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.

Acute Emergence of the Intestinal Pathobiome after Postinjury Pneumonia.

BACKGROUND: Previous preclinical studies have demonstrated sex-specific alterations in the gut microbiome following traumatic injury or sepsis alone; however, the impact of host sex on dysbiosis in the setting of postinjury sepsis acutely is unknown. We hypothesized that multicompartmental injury with subsequent pneumonia would result in host sex-specific dysbiosis. METHODS: Male and proestrus female Sprague-Dawley rats (n = 8/group) were subjected to either polytrauma (PT) (lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofracture), PT plus 2-hours daily restraint stress (PT/RS), PT with postinjury day 1 pseudomonas aeruginosa pneumonia (PT + PNA), PT/RS with pneumonia (PT/RS + PNA), or naive controls. Fecal microbiome was measured on days 0 and 2 using high-throughput 16S rRNA sequencing and QIIME2 bioinformatics analyses. Microbial α-diversity was assessed using Chao1 (number of different unique species) and Shannon (species richness and evenness) indices. ß-diversity was assessed using principal coordinate analysis. Significance was defined as p < 0.05. RESULTS: All groups had drastic declines in the Chao1 (α-diversity) index compared to naïve controls (p < 0.05). PT + PNA and PT/RS + PNA resulted in different ß-diversity arrays compared to uninfected counterparts (PT, PT/RS) (p = 0.001). Postinjury sepsis cohorts showed a loss of commensal bacteria along with emergence of pathogenic bacteria, with blooms of Proteus in PT + PNA and Escherichia-Shigella group in PT/RS + PNA compared to other cohorts. At day 2, PT + PNA resulted in ß-diversity which was unique between males and females (p = 0.004). Microbiome composition in PT + PNA males was dominated by Anaerostipes and Parasuterella whereas females had increased Barnesiella and Oscillibacter. PT/RS males had an abundance of Gastranaerophilales and Muribaculaceae. CONCLUSIONS: Multicompartmental trauma complicated by sepsis significantly diminishes diversity and alters microbial composition towards a severely dysbiotic state early after injury, which varies between males and females. These findings highlight the role of sex in postinjury sepsis and the pathobiome which may influence outcomes after severe trauma and sepsis. LEVEL OF EVIDENCE: Not applicable - basic science.

Posttraumatic pneumonia exacerbates bone marrow erythropoietic dysfunction.

INTRODUCTION: Pneumonia is a common complication after severe trauma that is associated with worse outcomes with increased mortality. Critically ill trauma patients also have persistent inflammation and bone marrow dysfunction that manifests as persistent anemia. Terminal erythropoiesis, which occurs in bone marrow structures called erythroblastic islands (EBIs), has been shown to be impacted by trauma. Using a preclinical model of polytrauma (PT) and pneumonia, we sought to determine the effect of infection on bone marrow dysfunction and terminal erythropoiesis. METHODS: Male and female Sprague-Dawley rats aged 9 to 11 weeks were subjected to either PT (lung contusion, hemorrhagic shock, cecectomy, and bifemoral pseudofracture) or PT with postinjury day 1 Pseudomonas pneumonia (PT-PNA) and compared with a naive cohort. Erythroblastic islands were isolated from bone marrow samples and imaged via confocal microscopy. Hemoglobin, early bone marrow erythroid progenitors, erythroid cells/EBI, and % reticulocytes/EBI were measured on day 7. Significance was defined as p < 0.05. RESULTS: Day 7 hemoglobin was significantly lower in both PT and PT-PNA groups compared with naive (10.8 ± 0.6 and 10.9 ± 0.7 vs. 12.1 ± 0.7 g/dL [ p < 0.05]). Growth of bone marrow early erythroid progenitors (colony-forming units-granulocyte, erythrocyte, monocyte, megakaryocyte; erythroid burst-forming unit; and erythroid colony-forming unit) on day 7 was significantly reduced in PT-PNA compared with both PT and naive. Despite a peripheral reticulocytosis following PT and PT-PNA, the percentage of reticulocytes/EBI was not different between naive, PT, and PT-PNA. However, the number of erythroblasts/EBI was significantly lower in PT-PNA compared with naive (2.9 ± 1.5 [ p < 0.05] vs. 8.9 ± 1.1 cells/EBI macrophage). In addition to changes in EBI composition, EBIs were also found to have significant structural changes following PT and PT-PNA. CONCLUSION: Multicompartmental PT altered late-stage erythropoiesis, and these changes were augmented with the addition of pneumonia. To improve outcomes following trauma and pneumonia, we need to better understand how alterations in EBI structure and function impact persistent bone marrow dysfunction and anemia.

