Integrin alpha 6 is upregulated and drives hepatocellular carcinoma progression through integrin α6ß4 complex.

Integrin α6 (ITGA6) forms integrin receptors with either integrin ß1 (ITGB1) or integrin ß4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6ß4 complex. Thus, integrin α6ß4 may be a therapeutic target for treating patients with HCC.

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.

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.

Modulation of dendritic cell differentiation in the bone marrow mediates sustained immunosuppression after polymicrobial sepsis.

Murine polymicrobial sepsis is associated with a sustained reduction of dendritic cell (DC) numbers in lymphoid organs and with a dysfunction of DC that is considered to mediate the chronic susceptibility of post-septic mice to secondary infections. We investigated whether polymicrobial sepsis triggered an altered de novo formation and/or differentiation of DC in the bone marrow. BrdU labeling experiments indicated that polymicrobial sepsis did not affect the formation of splenic DC. DC that differentiated from bone marrow (bone marrow-derived DC [BMDC]) of post-septic mice released enhanced levels of IL-10 but did not show an altered phenotype in comparison with BMDC from sham mice. Adoptive transfer experiments of BMDC into naive mice revealed that BMDC from post-septic mice impaired Th1 priming but not Th cell expansion and suppressed the innate immune defense mechanisms against Pseudomonas bacteria in the lung. Accordingly, BMDC from post-septic mice inhibited the release of IFN-γ from NK cells that are critical for the protection against Pseudomonas. Additionally, sepsis was associated with a loss of resident DC in the bone marrow. Depletion of resident DC from bone marrow of sham mice led to the differentiation of BMDC that were impaired in Th1 priming similar to BMDC from post-septic mice. Thus, in response to polymicrobial sepsis, DC precursor cells in the bone marrow developed into regulatory DC that impaired Th1 priming and NK cell activity and mediated immunosuppression. The absence of resident DC in the bone marrow after sepsis might have contributed to the modulation of DC differentiation.

Association Between Buprenorphine Dose and the Urine "Norbuprenorphine" to "Creatinine" Ratio: Revised.

Background: Utilizing a 1-year chart review as the data, Furo et al. conducted a research study on an association between buprenorphine dose and the urine "norbuprenorphine" to "creatinine" ratio and found significant differences in the ratio among 8-, 12-, and 16-mg/day groups with an analysis of variance (ANOVA) test. This study expands the data for a 2-year chart review and is intended to delineate an association between buprenorphine dose and the urine "norbuprenorphine" to "creatinine" ratio with a higher statistical power. Methods: This study performed a 2-year chart review of data for the patients living in a halfway house setting, where their drug administration was closely monitored. The patients were on buprenorphine prescribed at an outpatient clinic for opioid use disorder (OUD), and their buprenorphine prescription and dispensing information were confirmed by the New York Prescription Drug Monitoring Program (PDMP). Urine test results in the electronic health record (EHR) were reviewed, focusing on the "buprenorphine," "norbuprenorphine," and "creatinine" levels. The Kruskal-Wallis H and Mann-Whitney U tests were performed to examine an association between buprenorphine dose and the "norbuprenorphine" to "creatinine" ratio. Results: This study included 371 urine samples from 61 consecutive patients and analyzed the data in a manner similar to that described in the study by Furo et al. This study had similar findings with the following exceptions: (1) a mean buprenorphine dose of 11.0 ± 3.8 mg/day with a range of 2 to 20 mg/day; (2) exclusion of 6 urine samples with "creatinine" level <20 mg/dL; (3) minimum "norbuprenorphine" to "creatinine" ratios in the 8-, 12-, and 16-mg/day groups of 0.44 × 10-4 (n = 68), 0.1 × 10-4 (n = 133), and 1.37 × 10-4 (n = 82), respectively; however, after removing the 2 lowest outliers, the minimum "norbuprenorphine" to "creatinine" ratio in the 12-mg/day group was 1.6 × 10-4, similar to the findings in the previous study; and (4) a significant association between buprenorphine dose and the urine "norbuprenorphine" to "creatinine" ratios from the Kruskal-Wallis test (P < .01). In addition, the median "norbuprenorphine" to "creatinine" ratio had a strong association with buprenorphine dose, and this association could be formulated as: [y = 2.266 ln(x) + 0.8211]. In other words, the median ratios in 8-, 12-, and 16-mg/day groups were 5.53 × 10-4, 6.45 × 10-4, and 7.10 × 10-4, respectively. Therefore, any of the following features should alert providers to further investigate patient treatment compliance: (1) inappropriate substance(s) in urine sample; (2) "creatinine" level <20 mg/dL; (3) "buprenorphine" to "norbuprenorphine" ratio >50:1; (4) buprenorphine dose >24 mg/day; or (5) "norbuprenorphine" to "creatinine" ratios <0.5 × 10-4 in patients who are on 8 mg/day or <1.5 × 10-4 in patients who are on 12 mg/day or more. Conclusion: The results of the present study confirmed those of the previous study regarding an association between buprenorphine dose and the "norbuprenorphine" to "creatinine" ratio, using an expanded data set. Additionally, this study delineated a clearer relationship, focusing on the median "norbuprenorphine" to "creatinine" ratios in different buprenorphine dose groups. These results could help providers interpret urine test results more accurately and apply them to outpatient opioid treatment programs for optimal treatment outcomes.

