IL33-mediated ILC2 activation and neutrophil IL5 production in the lung response after severe trauma: A reverse translation study from a human cohort to a mouse trauma model.

BACKGROUND: The immunosuppression and immune dysregulation that follows severe injury includes type 2 immune responses manifested by elevations in interleukin (IL) 4, IL5, and IL13 early after injury. We hypothesized that IL33, an alarmin released early after tissue injury and a known regulator of type 2 immunity, contributes to the early type 2 immune responses after systemic injury. METHODS AND FINDINGS: Blunt trauma patients admitted to the trauma intensive care unit of a level I trauma center were enrolled in an observational study that included frequent blood sampling. Dynamic changes in IL33 and soluble suppression of tumorigenicity 2 (sST2) levels were measured in the plasma and correlated with levels of the type 2 cytokines and nosocomial infection. Based on the observations in humans, mechanistic experiments were designed in a mouse model of resuscitated hemorrhagic shock and tissue trauma (HS/T). These experiments utilized wild-type C57BL/6 mice, IL33-/- mice, B6.C3(Cg)-Rorasg/sg mice deficient in group 2 innate lymphoid cells (ILC2), and C57BL/6 wild-type mice treated with anti-IL5 antibody. Severely injured human blunt trauma patients (n = 472, average injury severity score [ISS] = 20.2) exhibited elevations in plasma IL33 levels upon admission and over time that correlated positively with increases in IL4, IL5, and IL13 (P < 0.0001). sST2 levels also increased after injury but in a delayed manner compared with IL33. The increases in IL33 and sST2 were significantly greater in patients that developed nosocomial infection and organ dysfunction than similarly injured patients that did not (P < 0.05). Mechanistic studies were carried out in a mouse model of HS/T that recapitulated the early increase in IL33 and delayed increase in sST2 in the plasma (P < 0.005). These studies identified a pathway where IL33 induces ILC2 activation in the lung within hours of HS/T. ILC2 IL5 up-regulation induces further IL5 expression by CXCR2+ lung neutrophils, culminating in early lung injury. The major limitations of this study are the descriptive nature of the human study component and the impact of the potential differences between human and mouse immune responses to polytrauma. Also, the studies performed did not permit us to make conclusions about the impact of IL33 on pulmonary function. CONCLUSIONS: These results suggest that IL33 may initiate early detrimental type 2 immune responses after trauma through ILC2 regulation of neutrophil IL5 production. This IL33-ILC2-IL5-neutrophil axis defines a novel regulatory role for ILC2 in acute lung injury that could be targeted in trauma patients prone to early lung dysfunction.

Computational Analysis Supports an Early, Type 17 Cell-Associated Divergence of Blunt Trauma Survival and Mortality.

OBJECTIVE: Blunt trauma patients may present with similar demographics and injury severity yet differ with regard to survival. We hypothesized that this divergence was due to different trajectories of systemic inflammation and utilized computational analyses to define these differences. DESIGN: Retrospective clinical study and experimental study in mice. SETTING: Level 1 trauma center and experimental laboratory. PATIENTS: From a cohort of 493 victims of blunt trauma, we conducted a pairwise, retrospective, case-control study of patients who survived over 24 hours but ultimately died (nonsurvivors; n = 19) and patients who, after ICU admission, went on to be discharged(survivors; n = 19). INTERVENTIONS: None in patients. Neutralizing anti-interleukin-17A antibody in mice. MEASUREMENTS AND MAIN RESULTS: Data on systemic inflammatory mediators assessed within the first 24 hours and over 7 days were analyzed with computational modeling to infer dynamic networks of inflammation. Network density among inflammatory mediators in nonsurvivors increased in parallel with organ dysfunction scores over 7 days, suggesting the presence of early, self-sustaining, pathologic inflammation involving high-mobility group protein B1, interleukin-23, and the Th17 pathway. Survivors demonstrated a pattern commensurate with a self-resolving, predominantly lymphoid response, including higher levels of the reparative cytokine interleukin-22. Mice subjected to trauma/hemorrhage exhibited reduced organ damage when treated with anti-interleukin-17A. CONCLUSIONS: Variable type 17 immune responses are hallmarks of organ damage, survival, and mortality after blunt trauma and suggest a lymphoid cell-based switch from self-resolving to self-sustaining inflammation.