Multicompartmental Trauma Induces Persistent Inflammation and Organ Injury.

INTRODUCTION: Previous preclinical models of multicompartmental injury have investigated its effects for durations of less than 72 h and the long-term effects have not been defined. We hypothesized that a model of multicompartmental injury would result in systemic inflammation and multiorgan dysfunction that persists at 1 wk. METHODS: Male and proestrus female Sprague-Dawley rats (n = 16/group) underwent polytrauma (PT) (unilateral right lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofractures) and were compared to naive controls. Weight, hemoglobin, plasma neutrophil gelatinase-associated lipocalin, and plasma toll-like receptor 4 were evaluated on days two and seven. Bilateral lungs were sectioned, stained and assessed for injury at day seven. Comparisons were performed in Graphpad with significance defined as ∗P <0.05. RESULTS: Rats who underwent PT had significant weight loss and anemia at day 2 (P = 0.001) compared to naïve rats which persisted at day 7 (P = 0.001). PT rats had elevated plasma neutrophil gelatinase-associated lipocalin at day 2 compared to naïve (P <0.0001) which remained elevated at day 7 (P <0.0001). Plasma toll-like receptor 4 was elevated in PT compared to naïve at day 2 (P = 0.03) and day 7 (P = 0.01). Bilateral lungs showed significant injury in PT cohorts at day 7 compared to naïve (P <0.0004). PT males had worse renal function at day seven compared to females (P = 0.02). CONCLUSIONS: Multicompartmental trauma induces systemic inflammation and multiorgan dysfunction without recovery by day seven. However, females demonstrate improved renal recovery compared to males. Long-term assessment of preclinical PT models are crucial to better understand and evaluate future therapeutic immunomodulatory and anti-inflammatory treatments.

Exosomal microRNA following severe trauma: Role in bone marrow dysfunction.

INTRODUCTION: Severe trauma disrupts bone marrow function and is associated with persistent anemia and altered hematopoiesis. Previously, plasma-derived exosomes isolated after trauma have been shown to suppress in vitro bone marrow function. However, the cargo contained in these vesicles has not been examined. We hypothesized that trauma plasma-derived exosomes exhibit microRNA (miRNA) changes that impact bone marrow function after severe injury. METHODS: Plasma was collected from a prospective cohort study of trauma patients (n = 15; 7 males, 8 females) with hip and/or femur fractures and an Injury Severity Score of ≥15; elective total hip arthroplasty (THA) patients (n = 8; 4 males, 4 females) served as operative controls. Exosomes were isolated from plasma with the Invitrogen Total Exosome Isolation Kit (Thermo Fisher Scientific, Waltham, MA), and RNA was isolated using a miRNeasy Mini Kit (Qiagen, Hilden, Germany). Direct quantification of miRNA was performed by NanoString Technologies on a human miRNA gene panel and analyzed with nSolver with significance defined as p < 0.05. RESULTS: There were no differences in age or sex distribution between trauma and THA groups; the average Injury Severity Score was 23. Trauma plasma-derived exosomes had 60 miRNA identities that were significantly downregulated and 3 miRNAs that were upregulated when compared with THA ( p < 0.05). Twelve of the downregulated miRNAs have a direct role in hematopoiesis regulation. Furthermore, male trauma plasma-derived exosomes demonstrated downregulation of 150 miRNAs compared with male THA ( p < 0.05). Female trauma plasma-derived exosomes demonstrated downregulation of only four miRNAs and upregulation of two miRNAs compared with female THA ( p < 0.05). CONCLUSION: We observed downregulation of 12 miRNAs linked to hematopoiesis along with sexual dimorphism in miRNA expression from plasma-derived exosomes following severe trauma. Understanding sexually dimorphic miRNA expression provides new insight into sex-based changes in postinjury systemic inflammation, immune system dysregulation, and bone marrow dysfunction and will aid us in more precise future potential therapeutic strategies. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Anemia Recovery After Lung Contusion, Hemorrhagic Shock, and Chronic Stress Is Gender-Specific in a Rat Model.