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).

Shock index and pulse pressure as triggers for massive transfusion.

BACKGROUND: Hemorrhage is the most common cause of preventable death in trauma patients. These mortalities might be prevented with prehospital transfusion. We sought to characterize injured patients requiring massive transfusion to determine the potential impact of a prehospital whole blood transfusion program. The primary goal of this analysis was to determine a method to identify patients at risk of massive transfusion in the prehospital environment. Many of the existing predictive models require laboratory values and/or sonographic evaluation of the patient after arrival at the hospital. Development of an algorithm to predict massive transfusion protocol (MTP) activation could lead to an easy-to-use tool for prehospital personnel to determine when a patient needs blood transfusion. METHODS: Using our Level I trauma center's registry, we retrospectively identified all adult trauma patients from January 2015 to August 2017 requiring activation of the MTP. Patients who were younger than 18 years, older than 89 years, prisoners, pregnant women, and/or with nontraumatic hemorrhage were excluded from the study. We retrospectively collected data including demographics, blood utilization, variable outcome data (survival, length of stay, intensive care unit days, ventilator days), prehospital vital signs, prehospital transport times, and Injury Severity Score. The independent-samples t test and χ test were used to compare the group who died to the group who survived. p < 0.05 was considered significant. Based on age and mechanism of injury, relative risk of death was calculated. Graphs were generated using Microsoft Excel software to plot patient variables. RESULTS: Our study population of 102 MTP patients had an average age of 42 years and average Injury Severity Score of 29, consisted of 80% males (82/102), and was 66% blunt trauma (67/102). The all-cause mortality was 67% (68/102). The positive predictive value of death for patients with pulse pressure of less than 45 and shock index of greater than 1 was 0.78 for all patients, but was 0.79 and 0.92 for blunt injury and elderly patients, respectively. CONCLUSIONS: Our data demonstrate a high mortality rate in trauma patients who require MTP despite short transport times, indicating the need for early intervention in the prehospital environment. Given our understanding that the most severely injured patients in hemorrhagic shock require blood resuscitation, this study demonstrates that this subset of trauma patients requiring massive transfusion can be identified in the prehospital setting. We recommend using Emergency Medical Services pulse pressure in combination with shock index to serve as a trigger for initiation of prehospital whole blood transfusion. LEVEL OF EVIDENCE: Therapeutic/care management, level V.

Toll-like receptor 2 and 4 expression after severe injury is not involved in the dysregulation of the innate immune system.

BACKGROUND: Severe injury after trauma is associated with a diminished production of different proinflammatory cytokines after stimulation with bacterial cell wall components. The cellular mechanisms, leading to a decreased responsiveness especially of monocytes after multiple injuries have not yet been elucidated in detail. The expression of Toll-like receptors (TLR) on leukocytes is essential for recognition of bacterial components. We investigated the expression of TLR2 and 4 in correlation with gram-negative and gram-positive stimuli-dependent cytokine liberation after severe injury in comparison with that in healthy volunteers. METHODS: In a prospective clinical experimental study, 12 trauma patients with an Injury Severity Score above 21 points and 14 healthy volunteers were analyzed. Heparinized whole blood samples of patients were collected within 48 hours after trauma and incubated in vitro with or without lipopolysaccharide (LPS) and peptidoglycan (PGN). TLR2 and TLR4 expression on monocytes was analyzed by flow cytometry. LPS- and PGN-induced tumor necrosis factor alpha (TNFalpha) and interleukin-8 production was measured by means of enzyme-linked immunosorbent assay. RESULTS: Both LPS- and PGN-induced TNFalpha liberation were significantly reduced in severely injured patients. The surface expression of TLR2 was also significantly decreased on monocytes collected from trauma patients, whereas the expression of TLR4 remained unchanged. There was only a negative correlation between TLR2 expression and the liberation of TNFalpha after stimulation with LPS or PGN. CONCLUSIONS: We conclude that diminished cytokine production after trauma cannot be explained simply by changes in TLR2 or TLR4 expression and that subsequent signaling cascades or additional receptors are involved in the blunted cytokine response after trauma.

