+DCS in regards to α-diversity (FDR = 0.052) and ß-diversity (FDR = 0.12), demonstrating a greater overall stability of their microbiota at 7 days despite the septic insult. The taxonomic changes in old mice undergoing CLP+DCS were dominated by decreased abundance of the order Clostridiales and genera Oscillospira. CONCLUSION: Young adult mice maintain an overall microbiome stability 7 days after CLP+DCS after compared with old adult mice. The lack of microbiome stability could contribute to PICS and worse long-term outcomes in older adult sepsis survivors. Further studies are warranted to elucidate mechanistic pathways and potential therapeutics.

Single-Cell RNA-seq of Human Myeloid-Derived Suppressor Cells in Late Sepsis Reveals Multiple Subsets With Unique Transcriptional Responses: A Pilot Study.

BACKGROUND: Increased circulating myeloid-derived suppressor cells (MDSCs) are independently associated with poor long-term clinical outcomes in sepsis. Studies implicate subsets of MDSCs having unique roles in lymphocyte suppression; however, characterization of these cells after sepsis remains incomplete. We performed a pilot study to determine the transcriptomic landscape in MDSC subsets in sepsis using single-cell RNAseq (scRNA-seq). METHODS: A mixture of whole blood myeloid-enriched and Ficoll-enriched PBMCs from two late septic patients on post-sepsis day 21 and two control subjects underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). RESULTS: We successfully identified the three MDSC subset clusters-granulocytic (G-), monocytic (M-), and early (E-) MDSCs. Sepsis was associated with a greater relative expansion of G-MDSCs versus M-MDSCs at 21 days as compared to control subjects. Genomic analysis between septic patients and control subjects revealed cell-specific and common differential expression of genes in both G-MDSC and M-MDSC subsets. Many of the common genes have previously been associated with MDSC proliferation and immunosuppressive function. Interestingly, there was no differential expression of several genes demonstrated in the literature to be vital to immunosuppression in cancer-induced MDSC. CONCLUSION: This pilot study successfully demonstrated that MDSCs maintain a transcriptomic profile that is immunosuppressive in late sepsis. Interestingly, the landscape in chronic critical illness is partially dependent on the original septic insult. Preliminary data would also indicate immunosuppressive MDSCs from late sepsis patients appear to have a somewhat unique transcriptome from cancer and/or other inflammatory diseases.

Aluminum Adjuvant Improves Survival Via NLRP3 Inflammasome and Myeloid Non-Granulocytic Cells in a Murine Model of Neonatal Sepsis.

ABSTRACT: Neonatal sepsis leads to significant morbidity and mortality with the highest risk of death occurring in preterm (<37 weeks) and low birth weight (<2,500 g) infants. The neonatal immune system is developmentally immature with well-described defects in innate and adaptive immune responses. Immune adjuvants used to enhance the vaccine response have emerged as potential therapeutic options, stimulating non-specific immunity and preventing sepsis mortality. Aluminum salts ("alum") have been used as immune adjuvants for over a century, but their mechanism of action remains poorly understood. This study aims to identify potential mechanisms by which pretreatment with alum induces host protective immunity to polymicrobial sepsis in neonatal mice. Utilizing genetic and cell-depletion studies, we demonstrate here that the prophylactic administration of aluminum adjuvants in neonatal mice improves sepsis survival via activation of the nucleotide oligomerization domain-like receptor family, pyrin-domain-containing 3 inflammasome and dendritic cell activation. Furthermore, this beneficial effect is dependent on myeloid, non-granulocytic Gr1-positive cells, and MyD88-signaling pathway activation. These findings suggest a promising therapeutic role for aluminum-based vaccine adjuvants to prevent development of neonatal sepsis and improve mortality in this highly vulnerable population.

Transcriptomic responses from improved murine sepsis models can better mimic human surgical sepsis.

