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.

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.

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.

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.

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.

Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot.

BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.

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.

Adverse Long-Term Outcomes and an Immune Suppressed Endotype in Sepsis Patients with Reduced Interferon-γELISpot: A Multicenter, Prospective Observational Study.

BACKGROUND: Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. METHODS: An ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-γ (IFN-γ) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. RESULTS: Compared with 46 healthy subjects, unstimulated and stimulated whole blood IFNγ expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFNγ expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFNγ producing cells and amount of IFNγ produced per cell (all p<0.05). Importantly, IFNγ total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFNγ expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. CONCLUSIONS: A whole blood IFNγ ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients.

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.

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.

Daily propranolol administration reduces persistent injury-associated anemia after severe trauma and chronic stress.

BACKGROUND: After severe trauma, patients develop a norepinephrine-mediated persistent, injury-associated anemia. This anemia is associated with suppression of bone marrow (BM) erythroid colony growth, along with decreased iron levels, and elevated erythropoietin (EPO) levels, which are insufficient to promote effective erythropoiesis. The impact of norepinephrine on iron regulators, such as ferroportin, transferrin, and transferrin receptor-1 (TFR-1), is unknown. Using a clinically relevant rodent model of lung contusion (LC), hemorrhagic shock (HS), and chronic stress (CS), we hypothesize that daily propranolol (BB), a nonselective ß blocker, restores BM function and improves iron homeostasis. METHODS: Male Sprague-Dawley rats were subjected to LCHS ± BB and LCHS/CS ± BB. BB was achieved with propranolol (10 mg/kg) daily until the day of sacrifice. Hemoglobin, plasma EPO, plasma hepcidin, BM cellularity and BM erythroid colony growth were assessed. RNA was isolated to measure transferrin, TFR-1 and ferroportin expression. Data are presented as mean ± SD; *p < 0.05 versus untreated counterpart by t test. RESULTS: The addition of CS to LCHS leads to persistent anemia on posttrauma day 7, while the addition of BB improved hemoglobin levels (LCHS/CS: 10.6 ± 0.8 vs. LCHS/CS + BB: 13.9 ± 0.4* g/dL). Daily BB use after LCHS/CS improved BM cellularity, colony-forming units granulocyte, erythrocyte, monocyte megakaryocyte, burst-forming unit erythroid and colony-forming unit erythroid cell colony growth. LCHS/CS + BB significantly reduced plasma EPO levels and increased plasma hepcidin levels on day 7. The addition of CS to LCHS resulted in decreased liver ferroportin expression as well as decreased BM transferrin and TFR-1 expression, thus, blocking iron supply to erythroid cells. However, daily BB after LCHS/CS improved expression of all iron regulators. CONCLUSION: Daily propranolol administration after LCHS/CS restored BM function and improved anemia after severe trauma. In addition, iron regulators are significantly reduced after LCHS/CS, which may contribute to iron restriction after injury. However, daily propranolol administration after LCHS/CS improved iron homeostasis.

Mesenchymal stem cells enhance lung recovery after injury, shock, and chronic stress.

BACKGROUND: Normal lung healing is impaired when lung contusion (LC) is followed by hemorrhagic shock (HS), and chronic stress (CS). Mesenchymal stem cells (MSCs) are immunomodulatory, pluripotent cells that are under investigation for use in wound healing and tissue regeneration. We hypothesized that treatment with MSCs can reverse the impaired healing seen after LC combined with HS and CS (LCHS/CS). METHODS: Male Sprague-Dawley rats (n = 6/group) underwent LCHS with or without a single intravenous dose of 5 × 10(6) Sprague-Dawley rat MSCs after resuscitation. Thereafter, rats were subjected to 2 hours of CS daily on days 1-6 and were humanely killed on day 7. Lung histology was scored according to a well-established lung injury score (LIS) that included interstitial and pulmonary edema, alveolar integrity, and inflammatory cells. Scoring ranges from 0 (normal lung) to 11 (most severely injured). Whole blood was analyzed for the presence of CD4(+)CD25(+)FoxP3(+) T-regulatory cells (Treg) by flow cytometry. RESULTS: Seven days after isolated LC, LIS had returned to 0.8 ± 0.4; however, after LCHS/CS healing is significantly delayed (7.2 ± 2.2; P < .05). Addition of MSC to LCHS/CS decreased LIS to 2.0 ± 1.3 (P < .05) and decreased all subgroup scores (inflammatory cells, interstitial and pulmonary edema, and alveolar integrity) significantly compared with LCHS/CS (P < .05). The percentage of Tregs found in the peripheral blood of animals undergoing LCHS/CS did not change from LC alone (10.5 ± 3.3% vs 6.7 ± 1.7%; P > .05). Treatment with MSCs significantly increased the Treg population compared with LCHS/CS alone (11.7 ± 2.7% vs 6.7 ± 1.7%; P < .05) CONCLUSION: In this model, severe impairment of wound healing observed 1 week after LCHS/CS is reversed by a single treatment with MSCs immediately after resuscitation. This improvement in lung healing is associated with a decrease in the number of inflammatory cells and lung edema and a significant increase in peripheral Tregs. Further study into the timing of administration and mechanisms by which cell-based therapy using MSCs modulate the immune system and improve wound healing is warranted.

