Trauma-induced lung injury is associated with infiltration of activated TLR expressing myeloid cells.

INTRODUCTION: Traumatic injury with hemorrhage (TH) induces an inflammatory response in the lung resulting in lung injury involving activation of immune cells including myeloid cells (i.e., monocytes, granulocytes and macrophages), in part through TLRs. TLRs, via the recognition of damage associated molecular patterns (DAMPs), are a key link between tissue injury and inflammation. Nonetheless, the role of TLRs in myeloid cell activation and TH-induced lung injury remains ill defined. METHODS: C57BL/6 male mice were subjected to TH or sham treatment (n = 4-6 /group). Lung tissues were collected two hrs. after injury. Single cells were isolated from the lungs by enzymatic digestion and myeloid cell TLR expression and activation (i.e., cytokine production) were assessed using flow cytometry techniques. RESULTS: The injury was associated with a profound change in the lung myeloid cell population. TH markedly increased lung CD11b+ monocyte numbers and Gr1+ granulocyte numbers as compared to sham mice. The number of cells expressing TLR2, TLR4, and TLR9 were increased 4-7 fold in the TH mice. Activation for elevated cytokine (TNFα, IL-10) production was observed in the lung monocyte population of the TH mice. CONCLUSIONS: Trauma-induced lung injury is associated with infiltration of the lungs with TLR expressing myeloid cells that are activated for elevated cytokine responses. This elevation in TLR expression may contribute to DAMP-mediated pulmonary complications of an inflammatory nature and warrants further investigation.

Burn injury is associated with an infiltration of the wound site with myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) have been identified in the burn wound, however their characterization is incomplete. To study this, mice were subjected to a major burn and skin cells were isolated 3 days thereafter for analysis. Significant infiltration of the burn wound with MDSCs was observed as compared with uninjured skin. The skin of naïve mice did not contain MDSCs. Characterization of the cells showed that 33% of MDSCs in the wound were monocytic (M)-MDSCs, which was significantly less than that found in uninjured skin (52%). In contrast, polymorphonuclear (PMN)-MDSCs were greater in the burn wound as compared with uninjured skin. Burn wound TLR expression by both MDSCs subsets was decreased as compared with uninjured skin. Wound MDSCs produced pro- and anti-inflammatory cytokines and iNOS was present in both MDSC subsets, whereas ARG1 was only present in M-MDSCs. In conclusion, both M- and PMN-MDSCs infiltrate burn wound with after injury, however, they displayed decreased TLR expression, suggesting receptor down-regulation.

Toll-like receptor responses are suppressed in trauma ICU patients.

BACKGROUND: Inflammation and activation of the innate immune system are often associated with traumatic injury and may involve alterations in toll-like receptor (TLR)-mediated responses. METHODS: A prospective observational study was designed and conducted. Twenty-one severely injured (ISS = 16-41) trauma intensive care unit (ICU) patients and six healthy volunteers that served as controls were enrolled. Anticoagulated whole blood was collected at 2-12 d after ICU admission and incubated in the presence of media alone (baseline), zymosan (TLR2 agonist) or lipopolysaccharide (LPS; TLR4 agonist) for 3 h. Supernatant levels of inflammatory cytokines (IL-1ß, IL-6, IL-10, and TNFα) were determined. RESULTS: TLR2-mediated and TLR4-mediated activation of whole blood cell cultures from both healthy volunteers and subjects-induced elevated cytokine levels over that observed in unstimulated cultures. Baseline values of IL-6 were significantly elevated in subject cultures as compared to healthy volunteers. Healthy volunteer cultures had 2-3-fold greater levels of IL-6 and TNFα than subject cultures when stimulated with zymosan (TLR2 agonist) or LPS (TLR4 agonist). IL-1ß and IL-10 levels did not differ significantly between healthy volunteers and subjects. CONCLUSIONS: The ability of circulating leukocytes from trauma ICU patients to be activated by TLR agonists is markedly suppressed and may play a role in the development of subsequent infectious complications.

The association between the Th-17 immune response and pulmonary complications in a trauma ICU population.