Historically, murine models of inflammation in biomedical research have been shown to minimally correlate with genomic expression patterns from blood leukocytes in humans. In 2019, our laboratory reported an improved surgical sepsis model of cecal ligation and puncture (CLP) that provides additional daily chronic stress (DCS), as well as adhering to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. This model phenotypically recapitulates the persistent inflammation, immunosuppression, and catabolism syndrome observed in adult human surgical sepsis survivors. Whether these phenotypic similarities between septic humans and mice are replicated at the circulating blood leukocyte transcriptome has not been demonstrated. Our analysis, in contrast with previous findings, demonstrated that genome-wide expression in our new murine model more closely approximated human surgical sepsis patients, particularly in the more chronic phases of sepsis. Importantly, our new model of murine surgical sepsis with chronic stress did not reflect well gene expression patterns from humans with community-acquired sepsis. Our work indicates that improved preclinical murine sepsis modeling can better replicate both the phenotypic and transcriptomic responses to surgical sepsis, but cannot be extrapolated to other sepsis etiologies. Importantly, these improved models can be a useful adjunct to human-focused and artificial intelligence-based forms of research in order to improve septic patients' morbidity and mortality.

Myeloid-derived suppressor cell function and epigenetic expression evolves over time after surgical sepsis.

BACKGROUND: Sepsis is an increasingly significant challenge throughout the world as one of the major causes of patient morbidity and mortality. Central to the host immunologic response to sepsis is the increase in circulating myeloid-derived suppressor cells (MDSCs), which have been demonstrated to be present and independently associated with poor long-term clinical outcomes. MDSCs are plastic cells and potentially modifiable, particularly through epigenetic interventions. The objective of this study was to determine how the suppressive phenotype of MDSCs evolves after sepsis in surgical ICU patients, as well as to identify epigenetic differences in MDSCs that may explain these changes. METHODS: Circulating MDSCs from 267 survivors of surgical sepsis were phenotyped at various intervals over 6 weeks, and highly enriched MDSCs from 23 of these samples were co-cultured with CD3/CD28-stimulated autologous T cells. microRNA expression from enriched MDSCs was also identified. RESULTS: We observed that MDSC numbers remain significantly elevated in hospitalized sepsis survivors for at least 6 weeks after their infection. However, only MDSCs obtained at and beyond 14 days post-sepsis significantly suppressed T lymphocyte proliferation and IL-2 production. These same MDSCs displayed unique epigenetic (miRNA) expression patterns compared to earlier time points. CONCLUSIONS: We conclude that in sepsis survivors, immature myeloid cell numbers are increased but the immune suppressive function specific to MDSCs develops over time, and this is associated with a specific epigenome. These findings may explain the chronic and persistent immune suppression seen in these subjects.

Old Mice Demonstrate Organ Dysfunction as well as Prolonged Inflammation, Immunosuppression, and Weight Loss in a Modified Surgical Sepsis Model.

OBJECTIVES: Our goal was to "reverse translate" the human response to surgical sepsis into the mouse by modifying a widely adopted murine intra-abdominal sepsis model to engender a phenotype that conforms to current sepsis definitions and follows the most recent expert recommendations for animal preclinical sepsis research. Furthermore, we aimed to create a model that allows the study of aging on the long-term host response to sepsis. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Young (3-5 mo) and old (18-22 mo) C57BL/6j mice. INTERVENTIONS: Mice received no intervention or were subjected to polymicrobial sepsis with cecal ligation and puncture followed by fluid resuscitation, analgesia, and antibiotics. Subsets of mice received daily chronic stress after cecal ligation and puncture for 14 days. Additionally, modifications were made to ensure that "Minimum Quality Threshold in Pre-Clinical Sepsis Studies" recommendations were followed. MEASUREMENTS AND MAIN RESULTS: Old mice exhibited increased mortality following both cecal ligation and puncture and cecal ligation and puncture + daily chronic stress when compared with young mice. Old mice developed marked hepatic and/or renal dysfunction, supported by elevations in plasma aspartate aminotransferase, blood urea nitrogen, and creatinine, 8 and 24 hours following cecal ligation and puncture. Similar to human sepsis, old mice demonstrated low-grade systemic inflammation 14 days after cecal ligation and puncture + daily chronic stress and evidence of immunosuppression, as determined by increased serum concentrations of multiple pro- and anti-inflammatory cytokines and chemokines when compared with young septic mice. In addition, old mice demonstrated expansion of myeloid-derived suppressor cell populations and sustained weight loss following cecal ligation and puncture + daily chronic stress, again similar to the human condition. CONCLUSIONS: The results indicate that this murine cecal ligation and puncture + daily chronic stress model of surgical sepsis in old mice adhered to current Minimum Quality Threshold in Pre-Clinical Sepsis Studies guidelines and met Sepsis-3 criteria. In addition, it effectively created a state of persistent inflammation, immunosuppression, and weight loss, thought to be a key aspect of chronic sepsis pathobiology and increasingly more prevalent after human sepsis.