Background: Severe trauma and hemorrhagic shock lead to persistent anemia. Although biologic gender is known to modulate inflammatory responses after critical illness, the impact of gender on anemia recovery after injury remains unknown. The aim of this study was to identify gender-specific differences in anemia recovery after critical illness. Materials and Methods: Male and proestrus female Sprague-Dawley rats (n = 8-9 per group) were subjected to lung contusion and hemorrhagic shock (LCHS) or LCHS with daily chronic stress (LCHS/CS) compared with naïve. Hematologic data, bone marrow progenitor growth, and bone marrow and liver gene transcription were analyzed on day seven. Significance was defined as p < 0.05. Results: Males lost substantial weight after LCHS and LCHS/CS compared with naïve males, while female LCHS rats did not compared with naive counterparts. Male LCHS rats had a drastic decrease in hemoglobin from naïve males. Male LCHS/CS rats had reduced colony-forming units-granulocyte, -erythrocyte, -monocyte, -megakaryocyte (CFU-GEMM) and burst-forming unit-erythroid (BFU-E) when compared with female counterparts. Naïve, LCHS, and LCHS/CS males had lower serum iron than their respective female counterparts. Liver transcription of BMP4 and BMP6 was elevated after LCHS and LCHS/CS in males compared with females. The LCHS/CS males had decreased expression of bone marrow pro-erythroid factors compared with LCHS/CS females. Conclusions: After trauma with or without chronic stress, male rats demonstrated increased weight loss, substantial decrease in hemoglobin level, dysregulated iron metabolism, substantial suppression of bone marrow erythroid progenitor growth, and no change in transcription of pro-erythroid factors. These findings confirm that gender is an important variable that impacts anemia recovery and bone marrow dysfunction after traumatic injury and shock in this rat model.

Sex-specific intestinal dysbiosis persists after multicompartmental injury.

BACKGROUND: Preclinical studies of the gut microbiome after severe traumatic injury have demonstrated severe dysbiosis in males, with sex-specific microbial differences up to 2 days after injury. However, the impact of host sex on injury-driven dysbiosis over time remains unknown. We hypothesized that sex-specific differences in intestinal microbiome diversity and composition after traumatic injury with and without stress would persist after 7 days. METHODS: Male and proestrus female Sprague-Dawley rats (n = 8/group) were subjected to either polytrauma (lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofractures), polytrauma plus chronic restraint stress, or naïve controls. The fecal microbiome was measured on days 0, 3, and 7 using 16S rRNA sequencing and Quantitative Insights into Microbial Ecology bioinformatics analyses. Microbial alpha-diversity (Chao1 and Shannon indices) and beta-diversity were assessed. Analyses were performed in GraphPad and "R," with significance defined as P < .05. RESULTS: Polytrauma and polytrauma plus chronic restraint stress reduced alpha-diversity (Chao1, Shannon) within 3 days postinjury, which persisted up to day 7 in both sexes; polytrauma and polytrauma plus chronic restraint stress females had significantly decreased Chao1 compared to male counterparts at day 7 (P = .02). At day 7, the microbiome composition in polytrauma females had higher proportion of Mucispirillum, whereas polytrauma plus chronic restraint stress males demonstrated elevated abundance of Ruminococcus and Akkermansia. CONCLUSION: Multicompartmental trauma induces intestinal dysbiosis that is sex-specific with persistence of decreased diversity and unique "pathobiome" signatures in females after 1 week. These findings underline sex as an important biological variable that may influence variable host-specific responses and outcomes after severe trauma and critical illness. This underscores the need to consider precision medicine strategies to ameliorate these outcomes.

The Intestinal Microbiome after Traumatic Injury.

The intestinal microbiome plays a critical role in host immune function and homeostasis. Patients suffering from-as well as models representing-multiple traumatic injuries, isolated organ system trauma, and various severities of traumatic injury have been studied as an area of interest in the dysregulation of immune function and systemic inflammation which occur after trauma. These studies also demonstrate changes in gut microbiome diversity and even microbial composition, with a transition to a pathobiome state. In addition, sex has been identified as a biological variable influencing alterations in the microbiome after trauma. Therapeutics such as fecal transplantation have been utilized to ameliorate not only these microbiome changes but may also play a role in recovery postinjury. This review summarizes the alterations in the gut microbiome that occur postinjury, either in isolated injury or multiple injuries, along with proposed mechanisms for these changes and future directions for the field.

NARROWING THE GAP: PRECLINICAL TRAUMA WITH POSTINJURY SEPSIS MODEL WITH INCREASED CLINICAL RELEVANCE.

