Persistence and Sexual Dimorphism of Gut Dysbiosis and Pathobiome after Sepsis and Trauma.

OBJECTIVE: To evaluate the persistence of intestinal microbiome dysbiosis and gut-plasma metabolomic perturbations following severe trauma or sepsis weeks after admission in patients experiencing chronic critical illness (CCI). SUMMARY: Trauma and sepsis can lead to gut dysbiosis and alterations in the plasma and fecal metabolome. However, the impact of these perturbations and correlations between gut dysbiosis and the plasma metabolome in chronic critical illness have not been studied. METHODS: A prospective observational cohort study was performed with healthy subjects, severe trauma patients, and patients with sepsis residing in an intensive care unit for 2 to 3 weeks. A high-throughput multi-omics approach was utilized to evaluate the gut microbial and gut-plasma metabolite responses in critically ill trauma and sepsis patients 14 to 21 days after intensive care unit admission. RESULTS: Patients in the sepsis and trauma cohorts demonstrated strikingly depleted gut microbiome diversity, with significant alterations and specific pathobiome patterns in the microbiota composition compared to healthy subjects. Further subgroup analyses based on sex revealed resistance to changes in microbiome diversity among female trauma patients compared to healthy counterparts. Sex--specific changes in fecal metabolites were also observed after trauma and sepsis, while plasma metabolite changes were similar in both males and females. CONCLUSIONS: Dysbiosis induced by trauma and sepsis persists up to 14 to 21 days after onset and is sex-specific, underscoring the implication of pathobiome and entero-septic microbial-metabolite perturbations in post-sepsis and posttrauma chronic critical illness. This indicates resilience to infection or injury in females' microbiome and should inform and facilitate future precision/personalized medicine strategies in the intensive care unit.

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.

Gut mycobiome dysbiosis after sepsis and trauma.

BACKGROUND: Sepsis and trauma are known to disrupt gut bacterial microbiome communities, but the impacts and perturbations in the fungal (mycobiome) community after severe infection or injury, particularly in patients experiencing chronic critical illness (CCI), remain unstudied. METHODS: We assess persistence of the gut mycobiome perturbation (dysbiosis) in patients experiencing CCI following sepsis or trauma for up to two-to-three weeks after intensive care unit hospitalization. RESULTS: We show that the dysbiotic mycobiome arrays shift toward a pathobiome state, which is more susceptible to infection, in CCI patients compared to age-matched healthy subjects. The fungal community in CCI patients is largely dominated by Candida spp; while, the commensal fungal species are depleted. Additionally, these myco-pathobiome arrays correlate with alterations in micro-ecological niche involving specific gut bacteria and gut-blood metabolites. CONCLUSIONS: The findings reveal the persistence of mycobiome dysbiosis in both sepsis and trauma settings, even up to two weeks post-sepsis and trauma, highlighting the need to assess and address the increased risk of fungal infections in CCI patients.

Mediators of Prolonged Hematopoietic Progenitor Cell Mobilization After Severe Trauma.

BACKGROUND: This study investigated the molecular mediators of prolonged hematopoietic progenitor cell mobilization a trauma and chronic stress and the role of propranolol in modifying this response. METHODS: Sprague-Dawley rats were randomized to lung contusion (LC), LC plus hemorrhagic shock (LCHS), or LCHS with daily restraint stress (LCHS/CS). Propranolol was administered daily. Bone marrow (BM) and lung expression of high mobility group box 1 (HMGB1), granulocyte colony-stimulating factor (G-CSF), neutrophil elastase, stromal cell-derived factor 1 (SDF-1)/CXR4, and vascular cell adhesion protein 1 (VCAM-1)/very late antigen-4 were measured by real-time polymerase chain reaction. RESULTS: Bone marrow HMGB1, G-CSF, and neutrophil elastase expression were significantly elevated two- to four-fold after LCHS/CS, and all were decreased with the use of propranolol. SDF-1 and VCAM-1 were both significantly decreased after LCHS/CS. CONCLUSIONS: The increased expression of HMGB1 and G-CSF and decreased expression of BM anchoring molecules, SDF-1 and VCAM-1, after LCHS/CS, likely mediates prolonged hematopoietic progenitor cell mobilization. Propranolol's ability to reduce HMGB1, G-CSF, and neutrophil elastase expression suggests that the mobilization of hematopoietic progenitor cells was driven by persistent hypercatecholaminemia.