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.

Mouse Injury Model of Polytrauma and Shock.

Severe injury and shock remain major sources of morbidity and mortality worldwide. Immunologic dysregulation following trauma contributes to these poor outcomes. Few, if any, therapeutic interventions have benefited these patients, and this is due to our limited understanding of the host response to injury and shock. The Food and Drug Administration requires preclinical animal studies prior to any interventional trials in humans; thus, animal models of injury and shock will remain the mainstay for trauma research. However, adequate animal models that reflect the severe response to trauma in both the acute and subacute phases have been limited. Here we describe a novel murine model of polytrauma and shock that combines hemorrhagic shock, cecectomy, long bone fracture, and soft-tissue damage. This model produces an equivalent Injury Severity Score associated with adverse outcomes in humans, and may better recapitulate the human leukocyte, cytokine, transcriptomic, and overall inflammatory response following injury and hemorrhagic shock.

Evidence for Persistent Immune Suppression in Patients Who Develop Chronic Critical Illness After Sepsis.

BACKGROUND: Many sepsis survivors develop chronic critical illness (CCI) and are assumed to be immunosuppressed, but there is limited clinical evidence to support this. We sought to determine whether the incidence of secondary infections and immunosuppressive biomarker profiles of patients with CCI differ from those with rapid recovery (RAP) after sepsis. METHODS: This prospective observational study evaluated 88 critically ill patients with sepsis and 20 healthy controls. Cohorts were defined based on clinical trajectory (early death, RAP, or CCI), whereas immunosuppression was clinically determined by the presence of a postsepsis secondary infection. Serial blood samples were collected for absolute lymphocyte counts (ALCs), monocytic human leukocyte antigen-DR (mHLA-DR) expression, and plasma-soluble programmed death-ligand 1 (sPD-L1) concentrations. RESULTS: Of the 88 patients with sepsis, 3 (3%) died within 14 days of sepsis onset, 50 (57%) experienced RAP, and 35 (40%) developed CCI. Compared with RAP patients, CCI patients exhibited a higher incidence and overall number of infections adjusted for hospital length of stay. ALC and mHLA-DR levels were dramatically reduced at the time of sepsis diagnosis when compared with healthy controls, whereas sPD-L1 concentrations were significantly elevated. There were no differences between RAP and CCI patients in ALC, sPD-L1, or mHLA-DR at the time of diagnosis or within 24 h after sepsis diagnosis. However, in contrast to the RAP group, CCI patients failed to exhibit any trend toward restoration of normal values of ALC, HLA-DR, and sPD-L1. CONCLUSIONS: Septic patients demonstrate clinical and biological evidence to suggest they are immunosuppressed at the time of sepsis diagnosis. Those who develop CCI have a greater incidence of secondary infections and persistently aberrant markers of impaired host immunity, although measurements at the time of sepsis onset did not distinguish between subjects with RAP and CCI.

LPS Stimulation of Cord Blood Reveals a Newborn-Specific Neutrophil Transcriptomic Response and Cytokine Production.