BACKGROUND: The overall immunopathology of the T-helper cell (Th)-17 immune response has been implicated in various inflammatory diseases including pulmonary inflammation; however its potential role in acute respiratory distress syndrome (ARDS) is not defined. This study aimed to evaluate the Th-17 response in bronchoalveolar lavage fluid (BALF) and blood and from trauma patients with pulmonary complications. METHODS: A total of 21 severely injured intensive care unit (ICU) subjects, who were mechanically ventilated and undergoing bronchoscopy, were enrolled. BALF and blood were collected and analyzed for Th-1 (interferon [IFN]γ), Th-2 (interleukin [IL]-4, -10), Th-17 (IL-17A, -17F, -22, 23) and pro-inflammatory (IL-1ß, IL-6, tumor necrosis factor [TNF]α) cytokine levels. RESULTS: Significant levels of the Th-17 cytokines IL-17A, -17F and -21 and IL-6 (which can be classified as a Th-17 cytokine) were observed in the BALF of all subjects. There were no significant differences in Th-17 cytokines between those subjects with ARDS and those without, with the exception of plasma and BALF IL-6, which was markedly greater in ARDS subjects, as compared with controls and non-ARDS subjects. CONCLUSIONS: Trauma patients with pulmonary complications exhibited a significant Th-17 response in the lung and blood, suggesting that this pro-inflammatory milieu may be a contributing factor to such complications.

Gamma delta (γδ) T-cells are critical in the up-regulation of inducible nitric oxide synthase at the burn wound site.

BACKGROUND: The high incidence of morbidity and mortality following major burn can in part be attributed to immune derangements and wound healing complications. Inflammation plays an important role in wound healing, of which inducible nitric oxide synthase (iNOS) derived nitric oxide is a central mediator. T-cells of the γδ TCR lineage have also been shown to be important in healing of the burn wound site. Nonetheless, the role of γδ T-cells in the regulation of the burn wound iNOS expression is unknown. METHODS: Wildtype (WT) and δ TCR(-/-) male C57BL/6 mice were subjected to burn (3rd degree, 12.5% TBSA) or sham treatment. Three days after injury, skin samples from non-injured and the burn wound were collected and analyzed for the expression of iNOS and cytokines and chemokine levels. In a second series of experiments, WT mice were subjected to burn and left untreated or treated with the iNOS inhibitor, L-Nil. Skin cytokine and chemokine levels were assessed 3days thereafter. RESULTS: Burn induced an 18-fold increase in iNOS expression at the wound site as compared to the uninjured skin of WT sham mice. In δ TCR(-/-) mice iNOS expression at the wound site was significantly lower than that of the WT group. Burn also induced increased levels of IL-1ß, IL-6, G-CSF, TNF-α, KC, MCP-1, MIP-1α and MIP-1ß at the wound site in WT and δ TCR(-/-) mice, but G-CSF, TNF-α, and MIP-1ß levels were greater in δ TCR(-/-) mice. Inhibition of iNOS activity in WT mice with L-Nil suppressed burn wound levels of IL-1ß, G-CSF, and MIP-1α, whereas IL-6, TNF-α, KC, MCP-1 and MIP-1ß were unaffected. CONCLUSIONS: T-cells of the γδ TCR lineage significantly contribute to the up-regulation of iNOS expression which contributes to wound inflammation.

Burn-induced alterations in toll-like receptor-mediated responses by bronchoalveolar lavage cells.

UNLABELLED: Burn is associated with profound inflammation and activation of the innate immune system in multiple organ beds, including the lung. Similarly, toll-like receptors (TLR) are associated with innate immune activation. Nonetheless, it is unclear what impact burn has on TLR-induced inflammatory responses in the lung. METHODS: Male C57BL/6 mice were subjected to burn (3rd degree, 25% TBSA) or sham procedure and 1, 3 or 7 days thereafter, bronchoalveolar lavage (BAL) fluid was collected and cells were isolated and cultured in vitro with specific TLR agonists as follows: Zymosan (TLR-2), LPS (TLR-4) and CpG-ODN (TLR-9). Supernatants were collected 48 h later and assayed for inflammatory cytokine levels (IL-1ß, IL-6, IL-10, IL-17, TNF-α, KC, MCP-1, MIP-1α, MIP-1ß and RANTES) by Bioplex. RESULTS: BAL fluid from sham and burn mice did not contain detectable cytokine levels. BAL cells, irrespective of injury, were responsive to TLR-2 and TLR-4 activation. Seven days after burn, TLR-2 and TLR-4 mediated responses by BAL cells were enhanced as evidenced by increased production of IL-6, IL-17, TNF-α, MCP-1, MIP-1ß and RANTES. CONCLUSIONS: Burn-induced changes in TLR-2 and TLR-4 reactivity may contribute to the development of post-burn complications, such as acute lung injury (ALI) and adult respiratory distress syndrome (ARDS).