Mesenchymal stem cells reverse bone marrow dysfunction following injury and stress.

BACKGROUND: Bone marrow (BM) dysfunction following experimental lung contusion (LC) resolves in 7 days; however, if followed by chronic stress (CS) following, BM dysfunction is persistent. Mesenchymal stem cells (MSCs) have protective immunomodulatory effects. We hypothesize that MSC can protect the BM against the deleterious effect of CS following LC. METHODS: Male Sprague-Dawley rats (n = 6-7 per group) underwent LC or LC/CS ± MSC injection. CS consisted of a daily 2-hour period of restraint with repositioning and alarming every 30 minutes to prevent habituation. A single intravenous dose of 5 × 10 MSCs was given within 10 minutes following LC. Animals were sacrificed at Day 7, and peripheral blood (PB) and BM were collected. Flow cytometry was used to assess hematopoietic progenitor cells (HPCs) mobilized to PB. Plasma granulocyte colony-stimulating factor (G-CSF) levels were measured by enzyme-linked immunosorbent assay. BM cellularity and growth of BM HPC colonies (colony-forming unit-erythroid [CFU-E], burst-forming unit-erythroid [BFU-E], colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte [CFU-GEMM]) were also evaluated. RESULTS: As previously reported, the addition of CS to LC resulted in a 32% decrease in BM cellularity; significant decreases in CFU-GEMM, BFU-E, and CFU-E; and marked increase in HPC in the PB as compared with the naive animals. The addition of MSC to LC/CS resulted in a 22% increase in BM cellularity and significant increases in CFU-GEMM, BFU-E, and CFU-E cultured from the BM. MSCs additionally reduced plasma G-CSF, prevented prolonged mobilization of HPC to PB, and restored colony growth to naive levels. CONCLUSION: CS following LC results in persistent BM dysfunction manifested by a significant decrease in cellularity, HPC colony growth, and increased G-CSF levels and HPC mobilization to the PB at 7 days following injury. The addition of a single dose of MSCs following acute traumatic injury reverses the deleterious effects of CS on BM function. Further study is warranted to better understand the mechanisms behind MSC-mediated protection of BM function in the setting of CS.

Daily propranolol prevents prolonged mobilization of hematopoietic progenitor cells in a rat model of lung contusion, hemorrhagic shock, and chronic stress.

INTRODUCTION: Propranolol has been shown previously to decrease the mobilization of hematopoietic progenitor cells (HPCs) after acute injury in rodent models; however, this acute injury model does not reflect the prolonged period of critical illness after severe trauma. Using our novel lung contusion/hemorrhagic shock/chronic restraint stress model, we hypothesize that daily administration of propranolol will decrease prolonged mobilization of HPCs without worsening lung healing. METHODS: Male Sprague-Dawley rats underwent 6 days of restraint stress after undergoing lung contusion or lung contusion/hemorrhagic shock. Restraint stress consisted of a daily 2-hour period of restraint interrupted every 30 minutes by alarms and repositioning. Each day after the period of restraint stress, the rats received intraperitoneal propranolol (10 mg/kg). On day 7, peripheral blood was analyzed for granulocyte-colony stimulating factor (G-CSF) and stromal cell-derived factor 1 via enzyme-linked immunosorbent assay and for mobilization of HPCs using c-kit and CD71 flow cytometry. The lungs were examined histologically to grade injury. RESULTS: Seven days after lung contusion and lung contusion/hemorrhagic shock, the addition of chronic restraint stress significantly increased the mobilization of HPC, which was associated with persistently increased levels of G-CSF and increased lung injury scores. The addition of propranolol to lung contusion/chronic restraint stress and lung contusion/hemorrhagic shock/chronic restraint stress models greatly decreased HPC mobilization and restored G-CSF levels to that of naïve animals without worsening lung injury scores. CONCLUSION: The daily administration of propranolol after both lung contusion and lung contusion/hemorrhagic shock subjected to chronic restraint stress decreased the prolonged mobilization of HPC from the bone marrow and decreased plasma G-CSF levels. Despite the decrease in mobilization of HPC, lung healing did not worsen. Alleviating chronic stress with propranolol may be a future therapeutic target to improve healing after severe injury.

