Meenakshi Rani

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 αβ 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-1β, 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.

Increased expression of cardiac IL-17 after burn.

BackgroundCardiac dysfunction is a common complication associated with major burns. While recent findings have linked the Th-17 T-cell response to the development of autoimmune myocarditis, the role of IL-17 and the Th-17 T-cell response in the development of post-burn cardiac dysfunction remains unknown.MethodsMale C57BL/6 mice were subjected to a major burn (3rd degree, 25% TBSA) or sham treatment. Three hours after injury plasma and tissue (i.e., heart, lung, liver, small intestine) samples were collected and analyzed for the expression of Th-17 cytokine (i.e., IL-6, IL-17, IL-22, IL-23, TGF-β) levels by ELISA.ResultsCardiac tissue levels of the Th-17 cytokines, IL-6, IL-17 and IL-22 were significantly elevated at 3 hrs after burn as compared to sham levels. IL-17 was analyzed 1, 3 and 7 days after burn and showed a return to baseline levels and without a difference in the burn group. Burn-induced alterations in the level of these cytokines in plasma or other tissues were not evident. The cardiac Th-17 cytokine response after burn injury was specific, as cardiac levels of Th-1 (IFN-γ) and Th-2 (IL-10) cytokines were not significantly affected after injury. The cardiac Th-17 response correlated with a significant increase in Troponin levels at 3 hr. after burn.ConclusionThese findings indicate that early after burn, cardiac tissue is associated with significantly elevated levels of Th-17 cytokines. The early Th-17 response after burn appears to be specific for cardiac tissue and may promote myocardial inflammation and dysfunction associated with this form of trauma.

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-1β, IL-6, IL-10, IL-17, TNF-α, KC, MCP-1, MIP-1α, MIP-1β 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-1β 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).

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

The ability to augment monocyte functions such as TNF-α-producing capacities confers a high immunostimulating potential to GM-CSF. In the present investigation, the mechanism of the GM-CSF-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-α 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-α release compared to unprimed controls. This was associated with increased phosphorylation of IκBα and elevated nuclear levels of the NF-κB components p50 and p65 and NF-κB binding to DNA. LPS-induced AP-1 binding to DNA was also enhanced in GM-CSF-pre-incubated cells. GM-CSF 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-α release and activation of both NF-κB and AP-1. It is concluded that a tyrosine kinase of the GM-CSF-triggered ERK1/2 pathway augments the LPS-induced NF-κB and AP-1 activation.

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

ABSTRACT Major burns induce immune complications, which are associated with myeloid cell activation by ill-defined mechanisms. Although &ggr;&dgr; 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 &ggr;&dgr; T cell–deficient (&dgr;TCR-/-) 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 &dgr;TCR-/- mice, burn increased the myeloid cell numbers approximately 5-fold. The increase in myeloid cells at the injury site of &dgr;TCR-/- 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 1&bgr;, interleukin 6, tumor necrosis factor-&agr;, macrophage inflammatory protein [MIP] 1&bgr;, etc). Tumor necrosis factor-&agr;, MIP-1&agr;, and MIP-1&bgr; levels were further elevated (2- to 3-fold) in the injured skin of &dgr;TCR-/- mice compared with those of WT mice. In conclusion, these data show that &ggr;&dgr; 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.

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-1β, IL-6, G-CSF, TNF-α, KC, MCP-1, MIP-1α and MIP-1β at the wound site in WT and δ TCR(-/-) mice, but G-CSF, TNF-α, and MIP-1β levels were greater in δ TCR(-/-) mice. Inhibition of iNOS activity in WT mice with L-Nil suppressed burn wound levels of IL-1β, G-CSF, and MIP-1α, whereas IL-6, TNF-α, KC, MCP-1 and MIP-1β 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 wound γδ T-cells support a Th2 and Th17 immune response.

Major burn triggers immune dysfunction, which is associated with wound healing complications. Gamma-&dgr; 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)&ggr; 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 &ggr;&dgr; T-cells positive for IFN&ggr;, 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 IFN&ggr; (ie, 9–20-fold vs 3-fold). Skin &agr;&bgr; T-cells preferentially produced IFN&ggr; (~20%), which was unaffected by burn injury. These data demonstrate that burn wound &ggr;&dgr; T-cells are activated for enhanced cytokine production and display a shift toward a Th2 and/or Th17 phenotype. In contrast, burn wound &agr;&bgr; T-cells were not activated for enhanced cytokine production.

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 αβ 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 αβ 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 αβ T-cells that likely facilitate the transition from the inflammatory to the proliferative phase of healing.

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.

Effect of inhaled tacrolimus on ischemia reperfusion injury in rat lung transplant model

OBJECTIVE Systemic tacrolimus therapy has been shown to protect against lung ischemia-reperfusion injury in animal models. We sought to investigate on a functional and cellular level if inhaled nanoparticle tacrolimus administered to the donor lung before procurement could similarly attenuate ischemia-reperfusion injury after lung transplant. METHODS An isogenic orthotopic rat model of single left lung transplant was used. Donor animals were pretreated with inhaled tacrolimus (treatment group) or inhaled lactose (controls) before lung procurement. Lung grafts were subjected to 3 hours of cold ischemia followed by 4 hours of reperfusion after graft implantation. Recipient animal arterial blood gas measurement and isograft wet to dry weight ratios were obtained. Macrophage, neutrophil, and T-cell accumulation and activation in lung isografts, including γδ T-cell, T-helper, and cytotoxic T-cell subtypes were analyzed by flow cytometry. Tacrolimus levels were measured in the lung isograft using liquid chromatography/mass spectrometry. Isograft cytokine levels were measured with commercial enzyme-linked immunosorbent assay and microbead array kits. RESULTS Oxygenation in treatment group animals was significantly higher than in controls. The presence of macrophages, neutrophils, and all T-cell subtypes in the isografts as well as isograft levels of inflammatory cytokines were all less in the treatment group versus controls, although no single variable achieved statistical significance. CONCLUSIONS Inhaled nanoparticle tacrolimus treatment of lung donors is associated with an attenuation of ischemia-reperfusion injury on a functional and cellular level in lung transplant.