Mechanism of the salutary effects of estrogen on kupffer cell phagocytic capacity following trauma-hemorrhage: pivotal role of Akt activation.

Kupffer cells are macrophages in the liver whose major role is to clear circulating pathogens. Decreased phagocytic capacity of Kupffer cells may result in severe systemic infection. We tested the hypothesis that the depressed Kupffer cell phagocytic capacity following trauma-hemorrhage is enhanced by estrogen administration and this occurs due to maintenance of Fc receptor expression and cellular ATP content via the activation of Akt. Male C3H/HeN mice were subjected to sham operation or trauma-hemorrhage and sacrificed 2 h thereafter. Estrogen, with or without an estrogen receptor antagonist (ICI 182,780), a PI3K inhibitor (Wortmannin), or vehicle, was injected during resuscitation. Kupffer cell phagocytic capacity was tested in vivo. The expression of Fc receptors, of Akt phosphorylation, of p38 MAPK phosphorylation, of DNA binding activity of NF-kappaB and ATP content of Kupffer cells were also determined. Trauma-hemorrhage suppressed Kupffer cell phagocytosis by decreasing Fc receptor expression and Akt activation; however, it induced p38 MAPK activation and increased NF-kappaB activity. Cellular ATP levels were also decreased following trauma-hemorrhage. Administration of estrogen following trauma-hemorrhage increased phospho-Akt levels and normalized all the parameters described as well as plasma levels of TNF-alpha, IL-6, and IL-10. Coadministration of ICI 182,780 or Wortmannin abolished the beneficial effects of estrogen in improving the phagocytic capacity of Kupffer cells following trauma-hemorrhage. Thus, activation of Akt plays a crucial role in mediating the salutary effect of estrogen in restoring trauma-hemorrhage-induced suppression of Kupffer cell phagocytosis.

Myeloid-Derived Suppressor Cells (MDSCs) and the Immunoinflammatory Response to Injury (Mini Review).

ABSTRACT: Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immature myeloid cells hallmarked by their potent immunosuppressive function in a vast array of pathologic conditions. MDSCs have recently been shown to exhibit marked expansion in acute inflammatory states including traumatic injury, burn, and sepsis. Although MDSCs have been well characterized in cancer, there are significant gaps in our knowledge of their functionality in trauma and sepsis, and their clinical significance remains unclear. It is suggested that MDSCs serve an important role in quelling profound inflammatory responses in the acute setting; however, MDSC accumulation may also predispose patients to developing persistent immune dysregulation with increased risk for nosocomial infections, sepsis, and multiorgan failure. Whether MDSCs may serve as the target for novel therapeutics or an important biomarker in trauma and sepsis is yet to be determined. In this review, we will discuss the current understanding of MDSCs within the context of specific traumatic injury types and sepsis. To improve delineation of their functional role, we propose a systemic approach to MDSC analysis including phenotypic standardization, longitudinal analysis, and expansion of clinical research.

Systemic T Cell Exhaustion Dynamics Is Linked to Early High Mobility Group Box Protein 1 (HMGB1) Driven Hyper-Inflammation in a Polytrauma Rat Model.