The composition of T-cell subsets are altered in the burn wound early after injury.

BACKGROUND: Burn-induced inflammation leads to impaired immune responses resulting in increased morbidity and mortality. T-cells are central in the immune response and circulating CD4 and CD8 T-cells have been used to evaluate immune status; however, the role of these T-cell subsets in the burn wound is unknown. METHODS: Male C57BL/6 mice were subjected to a major 3rd degree scald burn or sham treatment. Twenty-four hours later, full thickness skin samples from sham mice and the burn wounds were collected and single cells were isolated and analyzed for αß TCR, γδ TCR, CD3, CD4, CD8 and CD69 expressions by flow cytometry. RESULTS: The burn wound contained significantly greater numbers of T-cells than skin from sham mice, due to a profound infiltration of αß T-cells. These infiltrating αß T-cells were primarily suppressor T-cells with a CD8+ or CD8-CD4- phenotype. The 15-fold increase in CD8+ αß T-cells caused a decrease in the CD4:CD8 ratio from 0.7 in sham skin to 0.3 in the burn wound. In contrast, the majority of the γδ T-cells in sham skin were CD4-CD8-, which decreased 9-fold in the burn wound. CD69 expression was suppressed on burn wound αß T-cells, but increased on γδ T-cells in the burn wound. CONCLUSIONS: The infiltrating burn wound αß T-cells likely act to quell inflammation. In contrast wound γδ T-cells were activated with elevated CD4 and CD69 expression. Thus, these two distinct T-cell subsets likely differentially regulate the burn wound inflammatory response.

Damage-associated molecular patterns (DAMPs) released after burn are associated with inflammation and monocyte activation.

Burns are associated with activation of the innate immunity that can contribute to complications. Damage-associated molecular patterns (DAMPs) released after tissue injury play a critical role in the activation of the innate immunity, which appears to be mediated via toll-like receptors (TLRs). Previous findings have shown that TLRs and TLR-mediated responses are up-regulated after burn. Nonetheless, it is unclear what impact burn has on circulating levels of DAMPs. To study this, male C57BL/6 mice were subjected to a major burn or sham procedure. Three hours to 7days thereafter, plasma was collected and assayed for the representative DAMPs (i.e., HMGB1, cytochrome C, DNA and S100A) and extracellular cleavage products (fibronectin and hyaluronan). HMGB1, cytochrome C, fibronectin and hyaluronan levels were elevated in a time-dependent manner after burn as compared to sham levels. A significant elevation in TNF-α, IL-6 and IL-10 cytokine plasma levels was also found after burn. All cytokine levels were increased as early as 3h and remained elevated up to 24h. Circulating CD11b+ monocytes were increased at 24h after burn and showed increased expression of TLR-2. In conclusion, these findings support the concept that burn-induced elevations in circulating DAMPs are in part responsible for monocyte activation and the development of inflammatory complications under such conditions and warrants further investigation.

GM-CSF priming of human monocytes is dependent on ERK1/2 activation.

The ability to augment monocyte functions such as TNF-alpha-producing capacities confers a high immunostimulating potential to GM-CSF. In the present investigation, the mechanism of the GMCSF-mediated enhancement of monocyte cytokine production was analysed with regard to the involvement of intracellular signalling pathways. GM-CSF primes human monocytes dose- and time-dependently for enhanced LPS-stimulated TNF-alpha synthesis. Pre-incubation with 10 ng/ml GM-CSF for 6 h before LPS stimulation (10 ng/ml) caused a 3.4 +/- 1.9-fold increase in TNF-alpha release compared to unprimed controls. This was associated with increased phosphorylation of IkappaBalpha and elevated nuclear levels of the NF-kappaB components p50 and p65 and NF-kappaB binding to DNA. LPS-induced AP-1 binding to DNA was also enhanced in GM-CSF-pre-incubated cells. GMCSF treatment also caused a slight increase in TLR4 expression on monocytes while CD14 expression remained unchanged. GM-CSF-priming was unaffected by inhibitors of p38 MAPK (SB203580) and lipoxygenase (NDGA). In contrast, the broad-spectrum tyrosine kinase inhibitor genistein and the MEK-1 inhibitor (PD98059) abrogated GM-CSF priming of TNF-alpha release and activation of both NF-kappaB and AP-1. It is concluded that a tyrosine kinase of the GM-CSF-triggered ERK1/2 pathway augments the LPS-induced NF-kappaB and AP-1 activation.