BACKGROUND: The neonatal innate immune system differs to microbial infection both quantitatively and qualitatively when compared with adults. Here, we provide the first genome-wide ex-vivo expression profile of umbilical cord blood (UCB) neutrophils from full-term infants prior to and in response to whole-blood lipopolysaccharide (LPS) stimulation. Additionally, we provide cytokine expression prior to and following LPS stimulation. The genomic expression and cytokine profile are compared with LPS-stimulated whole blood from healthy adult subjects (HC). METHODS: Whole blood from UCB (n = 6) and HC (n = 6) was studied at baseline or was stimulated for 24 h with 100 ngs/mL of LPS. CD66b neutrophils were subsequently isolated with microfluidic techniques and genome-wide expression analyses were performed. Ingenuity Pathway Analysis (IPA) software was utilized to predict downstream functional effects. Additionally, cytokine concentrations in whole blood prior to and after 24 h of LPS incubation were determined. RESULTS: LPS stimulated whole blood from UCB demonstrated significant differences in both ex-vivo cytokine production and PMN gene expression. Mixed-effect modeling identified 1,153 genes whose expression changed significantly in UCB and HC after exposure to LPS (P < 0.001 with a minimum 1.5-fold change). IPA downstream predictions suggest that PMNs from UCB fail to effectively upregulate genes associated with activation, phagocytosis, and chemotaxis in response to LPS stimulation. Furthermore, whole blood from UCB showed increased interleukin (IL)-10 production to LPS, but failed to significantly increase several pro-inflammatory cytokines. CONCLUSIONS: LPS-stimulated whole blood from UCB exhibited a markedly suppressed inflammatory cytokine production and PMN innate immune genome response. These differences in gene expression and cytokine production may be an adaptive response to a prior fetal environment, but may also explain their increased susceptibility to infections. Characterization of these deficits is the first step toward developing prophylactic and therapeutic interventions.

Patterns of gene expression among murine models of hemorrhagic shock/trauma and sepsis.

Controversy remains whether the leukocyte genomic response to trauma or sepsis is dependent upon the initiating stimulus. Previous work illustrated poor correlations between historical models of murine trauma and sepsis (i.e., trauma-hemorrhage and lipopolysaccharide injection, respectively). The aim of this study is to examine the early genomic response in improved murine models of sepsis [cecal ligation and puncture (CLP)] and trauma [polytrauma (PT)] with and without pneumonia (PT+Pp). Groups of naïve, CLP, PT, and PT+Pp mice were killed at 2 h, 1 or 3 days. Total leukocytes were isolated for genome-wide expression analysis, and genes that were found to differ from control (false discovery rate adjusted P < 0.001) were assessed for fold-change differences. Spearman correlations were also performed. For all time points combined (CLP, PT, PT+Pp), there were 10,426 total genes that were found to significantly differ from naïve controls. At 2 h, the transcriptomic changes between CLP and PT showed a positive correlation (rs) of 0.446 (P < 0.0001) but were less positive thereafter. Correlations were significantly improved when we limited the analysis to common genes whose expression differed by a 1.5 fold-change. Both pathway and upstream analyses revealed the activation of genes known to be associated with pathogen-associated and damage-associated molecular pattern signaling, and early activation patterns of expression were very similar between polytrauma and sepsis at the earliest time points. This study demonstrates that the early leukocyte genomic response to sepsis and trauma are very similar in mice.

A Detailed Characterization of the Dysfunctional Immunity and Abnormal Myelopoiesis Induced by Severe Shock and Trauma in the Aged.