Prolonged Chronic Stress and Persistent Iron Dysregulation Prevent Anemia Recovery Following Trauma.

Introduction Following major trauma, persistent injury-associated anemia is associated with organ failure, increased length of stay and mortality. We hypothesize that prolonged adrenergic stimulation following trauma is directly responsible for persistent iron dysfunction that impairs anemia recovery. Materials and Methods Naïve rodents, lung contusion and hemorrhagic shock followed by daily handling for 13 d (LCHS), LCHS followed by 6 d of restraint stress and 7 d of daily handling (LCHS/CS-7) and LCHS/CS followed by 13 d of restraint stress with day and/or night disruption (LCHS/CS-14) were sacrificed on day 14. Hemoglobin, plasma, urine, bone marrow/liver inflammatory and erythropoietic markers were analyzed. Results LCHS/CS-14 led to a significant decline in weight gain and persistently elevated plasma and urine inflammatory markers. Liver IL-6, IL-1ß and hepcidin expression were significantly increased following LCHS/CS-14. LCHS/CS-14 also had impaired anemia recovery with reduced plasma transferrin and erythropoietin receptor expression. Conclusion Prolonged chronic stress following trauma/hemorrhagic shock led to sustained inflammation with increased expression of IL-1ß, IL-6 and hepcidin with decreased iron availability for uptake into erythroid progenitor cells and a lack of anemia recovery.

Impact of Injury Severity on the Inflammatory State and Severe Anemia.

BACKGROUND: Severe traumatic injury is a major cause of morbidity and mortality. Our goal was to analyze blunt traumatic injury by injury severity score (ISS) and compare with elective hip repair, as a transient injury, and healthy control with the hypothesis that more severe injury would lead to an increase in neuroendocrine activation, systemic inflammation, and worse anemia. MATERIALS AND METHODS: A prospective observational cohort study was performed at a level 1 trauma center, comparing blunt trauma patients (n = 37), elective hip replacement patients (n = 26), and healthy controls (n = 8). Bone marrow and plasma were assessed for hyperadrenergic state, erythropoiesis, and systemic inflammation. Trauma patient's ISS ranged from 4 to 41 and were broken down into quartiles for analysis. The ISS quartiles were 4-13, 14-20, 21-26, and 27-41. RESULTS: Plasma norepinephrine, interleukin-6, tumor necrosis factor-alpha, and hepcidin increased progressively as ISS increased. Hemoglobin significantly decreased as ISS increased and packed red blood cell (pRBC) transfusion increased as ISS increased. Elective hip replacement patients had an appropriate increase in the bone marrow expression of erythropoietin and the erythropoietin receptor, which was absent in all trauma patient groups. CONCLUSIONS: Increased neuroendocrine activation, systemic inflammation, and anemia correlated with worsening injury severity, lower age, and increased pRBC transfusions. Elective hip replacement patients have only minimal systemic inflammation with an appropriate bone marrow response to anemia. This study demonstrates a link between injury severity, neuroendocrine activation, systemic inflammation, and the bone marrow response to anemia.

Systemic Regulation of Bone Marrow Stromal Cytokines After Severe Trauma.