We previously reported an early surge in high mobility group box protein 1 (HMGB1) levels in a polytrauma (PT) rat model. This study investigates the association of HMGB1 levels in mediating PT associated dysregulated immune responses and its influence on the cellular levels of receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). Using the same PT rat model treated with anti-HMGB1 polyclonal antibody, we evaluated changes in circulating inflammatory cytokines, monocytes/macrophages and T cells dynamics and cell surface expression of RAGE and TLR4 at 1, 3, and 7 days post-trauma (dpt) in blood and spleen. Notably, PT rats demonstrating T helper (Th)1 and Th2 cells type early hyper-inflammatory responses also exhibited increased monocyte/macrophage counts and diminished T cell counts in blood and spleen. In blood, expression of RAGE and TLR4 receptors was elevated on CD68+ monocyte/macrophages and severely diminished on CD4+ and CD8+ T cells. Neutralization of HMGB1 significantly decreased CD68+ monocyte/macrophage counts and increased CD4+ and CD8+ T cells, but not γδ+TCR T cells in circulation. Most importantly, RAGE and TLR4 expressions were restored on CD4+ and CD8+ T cells in treated PT rats. Overall, findings suggest that in PT, the HMGB1 surge is responsible for the onset of T cell exhaustion and dysfunction, leading to diminished RAGE and TLR4 surface expression, thereby possibly hindering the proper functioning of T cells.

High-frequency percussive ventilation and low tidal volume ventilation in burns: a randomized controlled trial.

OBJECTIVES: In select burn intensive care units, high-frequency percussive ventilation is preferentially used to provide mechanical ventilation in support of patients with acute lung injury, acute respiratory distress syndrome, and inhalation injury. However, we found an absence of prospective studies comparing high-frequency percussive ventilation with contemporary low-tidal volume ventilation strategies. The purpose of this study was to prospectively compare the two ventilator modalities in a burn intensive care unit setting. DESIGN: Single-center, prospective, randomized, controlled clinical trial, comparing high-frequency percussive ventilation with low-tidal volume ventilation in patients admitted to our burn intensive care unit with respiratory failure. SETTING: A 16-bed burn intensive care unit at a tertiary military teaching hospital. PATIENTS: Adult patients ≥ 18 yrs of age requiring prolonged (> 24 hrs) mechanical ventilation were admitted to the burn intensive care unit. The study was conducted over a 3-yr period between April 2006 and May 2009. This trial was registered with ClinicalTrials.gov as NCT00351741. INTERVENTIONS: Subjects were randomly assigned to receive mechanical ventilation through a high-frequency percussive ventilation-based strategy (n = 31) or a low-tidal volume ventilation-based strategy (n = 31). MEASUREMENTS AND MAIN RESULTS: At baseline, both the high-frequency percussive ventilation group and the low-tidal volume ventilation group had similar demographics to include median age (interquartile range) (28 yrs [23-45] vs. 33 yrs [24-46], p = nonsignificant), percentage of total body surface area burn (34 [20-52] vs. 34 [23-50], p = nonsignificant), and clinical diagnosis of inhalation injury (39% vs. 35%, p = nonsignificant). The primary outcome was ventilator-free days in the first 28 days after randomization. Intent-to-treat analysis revealed no significant difference between the high-frequency percussive ventilation and the low-tidal volume ventilation groups in mean (± sd) ventilator-free days (12 ± 9 vs. 11 ± 9, p = nonsignificant). No significant difference was detected between groups for any of the secondary outcome measures to include mortality except the need for "rescue" mode application (p = .02). Nine (29%) in the low-tidal volume ventilation arm did not meet predetermined oxygenation or ventilation goals and required transition to a rescue mode. By contrast, two in the high-frequency percussive ventilation arm (6%) required rescue. CONCLUSIONS: A high-frequency percussive ventilation-based strategy resulted in similar clinical outcomes when compared with a low-tidal volume ventilation-based strategy in burn patients with respiratory failure. However, the low-tidal volume ventilation strategy failed to achieve ventilation and oxygenation goals in a higher percentage necessitating rescue ventilation.

Impact of thermal injury on wound infiltration and the dermal inflammatory response.