Haemorrhagic shock in mice--intracellular signalling and immunomodulation of peritoneal macrophages' LPS response.

Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mmHg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IkappaBalpha was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IkappaBalpha was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IkappaB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IkappaB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.

Dermal γδ T-Cells Can Be Activated by Mitochondrial Damage-Associated Molecular Patterns.

BACKGROUND: Gamma delta T-cells have been shown to be important to the early immunoinflammatory response to injury, independent of infection. This unique T-cell population acts to regulate cell trafficking and the release of cytokines and growth factors. We propose this sterile inflammatory response is in part associated with damage associated molecular patterns (DAMPs) generated by major injury, such as burn, and mediated via toll-like receptors (TLRs). It is unknown whether DAMPs can activate resident γδ T-cells that reside in skin. METHODS: Gamma delta T-cells were isolated from the skin of male C57BL/6 mice by enzymatic digestion. Mitochondrial DAMPs (MTDs) were generated from mitochondria isolated from mouse livers by sonication and centrifugation. Dermal γδ T-cells were incubated with MTDs (0-500 µg/ml) for 24 hr and cells and supernatants were collected for analysis. RESULTS: MTDs activated dermal γδ T-cells, as evidenced by increased TLR2 and TLR4 expression following in vitro exposure. MTDs also induced the production of inflammatory cytokines (IL-1ß, IL-6), and growth factors (PDGF and VEGF) by γδ T-cells. CONCLUSIONS: These findings herein support the concept that MTDs released after tissue/cellular injury are capable of activating dermal γδ T-cells. We propose that the activation of this unique T-cell population is central in the initiation of sterile inflammation and also contributes to the subsequent healing processes.

Gamma delta T cells regulate inflammatory cell infiltration of the lung after trauma-hemorrhage.

Trauma-hemorrhage (TH) promotes acute lung injury (ALI) and other pulmonary-related complications in part through an exaggerated inflammatory response. Studies have implicated γδ T cells in the development of inflammatory complications after major injury; however, it is unknown whether γδ T cells play a role in the development of ALI after TH. To study this, C57BL/6 wild-type (WT) and δ TCR mice were subjected to TH or sham treatment. Lung injury was clearly evident at 2 h after TH, as evidenced by increased lung permeability, myeloperoxidase levels, and proinflammatory cytokine/chemokine levels (interleukin-1ß [IL-1ß], IL-6, IL-10, keratinocyte chemokine, macrophage inflammatory protein 1α, macrophage inflammatory protein 1ß, and regulated upon activation normal T-cell expressed, secreted chemokine). Phenotypic analysis of lung cells showed an increase in T-cell numbers after TH. The vast majority of these cells were αß T cells, irrespective of injury. Although γδ T cells were a small percentage of the total T-cell infiltrate, their numbers did increase after injury. In mice lacking γδ T cells (δ TCR mice), TH-induced T-cell infiltration of the lung was markedly attenuated, whereas infiltration of other inflammatory cells was increased (i.e., monocytes, granulocytes, and myeloid-derived suppressor cells). In conclusion, these findings suggest that γδ T cells regulated the infiltration of the lung with inflammatory cells after injury.

Activated skin γδ T-cells regulate T-cell infiltration of the wound site after burn.

Burn induces an immunopathological response involving multiple immune cell types that includes γδ T-cells. Nonetheless, the role of γδ T-cells at the wound site after burn is not clearly defined. Wild type and γδ T-cell receptor deficient (δ TCR(-/-)) mice were subjected to a major burn or sham procedure. At 1-7 d thereafter, skin samples were collected and T-cell populations analyzed. The majority of T-cells in the skin of sham mice were γδ T-cells. After burn, however, an increase in the total T-cells was observed at the wound site and these cells were predominantly αß T-cells. Their influx was γδ T-cell dependent, as it was markedly reduced in injured δ TCR(-/-) mice. Burn wound γδ T-cells were activated with increased expression of TLRs and CD69. In contrast, the infiltrating αß T-cells TLR and CD69 expressions were attenuated after burn. Thus, burn is associated with of γδ T-cell activation at the injury site, which initiates a massive infiltration of the wound with αß T-cells that likely facilitate the transition from the inflammatory to the proliferative phase of healing.

Gamma delta T cells regulate wound myeloid cell activity after burn.