BACKGROUND: Traumatic injury generates a prolonged hypercatecholamine state that is associated with reduced growth of bone marrow erythroid progenitors mediated by the bone marrow stroma. The bone marrow stroma is made up of many cells including fibroblasts, which respond to inflammatory stimuli and alter the cytokine profile. We hypothesized that trauma plasma would increase bone marrow stromal fibroblast expression of interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), and activation of nuclear factor kappa-light-chain-enhancer of activated B cells and correlate with injury severity and anemia. MATERIALS AND METHODS: Plasma from 15 trauma patients was cultured with bone marrow fibroblast cells and compared with that from healthy volunteers. At 6, 24, and 48 h, the expression of IL-6, G-CSF, EPO, SCF, and the activation of nuclear factor kappa-light-chain-enhancer of activated B cells were measured using quantitative polymerase chain reaction. The influence of trauma plasma on cytokine expression was further stratified by injury severity score (ISS). RESULTS: The average hemoglobin significantly decreased from admission to discharge (10.7 ± 2.5 to 9.2 ± 1.1 g/dL, P < 0.04). The discharge hemoglobin significantly decreased by 14% from the admission hemoglobin. After 48 h, trauma plasma significantly increased IL-6, G-CSF, and EPO bone marrow fibroblast expression when compared with normal plasma. When stratified by ISS, IL-6, G-CSF, and EPO, bone marrow fibroblast expression was highest in the trauma plasma ISS 27-41 group and was significantly elevated compared with normal plasma. When SCF expression was stratified by ISS, there was a significant increase in expression in ISS 27-41. Higher ISS was also associated with a larger decrease in hemoglobin despite no difference in total blood transfusions. CONCLUSIONS: Severe trauma can systemically increase IL-6, G-CSF, and EPO expression in bone marrow stroma. Increased hematopoietic cytokine expression after traumatic injury correlated with a hypercatecholamine state, anemia, and injury severity.

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).

Effects of trauma, hemorrhagic shock, and chronic stress on lung vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) regulate vascular permeability and endothelial cell survival. We hypothesized that hemorrhagic shock (HS) and chronic stress (CS) would increase expression of lung VEGF and its receptors, potentiating pulmonary edema in lung tissue. MATERIALS AND METHODS: Male Sprague-Dawley rats aged 8-9 wk were randomized: naïve control, lung contusion (LC), LC followed by HS (LCHS), and LCHS with CS in a restraint cylinder for 2 h/d (LCHS/CS). Animals were sacrificed on days 1 and 7. Expressions of lung VEGF, VEGFR-1, and VEGFR-2 were determined by polymerase chain reaction. Lung Injury Score (LIS) was graded on light microscopy by inflammatory cell counts, interstitial edema, pulmonary edema, and alveolar integrity (range: 0 = normal; 8 = severe injury). RESULTS: Seven days after LC, lung VEGF and VEGFR-1 were increased, and lung tissue healed (LIS: 0.8 ± 0.8). However, 7 d after LCHS and LCHS/CS, lung VEGF and VEGFR-1 expressions were decreased. VEGFR-2 was also decreased after LCHS/CS. LIS was elevated 7 d after LCHS and LCHS/CS (6.5 ± 1.0 and 8.2 ± 0.8). Increased LIS after LCHS and LCHS/CS was because of higher inflammatory cell counts, increased interstitial edema, and loss of alveolar integrity, whereas pulmonary edema was unchanged. CONCLUSIONS: Elevation of lung VEGF and VEGFR-1 expressions after LC alone was associated with healing of injured lung tissue. Expressions of VEGF, VEGFR-1, and VEGFR-2 were reduced after LCHS and LCHS/CS, and injured lung tissue did not heal. Persistent lung injury after severe trauma was because of inflammation rather than pulmonary edema.

Persistent injury-associated anemia: the role of the bone marrow microenvironment.

BACKGROUND: The regulation of erythropoiesis involves hematopoietic progenitor cells, bone marrow stroma, and the microenvironment. Following severe injury, a hypercatecholamine state develops that is associated with increased mobilization of hematopoietic progenitor cells to peripheral blood and decreased growth of bone marrow erythroid progenitor cells that manifests clinically as a persistent injury-associated anemia. Changes within the bone marrow microenvironment influence the development of erythroid progenitor cells. Therefore, we sought to determine the effects of lung contusion, hemorrhagic shock, and chronic stress on the hematopoietic cytokine response. MATERIALS AND METHODS: Bone marrow was obtained from male Sprague-Dawley rats (n = 6/group) killed 7 d after lung contusion followed by hemorrhagic shock (LCHS) or LCHS followed by daily chronic restraint stress (LCHS/CS). End point polymerase chain reaction was performed for interleukin-1ß, interleukin-10, stem cell factor, transforming growth factor-ß, high-mobility group box-1 (HMGB-1), and B-cell lymphoma-extra large. RESULTS: Seven days following LCHS and LCHS/CS, bone marrow expression of prohematopoietic cytokines (interleukin-1ß, interleukin-10, stem cell factor, and transforming growth factor-ß) was significantly decreased, and bone marrow expression of HMGB-1 was significantly increased. B-cell lymphoma-extra large bone marrow expression was not affected by LCHS or LCHS/CS (naïve: 44 ± 12, LCHS: 44 ± 12, LCHS/CS: 37 ± 1, all P > 0.05). CONCLUSIONS: The bone marrow microenvironment was significantly altered following severe trauma in a rodent model. Prohematopoietic cytokines were downregulated, and the proinflammatory cytokine HMGB-1 had increased bone marrow expression. Modulation of the bone marrow microenvironment may represent a therapeutic strategy following severe trauma to alleviate persistent injury-associated anemia.