Healing of the burn wound is a critical component of the burn patient's successful recovery. While inflammation is a critical component of the healing process, it is unknown whether the inflammatory response differs between non-burn and burn wounds. To study this, mice were subjected to major burn injury or sham procedure. Wound cells were collected by implantation of polyvinyl alcohol sponges beneath the burn site in injured mice or beneath uninjured skin in sham mice (i.e., non-burn wound). Three days thereafter, skin, wound fluid, and infiltrating cells were collected for analysis. Significant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL-6), monocyte chemoattractant protein (MCP)-1, and keratinocyte-derived chemokine (KC) were observed in burn wound tissue and the wound fluid from both non-burn and burn wounds. Burn injury induced 3-fold higher levels of KC and 50-fold higher levels of IL-6 in the wound fluid compared with non-burn injury. Significant numbers of the cells from both burn and non-burn wounds were CD11b(+), GR1(+), and F4/80(+), suggestive of a myeloid suppressor cell phenotype, whereas CD3(+) T-cells were negligible under both conditions. LPS induced TNF-alpha, IL-6, IL-10, MCP-1, KC, and nitric oxide production in both cell populations, however, IL-6, IL-10, MCP-1, and KC levels were suppressed in burn wound cell cultures. These findings indicate that significant differences in the wound inflammatory response exist between burn and non-burn cutaneous wounds and that the unique characteristics of the inflammatory response at the burn site may be an important contributing factor to post-burn wound healing complications.

The role of MIP-1 alpha in the development of systemic inflammatory response and organ injury following trauma hemorrhage.

Although MIP-1alpha is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1alpha plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1alpha-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 +/- 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum alpha-glutathione transferase, TNF-alpha, IL-6, IL-10, MCP-1, and MIP-1alpha and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1alpha KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1alpha plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1alpha, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1alpha levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.

The gut microbiome distinguishes mortality in trauma patients upon admission to the emergency department.

BACKGROUND: Traumatic injury can lead to a compromised intestinal epithelial barrier, decreased gut perfusion, and inflammation. While recent studies indicate that the gut microbiome (GM) is altered early following traumatic injury, the impact of GM changes on clinical outcomes remains unknown. Our objective of this follow-up study was to determine if the GM is associated with clinical outcomes in critically injured patients. METHODS: We conducted a prospective, observational study in adult patients (N = 67) sustaining severe injury admitted to a level I trauma center. Fecal specimens were collected on admission to the emergency department, and microbial DNA from all samples was analyzed using the Quantitative Insights Into Microbial Ecology pipeline and compared against the Greengenes database. α-Diversity and ß-diversity were estimated using the observed species metrics and analyzed with t tests and permutational analysis of variance for overall significance, with post hoc pairwise analyses. RESULTS: Our patient population consisted of 63% males with a mean age of 44 years. Seventy-eight percent of the patients suffered blunt trauma with 22% undergoing penetrating injuries. The mean body mass index was 26.9 kg/m. Significant differences in admission ß-diversity were noted by hospital length of stay, intensive care unit hospital length of stay, number of days on the ventilator, infections, and acute respiratory distress syndrome (p < 0.05). ß-Diversity on admission differed in patients who died compared with patients who lived (mean time to death, 8 days). There were also significantly less operational taxonomic units in samples from patients who died versus those who survived. A number of species were enriched in the GM of injured patients who died, which included some traditionally probiotic species such as Akkermansia muciniphilia, Oxalobacter formigenes, and Eubacterium biforme (p < 0.05). CONCLUSION: Gut microbiome diversity on admission in severely injured patients is predictive of a variety of clinically important outcomes. While our study does not address causality, the GM of trauma patients may provide valuable diagnostic and therapeutic targets for the care of injured patients. LEVEL OF EVIDENCE: Prognostic and epidemiological, level III.

Trauma-hemorrhage and hypoxia differentially influence kupffer cell phagocytic capacity: role of hypoxia-inducible-factor-1alpha and phosphoinositide 3-kinase/Akt activation.

