Toll-like receptor 2 and 4 ligation results in complex altered cytokine profiles early and late after burn injury.

BACKGROUND: Toll-like receptors (TLR) 2 and TLR4 expressed on innate immune cells are important mediators of the immune response to pathogens. In this study, we hypothesized that burn injury results in altered cytokine secretion profiles after TLR2 or TLR4 ligation that is associated with altered TLR expression on innate immune cells. METHODS: Female C56BL/6 mice were subjected to 20% full thickness burn or sham injury. Three or 14 days after injury whole splenocytes or purified splenic macrophages were cultured with TLR2 ligand peptidoglycan or TLR4 ligand lipopolysaccharide. Supernatants were assayed for TNF-alpha, MCP-1, IL-6 and IL-10. Cell death was assessed using flow cytometry. Innate CD11b F4/80 macrophages were sorted 14 days after burn injury and TLR2 and 4 expression was determined by quantitative reverse-transcriptase polymerase chain reaction and flow cytometry. RESULTS: Burn injury results in a steady accumulation in the periphery of CD11bF4/80 macrophages. Macrophages purified early after burn injury upregulated TLR2 and 4, followed by a decrease of TLR2 and TLR4 expression late after burn injury. TLR2 and TLR4 ligation of an equivalent number of purified macrophages 3 days after burn injury revealed no significant differences in cytokine secretion compared with sham. Stimulation 14 days after burn injury revealed a significant reduction in tumor necrosis factor-alpha secretion by macrophages compared with sham mice. In contrast, interleukin-10 was significantly increased (mean, approximately 1.8-fold) late after burn injury after either TLR2 or TLR4 stimulation. Interleukin-6 and monocyte chemotactic protein-1 secretion was unchanged from sham levels. In contrast, whole splenocyte stimulation resulted in increased cytokine 3 days and 14 days after burn injury. This effect is likely caused by the accumulation of TLR macrophages, which are resistant to TLR-induced cell death. CONCLUSIONS: Cytokine secretion profiles after TLR2 and TLR4 ligation after burn injury are altered in a manner not clearly reflective of an anti-inflammatory or proinflammatory state and are associated with unique changes in the macrophage population. TLR2 and TLR4 ligation have complex and varied roles in mediating the immune response to burn injury.

Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin.

CD8+ cytotoxic T lymphocytes (CTLs) are important effector cells responsible for tissue destruction in several autoimmune and allograft-related diseases. To discover if pathogenic T cells could be selectively deleted, we investigated the ability of a toxin coupled to major histocompatibility complex (MHC) class I tetramers to kill antigen-specific CD8+ T cells. H2-D(b) tetramers were assembled using streptavidin conjugated to the ribosome-inactivating protein (RIP) saporin (SAP). These tetramers inhibited ribosome activity in vitro, retained the T-cell receptor (TCR)-binding specificity of their nontoxic counterparts, and were internalized by 100% of target cells, leading to cell death in 72 hours. Cytotoxicity was dependent on the tetramer dose and avidity for the T cell. A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact.

Increased Toll-like receptor 4 expression on T cells may be a mechanism for enhanced T cell response late after burn injury.

BACKGROUND: Burn injury is associated with a dynamic T cell response. We have previously reported an enhanced functional T cell response 14 days after burn injury. Toll-like receptors (TLR), primarily expressed on innate immune cells, have recently been identified on certain T cell subsets, including activated and memory T cells. Our hypothesis is that increased TLR4 expression on memory T cells may be a mechanism for enhanced T cell response 14 days after burn injury. METHODS: Splenocytes from wild-type C57Bl/6 mice were harvested 14 days after a 20% total body surface area (TBSA) scald burn or sham injury. Splenocytes ex vivo were surface stained either with monoclonal anti-CD3, anti-CD4, anti-CD8, or anti-CD44 antibodies or a two-step biotin-TLR4 monoclonal antibody-streptavidin-FITC surface stain and results analyzed by flow cytometry. RESULTS: TLR4 expression is successfully detected on CD4 and CD8 T cells. TLR4 expression is significantly (p < 0.05) increased on CD4 T cells and CD8 T cells 14 days after burn injury. There is a significant (p < 0.05) increase in CD44 (memory) CD4 and CD44 (memory) CD8 T cells 14 days after burn injury and this is associated with a significant (p < 0.05) increase of TLR4 expression in both T cell populations. CONCLUSIONS: This study demonstrates for the first time the potential role of TLR4 expression on memory T cells generated late after burn injury. Although further analysis is required, these data reiterate the importance of adaptive immunity and the complexity of the immune response to burn injury.