Postinjury Pneumonia Induces a Unique Blood Microbiome Signature.

BACKGROUND: Previous preclinical studies have demonstrated a pathobiome after traumatic injury; however, the impact of post-injury sepsis on gut epithelial permeability and bacterial translocation remains unknown. We hypothesized that polytrauma with post-injury pneumonia would result in impaired gut permeability leading to specific blood microbiome arrays. METHODS: Male and proestrus female Sprague-Dawley rats were subjected to either polytrauma (PT), PT plus 2-hours daily chronic restraint stress (PT/CS), PT with postinjury day 1 inoculation with pseudomonas pneumonia (PT + PNA), PT/CS + PNA, or naive controls. Whole blood microbiome was measured serially using high-throughput 16S rRNA sequencing and QIIME2 bioinformatics analyses. Microbial diversity was assessed using Chao1/Shannon indices and principle coordinate analysis. Intestinal permeability was evaluated by plasma occludin and lipopolysaccharide-binding protein (LBP) assays. RESULTS: PT/CS + PNA had increased intestinal permeability compared to uninfected counterparts (PT/CS) with significantly elevated occludin (p < 0.01). Bacteria was not detected in the blood of naïve controls, PT or PT/CS, but was present in both PT + PNA and PT/CS + PNA on days two and seven. The PT/CS + PNA blood biome showed dominance of Streptococcus compared to PT + PNA at day two (p < 0.05). Females PT/CS + PNA had a significant abundance of Staphylococcus at day two and Streptococcus at day seven in the blood biome compared to male counterparts (p < 0.05). CONCLUSIONS: Multicompartmental trauma with post-injury pneumonia results in increased intestinal permeability and bacteremia with a unique blood biome, with sexual dimorphisms evident in the blood biome composition. These findings suggest that post-injury sepsis has clinical significance and could influence outcomes after severe trauma and critical illness.

A rat model of multicompartmental traumatic injury and hemorrhagic shock induces bone marrow dysfunction and profound anemia.

BACKGROUND: Severe trauma is associated with systemic inflammation and organ dysfunction. Preclinical rodent trauma models are the mainstay of postinjury research but have been criticized for not fully replicating severe human trauma. The aim of this study was to create a rat model of multicompartmental injury which recreates profound traumatic injury. METHODS: Male Sprague-Dawley rats were subjected to unilateral lung contusion and hemorrhagic shock (LCHS), multicompartmental polytrauma (PT) (unilateral lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofracture), or naïve controls. Weight, plasma toll-like receptor 4 (TLR4), hemoglobin, spleen to body weight ratio, bone marrow (BM) erythroid progenitor (CFU-GEMM, BFU-E, and CFU-E) growth, plasma granulocyte colony-stimulating factor (G-CSF) and right lung histologic injury were assessed on day 7, with significance defined as p values <0.05 (*). RESULTS: Polytrauma resulted in markedly more profound inhibition of weight gain compared to LCHS (p = 0.0002) along with elevated plasma TLR4 (p < 0.0001), lower hemoglobin (p < 0.0001), and enlarged spleen to body weight ratios (p = 0.004). Both LCHS and PT demonstrated suppression of CFU-E and BFU-E growth compared to naïve (p < 0.03, p < 0.01). Plasma G-CSF was elevated in PT compared to both naïve and LCHS (p < 0.0001, p = 0.02). LCHS and PT demonstrated significant histologic right lung injury with poor alveolar wall integrity and interstitial edema. CONCLUSIONS: Multicompartmental injury as described here establishes a reproducible model of multicompartmental injury with worsened anemia, splenic tissue enlargement, weight loss, and increased inflammatory activity compared to a less severe model. This may serve as a more effective model to recreate profound traumatic injury to replicate the human inflammatory response postinjury.