OBJECTIVE: We investigated whether Kupffer cell phagocytosis is differentially regulated following hypoxia (by breathing hypoxic gas) and trauma-hemorrhage. We hypothesized that the differences might result from a differential activation of hypoxia-inducible factor (HIF)-1alpha and phosphoinositide 3-kinase (PI3K)/Akt pathway under those conditions. BACKGROUND: HIF-1alpha is a biologic O2 sensor enabling adaptation to hypoxia. Studies have shown that under hypoxic conditions, HIF-1alpha enhances macrophage phagocytosis. Trauma-hemorrhage also produces a hypoxic insult with HIF-1alpha activation; however, macrophage phagocytosis is suppressed under those conditions. Thus, signaling molecules other than HIF-1alpha should be taken into consideration in the regulation of macrophage phagocytosis following cellular hypoxia or trauma-hemorrhage. METHODS: Male C3H/HeN mice were subjected to sham operation, trauma-hemorrhage (laparotomy, 90 minutes hemorrhagic shock, MAP 35 +/- 5 mm Hg followed by resuscitation) or hypoxia (5% O2 for 120 minutes). The trauma-hemorrhage and hypoxia groups received Wortmannin (PI3K inhibitor), YC-1 (HIF-1alpha inhibitor) or vehicle at the time of maximum bleedout in the trauma-hemorrhage group or at a PaO2 of 30 mm Hg during hypoxic air inhalation. Mice were killed 2 hours later and samples/cells collected. RESULTS: While the systemic and Kupffer cell hypoxic states were similar in the trauma-hemorrhage and hypoxia groups, phagocytic capacity was suppressed following trauma-hemorrhage but enhanced in the hypoxia group. Kupffer cells from both groups showed increased HIF-1alpha activation, which was prevented by Wortmannin or YC-1 treatment. The increase in Kupffer cell phagocytosis following hypoxemia was also prevented by Wortmannin or YC-1 treatment. Akt activation was suppressed in the trauma-hemorrhage group, but enhanced in the hypoxia group. Wortmannin and YC-1 treatment prevented the increase in Akt activation. CONCLUSIONS: These findings indicate that the suppression of Kupffer cell phagocytosis following trauma-hemorrhage is independent of cellular hypoxia and activation of HIF-1alpha, but it is possibly related to suppression of the Akt activation.

17beta-Estradiol's salutary effects on splenic dendritic cell functions following trauma-hemorrhage are mediated via estrogen receptor-alpha.

Although 17beta-estradiol administration following trauma-hemorrhage attenuates Kupffer cell, splenic and peritoneal macrophage functions, it remains unknown whether 17beta-estradiol has any salutary effects on splenic dendritic cell (DC) functions and if so, whether such effects are mediated via the estrogen receptors (ER). We hypothesized that 17beta-estradiol administration following trauma-hemorrhage has salutary effects on splenic DC functions. Male C3H/HeN (6-8 weeks) mice were randomly assigned to sham operation or trauma-hemorrhage. Trauma-hemorrhage was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure [BP] 35 mmHg), followed by fluid resuscitation (4x the shed blood volume in the form of Ringer's lactate). Estrogen receptor (ER)-alpha agonist propyl pyrazole triol (PPT; 5microg/kg), ER-beta agonist diarylpropionitrile (DPN; 5microg/kg), 17beta-estradiol (50microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Two hours later, the mice were sacrificed, splenic DCs were isolated and the changes in their apoptosis, co-stimulating factors and MHC class II expression, ability to produce cytokines, and antigen presentation capacity were measured. Apoptosis of splenic DC increased following trauma-hemorrhage; however, 17beta-estradiol administration after trauma-hemorrhage normalized the rate of apoptosis. Moreover, splenic DC cytokines production, co-stimulating factors and MHC class II expression, and antigen presentation capacity were significantly decreased following trauma-hemorrhage; however, 17beta-estradiol as well as PPT also prevented these depressions. In contrast, DPN did not attenuate splenic DC functions following trauma-hemorrhage. Since PPT administration following trauma-hemorrhage was more effective in normalizing splenic DC functions than DPN, the salutary effects of 17beta-estradiol on splenic DC functions are mediated predominantly via ER-alpha.

Moderate Traumatic Brain Injury Alters the Gastrointestinal Microbiome in a Time-Dependent Manner.

The microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that colonize the human body, and alterations have been associated with a number of disease states. Changes in gut commensals can influence the neurologic system via the brain-gut axis, and systemic insults such as trauma or traumatic brain injury (TBI) may alter the gut microbiome. The objective of this study was to evaluate the gut microbiome in a preclinical TBI cortical impact model. Male rats underwent craniotomy and randomized to a sham group (n = 4), or a moderate TBI (n = 10) using a pneumatic impactor. MRI and behavioral assessments were performed pre-TBI and on days 2, 7, and 14 days thereafter. Microbiome composition was determined with 16s rRNA sequencing from fecal sample DNA pre-TBI and 2 hrs, 1, 3, and 7 days afterward. Alpha- and ß-bacterial diversity, as well as organizational taxonomic units (OTUs), were determined. Significant changes in the gut microbiome were evident as early as 2 h after TBI as compared with pre-injured samples and sham rats. While there were varying trends among the phylogenetic families across time, some changes persisted through 7 days in the absence of therapeutic intervention. While large structural lesions and behavioral deficits were apparent post-TBI, there were modest but significant decreases in α-diversity. Moreover, both changes in representative phyla and α-diversity measures were significantly correlated with MRI-determined lesion volume. These results suggest that changes in the microbiome may represent a novel biomarker to stage TBI severity and predict functional outcome.

A prospective study in severely injured patients reveals an altered gut microbiome is associated with transfusion volume.

BACKGROUND: Traumatic injury can lead to a compromised intestinal epithelial barrier and inflammation. While alterations in the gut microbiome of critically injured patients may influence clinical outcomes, the impact of trauma on gut microbial composition is unknown. Our objective was to determine if the gut microbiome is altered in severely injured patients and begin to characterize changes in the gut microbiome due to time and therapeutic intervention. METHODS: We conducted a prospective, observational study in adult patients (n = 72) sustaining severe injury admitted to a Level I Trauma Center. Healthy volunteers (n = 13) were also examined. Fecal specimens were collected on admission to the emergency department and at 3, 7, 10, and 13 days (±2 days) following injury. Microbial DNA was isolated for 16s rRNA sequencing, and α and ß diversities were estimated, according to taxonomic classification against the Greengenes database. RESULTS: The gut microbiome of trauma patients was altered on admission (i.e., within 30 minutes following injury) compared to healthy volunteers. Patients with an unchanged gut microbiome on admission were transfused more RBCs than those with an altered gut microbiome (p < 0.001). Although the gut microbiome started to return to a ß-diversity profile similar to that of healthy volunteers over time, it remained different from healthy controls. Alternatively, α diversity initially increased postinjury, but subsequently decreased during the hospitalization. Injured patients on admission had a decreased abundance of traditionally beneficial microbial phyla (e.g., Firmicutes) with a concomitant decrease in opportunistic phyla (e.g., Proteobacteria) compared to healthy controls (p < 0.05). Large amounts of blood products and RBCs were both associated with higher α diversity (p < 0.001) and a ß diversity clustering closer to healthy controls. CONCLUSION: The human gut microbiome changes early after trauma and may be aided by early massive transfusion. Ultimately, the gut microbiome of trauma patients may provide valuable diagnostic and therapeutic insight for the improvement of outcomes postinjury. LEVEL OF EVIDENCE: Prognostic and Epidemiological, level III.

Burn injury-induced alterations in wound inflammation and healing are associated with suppressed hypoxia inducible factor-1alpha expression.

A major complication associated with burn injury is delayed wound healing. While healing of the burn injury site is essential, healing of distal injury sites caused by surgical interventions and other processes also is important. The impact of burn injury on healing of these distal wound sites is not understood clearly. To study this, mice were subjected to major burn injury or a sham procedure. Immediately following, excisional wounds were made on the dorsal surface caudal to the burn site and wound closure was monitored over a 7-d period by planimetry. In a second series of experiments, plasma and excisional wounds were collected for in vitro analysis of cyto- and chemokine levels, L-arginine metabolism, and hypoxia-inducible factor (HIF)-1alpha expression. At 1-7 d post-injury, a significant inflammatory response was evident in both groups, but the healing process was delayed in the burn-injured mice. At 3 d post-injury, wound levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and keratinocyte-derived chemokine were suppressed in the burn group. This difference in the wound inflammatory response was independent of changes in L-arginine metabolism (nitrate levels, inducible nitric oxide synthase expression, arginase activity), but correlated with a marked reduction in HIF-1alpha protein levels. In conclusion, these findings suggest that HIF-1alpha and the inflammatory response play a significant role in wound healing, and reduced levels of HIF-1alpha contribute to the impaired healing response post-burn.