ABSTRACT: Background : Overall outcomes for trauma patients have improved over time. However, mortality for postinjury sepsis is unchanged. The use of relevant preclinical studies remains necessary to understand mechanistic changes after injury and sepsis at the cellular and molecular level. We hypothesized that a preclinical rodent model of multicompartmental injury with postinjury pneumonia and chronic stress would replicate inflammation and organ injury similar to trauma patients in the intensive care unit. Methods : Male and proestrus female Sprague-Dawley rats ( n = 16/group) were subjected to either polytrauma (PT) (lung contusion, hemorrhagic shock, cecectomy, and bifemoral pseudofracture), PT with daily chronic restraint stress (PT/CS), PT with postinjury day one Pseudomonas pneumonia (PT + PNA), PT/CS with pneumonia (PT/CS + PNA) or naive controls. Weight, white blood cell count, plasma toll-like receptor 4 (TLR4), urine norepinephrine (NE), hemoglobin, serum creatinine, and bilateral lung histology were evaluated. Results : PT + PNA and PT/CS + PNA groups lost more weight compared with those without sepsis (PT, PT/CS) and naive rats ( P < 0.03). Similarly, both PT + PNA and PT/CS + PNA had increased leukocytosis and plasma TLR4 compared with uninfected counterparts. Urine NE was elevated in PT + PNA and PT/CS + PNA compared with naive ( P < 0.03), with PT/CS + PNA exhibiting the highest levels. PT/CS + PNA exhibited worse acute kidney injury with elevated serum creatinine compared with PT/CS ( P = 0.008). PT/CS + PNA right and left lung injury scores were worse than PT + PNA ( P < 0.01). Conclusions : Sepsis, with postinjury pneumonia, induced significant systemic inflammation, organ dysfunction following polytrauma and chronic stress. Advanced animal models that replicate the critically ill human condition will help overcome the classic limitations of previous experimental models and enhance their translational value.

Multicompartmental traumatic injury induces sex-specific alterations in the gut microbiome.

BACKGROUND: Previous preclinical studies have demonstrated an altered gut microbiome after traumatic injury; however, the impact of sex on dysbiosis remains unknown. We hypothesized that the "pathobiome" phenotype induced by multicompartmental injuries and chronic stress is host sex specific with unique microbiome signatures. METHODS: Male and proestrus female Sprague-Dawley rats (n = 8/group) aged 9 weeks to 11 weeks were subjected to either multicompartmental injury (PT) (lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofractures), PT plus 2 hours daily chronic restraint stress (PT/CS) or naive controls. Fecal microbiome was measured on Days 0 and 2 using high-throughput 16S rRNA sequencing and Quantitative Insights Into Microbial Ecology bioinformatics analyses. Microbial alpha-diversity was assessed using Chao1 (number of different unique species) and Shannon (species richness and evenness) indices. Beta-diversity was assessed using principle coordinate analysis. Intestinal permeability was evaluated by plasma occludin and lipopolysaccharide binding protein. Histologic evaluation of ileum and colon tissues was scored for injury by a blinded pathologist. Analyses were performed in GraphPad and R, with significance defined as p < 0.05 between males versus females. RESULTS: At baseline, females had significantly elevated alpha-diversity (Chao1, Shannon indices) compared with males ( p < 0.05) which was no longer present 2 days postinjury in PT and PT/CS. Beta-diversity also differed significantly between males and females after PT ( p = 0.01). At Day 2, the microbial composition in PT/CS females was dominated by Bifidobacterium , whereas PT males demonstrated elevated levels of Roseburia ( p < 0.01). The PT/CS males had significantly elevated ileum injury scores compared with females ( p = 0.0002). Plasma occludin was higher in PT males compared with females ( p = 0.004); plasma lipopolysaccharide binding protein was elevated in PT/CS males ( p = 0.03). CONCLUSION: Multicompartmental trauma induces significant alterations in microbiome diversity and taxa, but these signatures differ by host sex. These findings suggest that sex is an important biological variable that may influence outcomes after severe trauma and critical illness.

EFFECTS OF TRAUMA PLASMA-DERIVED EXOSOMES ON HEMATOPOIETIC PROGENITOR CELLS.