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.

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 multicompartmental trauma (PT) (lung contusion, hemorrhagic shock, cecectomy, bifemoral pseudofracture), PT plus 2-hour 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 Quantitative Insights Into Microbial Ecology 2 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 with naive controls ( p < 0.05). Groups PT-PNA and PT/RS-PNA resulted in different ß-diversity arrays compared with 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 with 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 . The PT/RS males had an abundance of Gastranaerophilales and Muribaculaceae . CONCLUSION: Multicompartmental trauma complicated by sepsis significantly diminishes diversity and alters microbial composition toward 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.

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.

Multicompartmental trauma alters bone marrow erythroblastic islands.

BACKGROUND: Trauma is associated with widespread inflammation, neuroendocrine activation, and an inadequate bone marrow response to anemia. During late-stage erythropoiesis, erythroid progenitors/erythroblasts form clusters on the surface of specialized bone marrow macrophages where they are supported through terminal differentiation and enucleation. We hypothesized that these erythroblastic islands (EBIs) are adversely impacted by severe trauma. METHODS: Male Sprague-Dawley rats (n = 8/group) were subjected to either multiple injuries (PT) (lung contusion, hemorrhagic shock, cecectomy, and bifemoral pseudofractures), PT plus 2 hours of daily chronic restraint stress (PT/CS), or naive controls. Bone marrow was harvested on days 2 and 7. Nuclear-stained, enriched bone marrow EBIs were fixed and stained for CD71, VCAM-1, and CD163, and confocal images were obtained at 20 times magnification. Numbers of erythroid cells/EBI and ratio of reticulocytes/EBI were counted by a blinded observer. Differences were compared using analysis of variance, with significance defined as p < 0.05. RESULTS: PT and PT/CS had significantly reduced numbers of erythroid cells per EBI on day 2 when compared with naive (PT: 5.9 ± 1.0 cells [ p < 0.05], PT/CS: 6.8 ± 0.8 cells [ p < 0.05] vs. naive: 8.5 ± 0.8 cells). On day 7, the number of erythroid cells/EBI increased following PT (8.3 ± 0.4 cells) but remained reduced following PT/CS (5.9 ± 0.5 cells [ p < 0.05]). This correlated with an increased proportion of reticulocytes/EBI on day 7 following PT, which was not present following PT/CS (PT: 54% [ p < 0.05] vs. PT/CS: 28%). CONCLUSION: Late-stage erythropoiesis was altered following multicompartmental PT early after injury, and these alterations persisted with the addition of daily chronic stress. Alterations in EBI structure and function after severe trauma and critical illness may serve as a promising new area of study to improve mechanistic understanding of persistent anemia after trauma.

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.

Bone Marrow Adipokine Expression Was Associated With Decreased Erythroid Colony Growth After Trauma.

BACKGROUND: Proinflammatory and immunomodulatory adipokines are linked to inflammation in critically ill patients but are poorly studied after injury. We hypothesized that trauma would induce systemic adipokine release and influence erythroid suppression. METHODS: Blood and bone marrow (BM) were collected from trauma patients (ISS > 15, n = 90) and compared to patients undergoing elective hip replacement (n = 37). Plasma adipokine levels were measured, and BM was assayed for adipokine transcription and erythroid progenitor growth potential. Differences were detected using t-tests and correlations using simple linear regression. RESULTS: Trauma patients exhibited decreased adiponectin (1.8* vs 3.4 mg/mL) and increased leptin (7.8* vs 4.6 ng/mL) and resistin (3.1* vs 2.5 ng/mL), with sex- and age-specific differences. They also showed increased BM visfatin transcription. Adipokine transcription negatively correlated with erythroid progenitor growth. CONCLUSION: Adipose tissue activity is linked to inflammatory responses after injury, with variability by age and sex. Bone marrow adipose tissue may influence erythroid recovery after trauma.

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.