Interleukin 37; a possible marker of arterial stiffness in Behçet's disease.

BACKGROUND: Interleukin 37 (IL-37) is an anti-inflammatory cytokine previously studied in Behçet's disease (BD) and atherosclerosis. However, little is known about its relation to macro and microcirculations in BD. Previous studies relied mainly on common carotid artery (CCA) intima-media thickness (IMT) and ankle brachial index (ABI) to study atherosclerosis in BD with conflicting results. This study evaluated flow parameters of CCA, ABI and nailfold videocapillaroscopy in relation to serum IL-37 in BD. METHODS: Forty BD patients and 30 healthy controls were included. IMT, peak-systolic, end-diastolic velocities, resistivity index of CCA and ABI were measured by duplex ultrasound. Capillary loop, length, diameter and morphology were recorded by nailfold videocapillaroscopy. Serum IL-37 levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to controls, patients had higher mean CCA IMT (p < 0.0001), resistivity index (p < 0.001) and peak-systolic velocity (p=0.09) and lower mean CCA end-diastolic velocity (p=0.002), capillary loop, length, arterial, venous limbs diameter and serum IL-37 (p < 0.001). Patients with ABI ≥ 1.4 "indicating stiff arteries" had higher serum IL-37 (p < 0.05 on left, p>0.05 right sides). Serum IL-37 correlated negatively with left CCA end-diastolic velocity "denoting atherosclerosis" and positively with left posterior tibial artery ABI and CRP (p < 0.03) "denoting inflammation". Multiple regression analysis showed only association with left CCA end-diastolic velocity. CONCLUSIONS: IL-37 may be related to arterial stiffness in BD and could be used as a possible marker of arteriosclerosis in the disease for further investigations. Changes of CCA peak-systolic, end-diastolic velocities, resistivity index and IMT refer to increased atherosclerosis in larger elastic arteries. In smaller muscular "crural" arteries, vasculitis with possible medial disease may be more evident.

Spatially controlled delivery of neurotrophic factors in silk fibroin-based nerve conduits for peripheral nerve repair.

Restoration with sufficient functional recovery after long-gap peripheral nerve damage remains a clinical challenge. Silk has shown clinical promise for numerous tissue engineering applications due to its biocompatibility, impressive mechanical properties, and Food and Drug Administration approval. The aim of this study was to evaluate the efficacy of silk fibroin--based nerve guides containing glial cell line-derived neurotrophic factor (GDNF) in a long-gap sized (15 mm) rat sciatic nerve defect model. Four groups of nerve conduits were prepared: (1) silk conduits with empty silk microspheres, (2) silk conduits with GDNF-loaded silk microspheres uniformly distributed in the conduit wall, (3) silk conduits with GDNF-loaded silk microspheres in a controlled manner with the highest GDNF concentration at the distal end, and (4) isograft. After 6 weeks, the nerve grafts were explanted, harvested, and fixed for histologic analysis. Nerve tissue stained with the S-100, and neuroendocrine marker PGP 9.5 antibodies demonstrated a significantly increased density of nerve tissue in the GDNF-treated groups compared with the empty microsphere (control) group (P < 0.05). GDNF-treated animals with a higher concentration of GDNF in the distal portion possessed a significantly higher density of PGP 9.5 protein middle conduit part than comparison to GDNF uniform-treated animals (P < 0.05). Silk-based nerve conduits possess optimal mechanical and degradative properties, rendering them potentially useful in peripheral nerve repair. This study demonstrates that novel, porous silk fibroin--based nerve conduits, infused with GDNF in a controlled manner, represent a potentially viable conduit for Schwann cell migration and proliferation in the regeneration of peripheral nerves.