Lymphopenia-induced homeostatic proliferation of CD8+ T cells is a mechanism for effective allogeneic skin graft rejection following burn injury.

Burn patients are immunocompromised yet paradoxically are able to effectively reject allogeneic skin grafts. Failure to close a massive burn wound leads to sepsis and multiple system organ failure. Immune suppression early (3 days) after burn injury is associated with glucocorticoid-mediated T cell apoptosis and anti-inflammatory cytokine responses. Using a mouse model of burn injury, we show CD8+ T cell hyperresponsiveness late (14 days) after burn injury. This is associated with a CD8+ T cell pro- and anti-inflammatory cytokine secretion profile, peripheral lymphopenia, and accumulation of a rapidly cycling, hyperresponsive memory-like CD8+CD44+ IL-7R- T cells which do not require costimulation for effective Ag response. Adoptive transfer of allospecific CD8+ T cells purified 14 days postburn results in enhanced allogeneic skin graft rejection in unburned recipient mice. Chemical blockade of glucocorticoid-induced lymphocyte apoptosis early after burn injury abolishes both the late homeostatic accumulation of CD8+ memory-like T cells and the associated enhanced proinflammatory CD8+ T cell response, but not the late enhanced CD8+ anti-inflammatory response. These data suggest a mechanism for the dynamic CD8+ T cell response following injury involving an interaction between activation, apoptosis, and cellular regeneration with broad clinical implications for allogeneic skin grafting and sepsis.

Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children.

OBJECTIVE: Open-label studies indicate that oral dichloroacetate (DCA) may be effective in treating patients with congenital lactic acidosis. We tested this hypothesis by conducting the first double-blind, randomized, control trial of DCA in this disease. METHODS: Forty-three patients who ranged in age from 0.9 to 19 years were enrolled. All patients had persistent or intermittent hyperlactatemia, and most had severe psychomotor delay. Eleven patients had pyruvate dehydrogenase deficiency, 25 patients had 1 or more defects in enzymes of the respiratory chain, and 7 patients had a mutation in mitochondrial DNA. Patients were preconditioned on placebo for 6 months and then were randomly assigned to receive an additional 6 months of placebo or DCA, at a dose of 12.5 mg/kg every 12 hours. The primary outcome results were (1) a Global Assessment of Treatment Efficacy, which incorporated tests of neuromuscular and behavioral function and quality of life; (2) linear growth; (3) blood lactate concentration in the fasted state and after a carbohydrate meal; (4) frequency and severity of intercurrent illnesses and hospitalizations; and (5) safety, including tests of liver and peripheral nerve function. OUTCOME: There were no significant differences in Global Assessment of Treatment Efficacy scores, linear growth, or the frequency or severity of intercurrent illnesses. DCA significantly decreased the rise in blood lactate caused by carbohydrate feeding. Chronic DCA administration was associated with a fall in plasma clearance of the drug and with a rise in the urinary excretion of the tyrosine catabolite maleylacetone and the heme precursor delta-aminolevulinate. CONCLUSIONS: In this highly heterogeneous population of children with congenital lactic acidosis, oral DCA for 6 months was well tolerated and blunted the postprandial increase in circulating lactate. However, it did not improve neurologic or other measures of clinical outcome.