Major burns induce immune complications, which are associated with myeloid cell activation by ill-defined mechanisms. Although γδ T cells have been shown to be important in postinjury inflammation and wound healing, their role in the regulation of myeloid cells remains unknown. To study this, wild-type (WT) and γδ T cell-deficient (δTCR) mice were subjected to major burn (25% total body surface area, third degree) or sham treatment. At 3 days thereafter, skin samples were assayed for cytokine content or used to isolate single cells that were used for myeloid cell characterization by flow cytometry. The number of CD11b myeloid cells increased by approximately 75% in the wound skin of WT mice. This influx was caused by increased myeloid-derived suppressor cells (CD11b GR1) whose numbers increased 19-fold compared with those of sham skin. In contrast, macrophage (MØ; CD11b F4/80) numbers decreased by approximately 50% after burn. In δTCR mice, burn increased the myeloid cell numbers approximately 5-fold. The increase in myeloid cells at the injury site of δTCR mice was caused by both a myeloid-derived suppressor cell (50-fold) and a MØ (2-fold) influx. Burn increased skin cytokine levels for a number of prototypic inflammatory cytokines (interleukin 1ß, interleukin 6, tumor necrosis factor-α, macrophage inflammatory protein [MIP] 1ß, etc). Tumor necrosis factor-α, MIP-1α, and MIP-1ß levels were further elevated (2- to 3-fold) in the injured skin of δTCR mice compared with those of WT mice. In conclusion, these data show that γδ T cells regulate myeloid cell infiltration of the wound site and act to quell inflammation, thereby promoting the transition to the proliferative phase of wound healing.

Burn wound γδ T-cells support a Th2 and Th17 immune response.

Major burn triggers immune dysfunction, which is associated with wound healing complications. Gamma-δ T-cells have been shown to be important in postburn inflammation and wound healing; however, their cytokine phenotype at the burn wound site is unknown. C57BL/6 male mice were subjected to a major burn (25% TBSA, third degree) or sham treatment. At 3 hours, 3 days, and 7 days thereafter, skin samples were collected and subjected to dispase and trypsin digestion to isolate single cells. The cells were phenotyped and evaluated for cytokine profiles by flow cytometry. Th1 cells were defined as interferon (IFN)γ positive, Th2 cells were defined as interleukin (IL)-10 positive, and Th17 cells were defined as IL-17 positive. At 7 days after burn a shift toward Th2 and Th17 positive T-cells at the wound site was observed. Further analysis revealed that at 3-hour postinjury the percentage of γδ T-cells positive for IFNγ, IL-10, and IL-17 were comparable between sham and burn skin samples. At 3 days and 7 days postinjury the percentage of cells positive for each cytokine increased; however, the increase was significantly greater for IL-10 and IL-17, as compared with IFNγ (ie, 9-20-fold vs 3-fold). Skin αß T-cells preferentially produced IFNγ (~20%), which was unaffected by burn injury. These data demonstrate that burn wound γδ T-cells are activated for enhanced cytokine production and display a shift toward a Th2 and/or Th17 phenotype. In contrast, burn wound αß T-cells were not activated for enhanced cytokine production.

Mitochondrial damage-associated molecular patterns activate γδ T-cells.

Gamma delta T-cells have been shown to be important in the early immunoinflammatory response to injury, which can be independent of infection. This sterile inflammatory response is believed to be, in part, associated with danger-associated molecular patterns (DAMPs). Mitochondrial DAMPs (MTDs) have been shown to be important in trauma-induced neutrophil activation, but it is unknown whether MTDs activate other innate immune cells, such as γδ T-cells. To study this, splenic CD3(+) γδ T-cells were isolated from αß T-cell-deficient C57BL/6 mice and mitochondria isolated from wild type mouse livers. MTDs were isolated from mitochondria by sonication and centrifugation. Gamma delta T-cells were incubated with various concentrations of MTDs (0-500 µg/ml) for 24 h. T-cells were phenotyped for TLR expression by flow cytometry and the supernatants assayed for cytokine and growth factor content. MTDs caused a dose-dependent increase in TLR2 and TLR4 expression by γδ T-cells. Both the percentage of cells positive for TLRs and the degree of expression increased. MTDs also induced the production of IL-1ß, IL-6, IL-10, RANTES, fibroblast growth factor-basic and vascular endothelial growth factor by γδ T-cells. These findings support the concept that the MTDs released after tissue/cellular injury are capable of activating γδ T-cells, thus initiating sterile inflammation, as well as subsequent healing processes.