The elderly are particularly susceptible to trauma, and their outcomes are frequently dismal. Such patients often have complicated clinical courses and ultimately die of infection and sepsis. Recent research has revealed that although elderly subjects have increased baseline inflammation as compared with their younger counterparts, the elderly do not respond to severe infection or injury with an exaggerated inflammatory response. Initial retrospective analysis of clinical data from the Glue Grant trauma database demonstrated that despite a similar frequency, elderly trauma patients have worse outcomes to pneumonia than younger subjects do. Subsequent analysis with a murine trauma model also demonstrated that elderly mice had increased mortality after posttrauma Pseudomonas pneumonia. Blood, bone marrow, and bronchoalveolar lavage sample analyses from juvenile and 20-24-mo-old mice showed that increased mortality to trauma combined with secondary infection in the aged are not due to an exaggerated inflammatory response. Rather, they are due to a failure of bone marrow progenitors, blood neutrophils, and bronchoalveolar lavage cells to initiate and complete an emergency myelopoietic response, engendering myeloid cells that fail to clear secondary infection. In addition, elderly people appeared unable to resolve their inflammatory response to severe injury effectively.

Improved emergency myelopoiesis and survival in neonatal sepsis by caspase-1/11 ablation.

Over one million newborns die annually from sepsis with the highest mortality in premature and low-birthweight infants. The inflammasome plays a central role in the regulation of innate immunity and inflammation, and is presumed to be involved in protective immunity, in large part through the caspase-1-dependent activation of interleukin-1ß (IL-1ß) and IL-18. Studies in endotoxic shock, however, suggest that endogenous caspase-1 activity and the inflammasome contribute to mortality primarily by promoting excessive systemic inflammatory responses. We examined whether caspase-1 and the inflammasome also regulate neonatal inflammation, host protective immunity and myelopoiesis during polymicrobial sepsis. Neonatal (5-7 days) C57BL/6 and caspase-1/11(-/-) mice underwent a low-lethality caecal slurry model of intra-abdominal sepsis (LD25-45 ). Ablation of caspase-1/11, but not apoptosis-associated speck-like protein containing a CARD domain or nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), improved neonatal survival following septic challenge compared with wild-type mice (P < 0·001), with decreased concentrations of inflammatory cytokines in the serum and peritoneum. Surprisingly, caspase-1/11(-/-) neonates also exhibited increased bone marrow and splenic haematopoietic stem cell expansion (P < 0·001), and increased concentrations of granulocyte and macrophage colony-stimulating factors in the peritoneum (P < 0·001) after sepsis. Ablation of caspase-1/11 signalling was also associated with increased recruitment of peritoneal macrophages and neutrophils (P < 0·001), increased phagocytosis by neutrophils (P = 0·003), and decreased bacterial colonization (P = 0·02) in the peritoneum. These findings suggest that endogenous caspase-1/11 activity, independent of the NLRP3 inflammasome, not only promotes the magnitude of the inflammatory response, but also suppresses protective immunity in the neonate, so contributing to innate immune dysfunction and poor survival in neonatal sepsis.

Host responses to sepsis vary in different low-lethality murine models.

INTRODUCTION: Animal models for the study of sepsis are being increasingly scrutinized, despite their essential role for early translational research. In particular, recent studies have suggested that at the level of the leukocyte transcriptome, murine models of burns, trauma and endotoxemia markedly differ from their human equivalents, and are only weakly similar amongst themselves. We compared the plasma cytokine and leukocyte transcriptome responses between two different low-lethality murine models of polymicrobial intra-abdominal sepsis. METHODS: Six to ten week male C57BL/6j mice underwent either the 'gold standard' cecal ligation and puncture (CLP) model of intra-abdominal sepsis or administration of a cecal slurry (CS), where cecal contents are injected intraperitoneally. Surviving mice were euthanized at two hours, one or three days after sepsis. RESULTS: The murine leukocyte transcriptomic response to the CLP and CS models of sepsis was surprisingly dissimilar at two hours, one, and three days after sepsis. The Pearson correlation coefficient for the maximum change in expression for the entire leukocyte transcriptome that changed significantly over time (n = 19,071) was R = 0.54 (R2 = 0.297). The CS model resulted in greater magnitude of early inflammatory gene expression changes in response to sepsis with associated increased production of inflammatory chemokines and cytokines. Two hours after sepsis, CLP had more significant expression of genes associated with IL-10 signaling pathways, whereas CS had greater expression of genes related to CD28, apoptosis, IL-1 and T-cell receptor signaling. By three days, the changes in gene expression in both sepsis models were returning to baseline in surviving animals. CONCLUSION: These analyses reveal that the murine blood leukocyte response to sepsis is highly dependent on which model of intra-abdominal sepsis is employed, despite their similar lethality. It may be difficult to extrapolate findings from one murine model to another, let alone to human sepsis.

