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.

Diabetic nephropathy after kidney transplantation in patients with pretransplantation type II diabetes: A retrospective case series study from a high-volume center in the United States.

BACKGROUND: Patients with type II diabetes mellitus (DM) undergoing renal transplantation are at risk of diabetic nephropathy (DN) in the transplanted kidney. The true risk of developing post-transplantation DN is unknown, and post-transplantation DN is poorly characterized in the literature. METHODS: The biopsy database at the University of Maryland Medical Center was queried for kidney transplant biopsies which demonstrated evidence of DN. The time from transplantation to biopsy-proven DN (time to diagnosis, TTD) was calculated and analyzed in the context of demographics, serum creatinine, and onset of diabetes. By extrapolating the total number of patients who developed DN in the last 2 years, we estimated the recurrence rate of DN. RESULTS: Sixty patients whose renal biopsies met criteria were identified. The mean age was 56.6 (±1.58) years, and the mean creatinine level at time of biopsy was 1.65 (±0.12) mg/dL. Simultaneous pathological diagnoses were frequent on kidney biopsy; rejection was present at variable rates: classes I, IIA, IIB, and III were 5.0%, 66.7%, 18.4%, and 10%, respectively. The mean TTD was 1456 (±206) days. TTD was significantly shorter for patients receiving a cadaveric vs living donor renal transplant (1118 ± 184 vs 2470 ± 547 days, P = 0.004). Older patients (r = 0.378, P = 0.003) and patients with higher serum creatinine (r = 0.282, P = 0.029) had shorter TTDs. Extrapolations showed that 74.7% of patients would be free of DN 10 years after renal transplantation. CONCLUSIONS: Diabetic nephropathy occurs after transplantation, and this appears to be due to both donor and recipient-derived factors. Encouragingly, our estimates suggest that as many as 75% of patients may be free of DN at 10 years following kidney transplantation.

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.

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.

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.

Chronic mesenteric ischemia-induced intestinal dysbiosis resolved after revascularization.

Objective: Chronic mesenteric ischemia (CMI) is a debilitating condition arising from intestinal malperfusion from mesenteric artery stenosis or occlusion. Mesenteric revascularization has been the standard of care but can result in substantial morbidity and mortality. Most of the perioperative morbidity has been secondary to postoperative multiple organ dysfunction, potentially from ischemia-reperfusion injury. The intestinal microbiome is a dense community of microorganisms in the gastrointestinal tract that help regulate pathways ranging from nutritional metabolism to the immune response. We hypothesized that patients with CMI will have microbiome perturbations that contribute to this inflammatory response and could potentially normalize in the postoperative period. Methods: We performed a prospective study of patients with CMI who had undergone mesenteric bypass and/or stenting from 2019 to 2020. Stool samples were collected at three time points: preoperatively at the clinic, perioperatively within 14 days after surgery, and postoperatively at the clinic at >30 days after revascularization. Stool samples from healthy controls were used for comparison. The microbiome was measured using 16S rRNA sequencing on an Illumina-MiSeq sequence platform and analyzed using the QIIME2 (quantitative insights into microbial ecology 2)-DADA2 bioinformatics pipeline with the Silva database. Beta-diversity was analyzed using a principal coordinates analysis and permutational analysis of variance. Alpha-diversity (microbial richness and evenness) was compared using the nonparametric Mann-Whitney U test. Microbial taxa unique to CMI patients vs controls were identified using linear discriminatory analysis effect size analysis. P < .05 was considered statistically significant. Results: Eight patients with CMI had undergone mesenteric revascularization (25% men; average age, 71 years). Nine healthy controls were also analyzed (78% men; average age, 55 years). Bacterial alpha-diversity (number of operational taxonomic units) was dramatically reduced preoperatively compared with that of the controls (P = .03). However, revascularization partially restored the species richness and evenness in the perioperative and postoperative phases. Beta-diversity was only different between the perioperative and postoperative groups (P = .03). Further analyses revealed increased abundance of Bacteroidetes and Clostridia taxa preoperatively and perioperatively compared with the controls, which was reduced during the postoperative period. Conclusions: The results from the present study have shown that patients with CMI have intestinal dysbiosis that resolves after revascularization. The intestinal dysbiosis is characterized by the loss of alpha-diversity, which is restored perioperatively and maintained postoperatively. This microbiome restoration demonstrates the importance of intestinal perfusion to sustain gut homeostasis and suggests that microbiome modulation could be a possible intervention to ameliorate acute and subacute postoperative outcomes in these patients.

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.

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.

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.

Anemia Recovery after Trauma: A Longitudinal Study.

Background: Post-injury inflammation and its correlation with anemia recovery after severe trauma is poorly described. Severe injury induces a systemic inflammatory response associated with critical illness and organ dysfunction, including disordered hematopoiesis, and anemia. This study sought to characterize the resolution of post-injury inflammation and anemia to identify risk factors associated with persistence of anemia. Patients and Methods: This single-institution study prospectively enrolled 73 trauma patients with an injury severity score >15, hemorrhagic shock, and a lower extremity long bone orthopedic injury. Blood was obtained at enrollment and after 14 days, one, three, and six months. Analytes were compared using Mann-Whitney U tests with correction for multiple comparisons. Results: Median age was 45 years and Injury Severity Score (ISS) was 27, with anemia rates of 97% at two weeks, 80% at one month, 52% at three months, and 30% at six months. Post-injury elevations in erythropoietin, interleukin-6, and C-reactive protein resolved by one month, three months, and six months, respectively. Median granulocyte colony-stimulating factor (G-CSF) and tumor necrosis factor (TNF)-α concentrations remained elevated throughout the six-month follow-up period. Patients with persistent anemia had longer intensive care unit and hospital lengths of stay, more infectious complications, and received more packed red blood cell transfusions compared to those with early anemia recovery. Conclusions: Severe trauma is associated with a prolonged inflammatory response, which is associated with increased transfusion requirements, lengths of stay, and persistent anemia. Further analysis is needed to identify correlations between prolonged inflammation and clinical outcomes after discharge.

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.

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.