ABSTRACT: Background: Severe trauma disrupts bone marrow function resulting in persistent anemia and immunosuppression. Exosomes are extracellular vesicles implicated in disease, cellular functions, and immunomodulation. The effects of trauma plasma-derived exosomes on bone marrow hematopoiesis are unstudied; we hypothesized that trauma plasma-derived exosomes suppress bone marrow hematopoietic progenitor cell (HPC) growth and contribute to increased inflammatory cytokines and HPC mobilization. Methods: Plasma was collected from a prospective, cohort study of trauma patients (n = 15) with hip and/or femur fractures and an ISS > 15 and elective total hip arthroplasty (THA) patients (n = 15). Exosomes were isolated from both groups using the Invitrogen Total Exosome Isolation Kit. Healthy bone marrow was cultured with 2% plasma, 50 µg, 100 µg, or 200 µg of exosomal protein and HPC (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units [CFU-GEMM], erythroid burst-forming units [BFU-E], and macrophage colony-forming units [CFU-GM]) growth assessed. After culturing healthy bone marrow stroma with 100 µg of exosomal protein, expression of cytokines and factors influencing HPC mobilization were assessed by qPCR. Differences were compared using the ANOVA, with significance defined as P < 0.05. Results: The only demographic difference was age; trauma patients were significantly younger than THA (mean 44 vs. 63 years). In vitro exposure to trauma plasma significantly decreased growth of all HPCs. In vitro exposure to 100 µg or 200 µg of trauma exosomal protein significantly decreased growth of BFU-E and CFU-GM, whereas 50 µg had no effect. Culture of trauma exosomal protein with bone marrow stromal cells resulted in increased expression of IFN-γ, IL-1α, TNF-α, G-CSF, CXCR4, SDF-1, and VCAM-1 in bone marrow stroma. Conclusions: Both plasma and plasma-derived exosomes from trauma patients adversely affect bone marrow function. Plasma-derived exosomes may contribute to altered hematopoiesis after severe trauma; analysis of exosomal content may improve our understanding of altered bone marrow function.

Multicompartmental traumatic injury and the microbiome: Shift to a pathobiome.

BACKGROUND: Previous animal models have demonstrated altered gut microbiome after mild traumatic injury; however, the impact of injury severity and critical illness is unknown. We hypothesized that a rodent model of severe multicompartmental injuries and chronic stress would demonstrate microbiome alterations toward a "pathobiome" characterized by an overabundance of pathogenic organisms, which would persist 1 week after injury. METHODS: Male Sprague-Dawley rats (n = 8 per group) were subjected to either multiple injuries (PT) (lung contusion, hemorrhagic shock, cecectomy, and bifemoral pseudofractures), PT plus daily chronic restraint stress for 2 hours (PT/CS), or naive controls. Fecal microbiome was measured on days 0, 3, and 7 using high-throughput 16S rRNA sequencing and Quantitative Insights Into Microbial Ecology 2 bioinformatics analysis. Microbial α diversity was assessed using Chao1 and Shannon indices, and ß diversity with principle coordinate analysis. Intestinal permeability was evaluated by plasma occludin; ileum and descending colon tissues were reviewed for injury. Analyses were performed in GraphPad (GraphPad Software, La Jolla, CA) and R (R Foundation for Statistical Computing, Vienna, Austria), with significance defined as p < 0.05. RESULTS: There were significant alterations in ß diversity at day 3 and between all groups. By day 3, both PT and PT/CS demonstrated significantly depleted bacterial diversity (Chao1) ( p = 0.01 and p = 0.001, respectively) versus naive, which persisted up to day 7 in PT/CS only ( p = 0.001). Anaerostipes and Rothia dominated PT and Lactobacillus bloomed in PT/CS cohorts by day 7. Plasma occludin was significantly elevated in PT/CS compared with naive ( p = 0.04), and descending colon of both PT and PT/CS showed significantly higher injury compared with naive ( p = 0.005, p = 0.006). CONCLUSIONS: Multiple injuries with and without chronic stress induces significant alterations in microbiome diversity and composition within 3 days; these changes are more prominent and persist for 1 week postinjury with stress. This rapid and persistent transition to a "pathobiome" phenotype represents a critical phenomenon that may influence outcomes after severe trauma and critical illness.

Foxc1 dependent mesenchymal signalling drives embryonic cerebellar growth.

Loss of Foxc1 is associated with Dandy-Walker malformation, the most common human cerebellar malformation characterized by cerebellar hypoplasia and an enlarged posterior fossa and fourth ventricle. Although expressed in the mouse posterior fossa mesenchyme, loss of Foxc1 non-autonomously induces a rapid and devastating decrease in embryonic cerebellar ventricular zone radial glial proliferation and concurrent increase in cerebellar neuronal differentiation. Subsequent migration of cerebellar neurons is disrupted, associated with disordered radial glial morphology. In vitro, SDF1α, a direct Foxc1 target also expressed in the head mesenchyme, acts as a cerebellar radial glial mitogen and a chemoattractant for nascent Purkinje cells. Its receptor, Cxcr4, is expressed in cerebellar radial glial cells and conditional Cxcr4 ablation with Nes-Cre mimics the Foxc1-/- cerebellar phenotype. SDF1α also rescues the Foxc1-/- phenotype. Our data emphasizes that the head mesenchyme exerts a considerable influence on early embryonic brain development and its disruption contributes to neurodevelopmental disorders in humans.