Estrogen ameliorates trauma-hemorrhage-induced lung injury via endothelial nitric oxide synthase-dependent activation of protein kinase G.

OBJECTIVE: In this study, we tested the hypothesis that 17beta-estradiol (E2) administration after trauma-hemorrhage reduces lung injury through a mechanism involving estrogen receptor (ER)-dependent activation of the endothelial nitric oxide (NO) synthase (eNOS)/protein kinase G (PKG)/vasodilator-stimulated phosphoprotein (VASP) pathway. BACKGROUND: Estrogen provides protection after injury via activation of multiple signaling cascades, including the cyclic GMP-dependent PKG pathway. Phosphorylation of VASP at Ser239 (p-VASP) can be used to assess PKG signaling activity. METHODS: Male Sprague-Dawley rats (275-325 g) underwent soft tissue trauma (midline laparotomy) and hemorrhagic shock (mean blood pressure 35-40 mm Hg for 90 minutes) followed by fluid resuscitation. Animals were pretreated with a nonselective NOS inhibitor (N(omega)-nitro-L-arginine methyl ester; 30 mg/kg), a soluble guanylyl cyclase (sGC) inhibitor [1H-(1, 2, 4) oxadiazolo (3, 4-alpha) quinoxalin-1-one; 10 mg/kg] or an ER antagonist (ICI 182,780; 3 mg/kg) 30 minutes before E2 (100 microg/kg) or vehicle administration. Animals were killed at 2 hours after resuscitation. RESULTS: Lung injury induced by trauma-hemorrhage is evidenced by edema (wet/dry ratio), neutrophil infiltration (myeloperoxidase activity), and with an increased expression of cytokines, chemokines, and adhesion molecules. E2 treatment after trauma-hemorrhage resulted in an increase in eNOS expression/phosphorylation, PKG-I activation, and VASP/p-VASP expression, which paralleled a decrease in lung injury. Inhibition of NOS and sGC abolished the E2-induced increase in PKG-I activity, VASP/p-VASP expression. Blockade of eNOS, PKG-I, and ER exacerbated lung inflammation and injury. CONCLUSIONS: These results collectively suggest that activation of the eNOS-PKG/VASP pathway by E2 protects against trauma-hemorrhage-induced lung injury.

Up-regulation of cell surface Toll-like receptors on circulating gammadelta T-cells following burn injury.

Burn injury is associated with profound inflammation and activation of the innate immune system involving gammadelta T-cells. Similarly, Toll-like receptors (TLR) are associated with activation of the innate immune response; however, it is unclear whether TLR expression is altered in gammadelta T-cells after major burn injury. To study this, male C57BL/6 mice were subjected to burn injury (25% TBSA) and 1 or 7 days thereafter, blood and spleen cells were isolated and subjected to FACs analysis for TLRs and other phenotypic markers (gammadelta TCR, alphabeta TCR, CD69, CD120b). A marked increase in the number of circulating gammadelta T-cells was observed at 24h post-burn (14% vs. 4%) and a higher percentage of these cells expressed TLR-2. TLR-4 expression was also increased post-burn, but to a lesser degree. These changes in TLR expression were not associated with altered CD69 or CD120b expression in gammadelta T-cells. The mobilization of, and increased TLR expression in, gammadelta T-cells was transient, as phenotypic changes were not evident at 7 days post-burn or in gammadelta T-cells from the circulation or spleen. The increases in TLR expression were not observed in alphabeta T-cells after burn injury. In conclusion, 24h after burn injury mobilization of gammadelta T-cells with increased TLR expression was observed. This finding suggests that this unique T-cell population is critical in the innate immune response to injury, possibly through the recognition of danger signals by TLRs.