Protective immunity and defects in the neonatal and elderly immune response to sepsis.

Populations encompassing extremes of age, including neonates and elderly, have greater mortality from sepsis. We propose that the increased mortality observed in the neonatal and elderly populations after sepsis is due to fundamental differences in host-protective immunity and is manifested at the level of the leukocyte transcriptome. Neonatal (5-7 d), young adult (6-12 wk), or elderly (20-24 mo) mice underwent a cecal slurry model of intra-abdominal sepsis. Both neonatal and elderly mice exhibited significantly greater mortality to sepsis (p < 0.05). Neonates in particular exhibited significant attenuation of their inflammatory response (p < 0.05), as well as reductions in cell recruitment and reactive oxygen species production (both p < 0.05), all of which could be confirmed at the level of the leukocyte transcriptome. In contrast, elderly mice were also more susceptible to abdominal peritonitis, but this was associated with no significant differences in the magnitude of the inflammatory response, reduced bacterial killing (p < 0.05), reduced early myeloid cell activation (p < 0.05), and a persistent inflammatory response that failed to resolve. Interestingly, elderly mice expressed a persistent inflammatory and immunosuppressive response at the level of the leukocyte transcriptome, with failure to return to baseline by 3 d. This study reveals that neonatal and elderly mice have profoundly different responses to sepsis that are manifested at the level of their circulating leukocyte transcriptome, although the net result of increased mortality is similar. Considering these differences are fundamental aspects of the genomic response to sepsis, interventional therapies will require individualization based on the age of the population.

A better understanding of why murine models of trauma do not recapitulate the human syndrome.

OBJECTIVE: Genomic analyses from blood leukocytes have concluded that mouse injury poorly reflects human trauma at the leukocyte transcriptome. Concerns have focused on the modest severity of murine injury models, differences in murine compared with human age, dissimilar circulating leukocyte populations between species, and whether similar signaling pathways are involved. We sought to examine whether the transcriptomic response to severe trauma in mice could be explained by these extrinsic factors, by utilizing an increasing severity of murine trauma and shock in young and aged mice over time, and by examining the response in isolated neutrophil populations. DESIGN: Preclinical controlled in vivo laboratory study and retrospective cohort study. SETTING: Laboratory of Inflammation Biology and Surgical Science and multi-institution level 1 trauma centers. SUBJECTS: Six- to 10-week-old and 20- to 24-month-old C57BL/6 (B6) mice and two cohorts of 167 and 244 severely traumatized (Injury Severity Score > 15) adult (> 18 yr) patients. INTERVENTIONS: Mice underwent one of two severity polytrauma models of injury. Total blood leukocyte and neutrophil samples were collected. MEASUREMENTS AND MAIN RESULTS: Fold expression changes in leukocyte and neutrophil genome-wide expression analyses between healthy and injured mice (p < 0.001) were compared with human total and enriched blood leukocyte expression analyses of severe trauma patients at 0.5, 1, 4, 7, 14, and 28 days after injury (Glue Grant trauma-related database). We found that increasing the severity of the murine trauma model only modestly improved the correlation in the transcriptomic response with humans, whereas the age of the mice did not. In addition, the genome-wide response to blood neutrophils (rather than total WBC) was also not well correlated between humans and mice. However, the expression of many individual gene families was much more strongly correlated after injury in mice and humans. CONCLUSIONS: Although overall transcriptomic association remained weak even after adjusting for the severity of injury, age of the animals, timing, and individual leukocyte populations, there were individual signaling pathways and ontogenies that were strongly correlated between mice and humans. These genes are involved in early inflammation and innate/adaptive immunity.