Combined effect of hydrogen sulfide and mesenchymal stem cells on mitigating liver fibrosis induced by bile duct ligation: Role of anti-inflammatory, anti-oxidant, anti-apoptotic, and anti-fibrotic biomarkers.

Objectives: Liver fibrosis eventually develops into cirrhosis and hepatic failure, which can only be treated with liver transplantation. We aimed to assess the potential role of hydrogen sulfide (H2S) alone and combined with bone marrow-derived mesenchymal stem cells (BM-MSCs) on hepatic fibrosis induced by bile-duct ligation (BDL) and to compare their effects to silymarin. Materials and Methods: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and alkaline phosphatase (ALP) were investigated in serum. Gene expression levels of CBS (cystathionine ß-synthase), CSE (cystathionine γ-lyase), and alpha-smooth muscle actin (α- SMA) were measured in liver tissues using RT-PCR. Hepatic protein kinase (Akt) was assessed by Western blot assay. Liver oxidative stress markers, malondialdehyde (MDA), and reduced glutathione (GSH) were analyzed by the colorimetric method. Lipocalin-2 (LCN2) and transforming growth factor-ß (TGF-ß) were measured using ELIZA. Liver tissues were examined by H&E and Masson trichome staining for detection of liver necrosis or fibrosis. Caspase 3 expression was evaluated by immunohistochemistry. Results: H2S and BM-MSCs ameliorated liver function and inhibited inflammation and oxidative stress detected by significantly decreased serum ALT, AST, ALP, TB, and hepatic MDA, Akt, TGF-ß, LCN2, and α-SMA expression and significantly increased CBS and CSE gene expression levels. They attenuated hepatic apoptosis evidenced by decreased hepatic caspase expression. Conclusion: Combined treatment with H2S and BM-MSCs could attenuate liver fibrosis induced by BDL through mechanisms such as anti-inflammation, anti-oxidation, anti-apoptosis, anti-fibrosis, and regenerative properties indicating that using H2S and MSCs may represent a promising approach for management of cholestatic liver fibrosis.

Analysis of hMSH2 mismatch repair protein expression in dysplasia, carcinoma in situ and invasive squamous cell carcinoma of the vocal folds.

UNLABELLED: The expression pattern of hMSH2 mismatch repair protein during the progression of benign epithelium to vocal fold invasive squamous cell carcinoma has not been previously described. Nor has the correlation between the hMSH2 protein expression and the clinicopathologic features of the vocal fold dysplasia and carcinoma been examined. HYPOTHESIS: "The progression of benign epithelium to invasive squamous cell carcinoma of the vocal folds is associated with reduction of the hMSH2 mismatch repair protein expression." METHODS: Vocal fold biopsies were obtained from 20 patients with mild and moderate dysplasia: 10 patients with severe dysplasia (squamous cell carcinoma in situ) and 20 patients with invasive squamous cell carcinoma. The expression pattern of hMSH2 protein was examined by using immunoperoxidase-staining methods and mouse monoclonal antibodies. The results were scored as the percentage of hMSH2 positively stained cells. RESULTS: The mean values of hMSH2 positively stained cells decreased gradually with the transitions from normal epithelium to dysplasia and finally to invasive squamous cell carcinoma. There was a negative correlation between the expression of hMSH2 and the degree of dysplasia, that is, as the severity of the dysplasia increases at the microscopic level, there was a decrease in the expression values of the hMSH2 protein. CONCLUSIONS: We report, for the first time, that the reduced expression of the hMSH2 mismatch repair protein is related to the progression of the benign epithelium to invasive squamous cell carcinoma of the vocal folds.

Innate-Like Lymphocytes Are Immediate Participants in the Hyper-Acute Immune Response to Trauma and Hemorrhagic Shock.