Mesenchymal stem cells reverse trauma and hemorrhagic shock-induced bone marrow dysfunction.

BACKGROUND: Lung contusion (LC) followed by hemorrhagic shock (HS) causes persistent bone marrow (BM) dysfunction lasting up to 7 d after injury. Mesenchymal stem cells (MSCs) are multipotent cells that can hasten healing and exert protective immunomodulatory effects. We hypothesize that MSCs can attenuate BM dysfunction after combined LCHS. MATERIALS AND METHODS: Male Sprague-Dawley rats (n = 5-6 per group) underwent LC plus 45 min of HS (mean arterial pressure of 30-35). Allogeneic MSCs (5 × 10(6) cells) were injected intravenously after resuscitation. At 7 d, BM was analyzed for cellularity and growth of hematopoietic progenitor cell (HPC) colonies (colony-forming unit-erythroid; burst-forming unit-erythroid; and colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte). Flow cytometry measured %HPCs in peripheral blood; plasma granulocyte colony-stimulating factor (G-CSF) levels were measured via enzyme-linked immunosorbent assay. Data were analyzed by one-way analysis of variance followed by the Tukey multiple comparison test. RESULTS: As previously shown, at 7 d, LCHS resulted in 22%, 30%, and 24% decreases in colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte, burst-forming unit-erythroid, and colony-forming unit-erythroid colony growth, respectively, versus naive. Treatment with MSCs returned all BM parameters to naive levels. There was no difference in %HPCs in peripheral blood between groups; however, G-CSF remained increased up to 7 d after LCHS. MSCs returned G-CSF to naive levels. Plasma from animals receiving MSCs was not suppressive to the BM. CONCLUSIONS: One week after injury, the persistent BM dysfunction observed in animals undergoing LCHS is reversed by treatment with MSCs with an associated return of plasma G-CSF levels to normal. Plasma from animals undergoing LCHS plus MSCs was not suppressive to BM cells in vitro. Treatment with MSCs after injury and shock reverses BM suppression and returns plasma G-CSF levels to normal.

Mesenchymal stem cells increase T-regulatory cells and improve healing following trauma and hemorrhagic shock.

BACKGROUND: Rodent lungs undergo full histologic recovery within 1 week following unilateral lung contusion (LC). However, when LC is followed by hemorrhagic shock (HS), healing is impaired. We hypothesize that the intravenous administration of mesenchymal stem cells (MSCs) in animals undergoing combined LC followed by HS (LCHS) will improve wound healing. METHODS: Male Sprague-Dawley rats (n = 5-6 per group) were subjected to LCHS with or without the injection of a single intravenous dose of 5 × 10 MSCs following return of shed blood after HS. Rats were sacrificed 7 days following injury. Flow cytometry was used to determine the T-regulatory cell (Treg) population in peripheral blood. Lung histology was graded using a well-established lung injury score (LIS). Components of the LIS include average inflammatory cells per high-power field over 30 fields, interstitial edema, pulmonary edema, and alveolar integrity, with total scores ranging from 0 to 11. Data were analyzed by analysis of variance followed by Tukey's multiple comparison test, expressed as mean (SD). p < 0.05 was considered significant. RESULTS: Seven days following isolated LC, animals demonstrated lung healing with an LIS unchanged from naive. The addition of HS resulted in a persistently elevated LIS score, whereas the addition of MSCs to LCHS decreased the LIS score back to naive levels. The change in LIS was driven by a significant decrease in edema scores. In rats undergoing LC alone, 10.5% (3.3%) of CD4 cells were Tregs. The addition of HS caused no significant change in Treg population (9.3% [0.7%]), whereas LCHS + MSC significantly increased the population to 18.2% (6.8%) in peripheral blood (p < 0.05 vs. LCHS). CONCLUSION: Impaired wound healing following trauma and HS is improved by a single dose of MSCs given immediately after injury. This enhanced healing is associated with an increase in the Treg population and a significant decrease in lung edema score as compared with animals undergoing LCHS. Further study into the role of Tregs in MSC-mediated wound healing is warranted.