Adverse outcomes following severe traumatic injury are frequently attributed to a state of immunological dysfunction acquired during treatment and recovery. Recent genomic evidence however, suggests that the trajectory toward development of multiple organ dysfunction syndrome (MODS) is already in play at admission (<2 h following injury). Improved understanding of the molecular events during the hyper-acute immunological response to trauma, <2 h following injury, may reveal opportunities to ameliorate organ injury and expedite recovery. Lymphocytes have not previously been considered key participants in this early response; however, two observations in human trauma patients namely, raised populations of circulating NKT and NK cells during the hyper-acute phase and persistent lymphopenia beyond 48 h show association with the development of MODS during recovery. These highlight the need for greater understanding of lymphocyte function in the hyper-acute phase of inflammation. An exploratory study was therefore conducted in a well-established murine model of trauma and hemorrhagic shock (T&HS) to investigate (1) the development of lymphopenia in the murine model and (2) the phenotypic and functional changes of three innate-like lymphocyte subsets, NK1.1+ CD3-, NK1.1+ CD3+, γδTCR+ CD3+ cells, focusing on the first 6 h following injury. Rapid changes in phenotype and function were demonstrated in these cells within blood and spleen, but responses in lung tissue lagged behind. This study describes the immediacy of the innate-like lymphocyte response to trauma in different body compartments and considers new lines for further investigation to develop our understanding of MODS pathogenesis.

NK1.1+ cells promote sustained tissue injury and inflammation after trauma with hemorrhagic shock.

Various cell populations expressing NK1.1 contribute to innate host defense and systemic inflammatory responses, but their role in hemorrhagic shock and trauma remains uncertain. NK1.1+ cells were depleted by i.p. administration of anti-NK1.1 (or isotype control) on two consecutive days, followed by hemorrhagic shock with resuscitation and peripheral tissue trauma (HS/T). The plasma levels of IL-6, MCP-1, alanine transaminase (ALT), and aspartate aminotransferase (AST) were measured at 6 and 24 h. Histology in liver and gut were examined at 6 and 24 h. The number of NK cells, NKT cells, neutrophils, and macrophages in liver, as well as intracellular staining for TNF-α, IFN-γ, and MCP-1 in liver cell populations were determined by flow cytometry. Control mice subjected to HS/T exhibited end organ damage manifested by marked increases in circulating ALT, AST, and MCP-1 levels, as well as histologic evidence of hepatic necrosis and gut injury. Although NK1.1+ cell-depleted mice exhibited a similar degree of organ damage as nondepleted animals at 6 h, NK1.1+ cell depletion resulted in marked suppression of both liver and gut injury by 24 h after HS/T. These findings indicate that NK1.1+ cells contribute to the persistence of inflammation leading to end organ damage in the liver and gut.

Keratin gel filler for peripheral nerve repair in a rodent sciatic nerve injury model.

BACKGROUND: Restoration with sufficient functional recovery after long-gap peripheral nerve damage remains a clinical challenge. In vitro, keratins, which are derived from human hair, enhance activity and gene expression of Schwann cells. The specific aim of the authors' study was to examine keratin gel as conduit filler for peripheral nerve regeneration in a rat sciatic nerve injury model. METHODS: Incorporation of glial cell line-derived, neurotrophic factor, double-walled microspheres into polycaprolactone nerve guides has demonstrated an off-the-shelf product alternative to promote nerve regeneration, and this conduit was filled with keratin gel and examined in a rat 15-mm sciatic nerve defect model. As an indicator of recovery, nerve sections were stained with S100 and protein gene product 9.5 antibody. RESULTS: The keratin-treated groups, compared with both saline and empty polycaprolactone (control) groups (p < 0.05), demonstrated a significantly increased density of Schwann cells and axons. Polycaprolactone-based nerve conduits possess optimal mechanical and degradative properties, rendering the biocompatible conduits potentially useful in peripheral nerve repair. CONCLUSION: From their studies, the authors conclude that polycaprolactone nerve guides with glial cell line-derived, neurotrophic factor-loaded, double-walled microspheres filled with keratin gel represent a potentially viable guiding material for Schwann cell and axon migration and proliferation in the treatment of peripheral nerve regeneration.