Activation of Toll-Like Receptor 2 Prevents Suppression of T-Cell Interferon γ Production by Modulating p38/Extracellular Signal-Regulated Kinase Pathways following Alcohol and Burn Injury
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Recent studies indicate that toll-like receptors (TLRs) are expressed on T cells and that these receptors directly or indirectly activate the adaptive immune system. We have shown previously that acute alcohol/ethanol (EtOH) intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell interleukin-2 (IL-2) and interferon γ (IFN-γ) production. We examined whether direct stimulation of T cells with TLR2, 4, 5 and 7 agonists modulates CD3-mediated T-cell IL-2/IFN-γ release following EtOH and burn injury. Male mice were gavaged with EtOH (2.9 gm/kg) 4 h prior to receiving an ~12.5% total body surface area sham or full-thickness burn injury. Animals were killed on d 1 after injury and T cells were purified from MLN and spleens. T cells were cultured with plate-bound anti-CD3 in the presence or absence of various TLR ligands. Although TLR2, 4 and 5 agonists potentiate anti-CD3-dependent IFN-γ by T cells, the TLR2 agonist alone induced IFN-γ production independent of CD3 stimulation. Furthermore, T cells were treated with inhibitors of myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), p38 and/or extracellular signal-regulated kinase (ERK) to determine the mechanism by which TLR2 mediates IL-2/IFN-γ production. IL-2 was not influenced by TLR agonists. MyD88 and TIRAP inhibitory peptides dose-dependently diminished the ability of T cells to release IFN-γ. p38 and ERK inhibitors also abolished TLR2-mediated T-cell IFN-γ. Together, our findings suggest that TLR2 directly modulates T-cell IFN-γ production following EtOH and burn injury, independent of antigen-presenting cells. Furthermore, we demonstrated that MyD88/TIRAP-dependent p38/ERK activation is critical to TLR2-mediated T-cell IFN-γ release following EtOH and burn injury.
Alcohol remains the most abused substance worldwide. It is a high risk factor for traumatic injury, including burns (1, 2, 3). Nearly one million burn injuries are reported in the United States every year, and 50% of these injuries occur in individuals under the influence of alcohol/ethanol (EtOH) (4, 5, 6, 7, 8). Studies indicate that intoxicated patients have higher rates of septic complications, longer hospital stays and increased mortality compared with patients who have a similar extent of burn injury but did not consume EtOH before injury (7, 8, 9, 10, 11). There is evidence that EtOH intoxication combined with burn injury abrogates the host immune system. Specifically, the combined insult of EtOH and burn injury suppresses T-cell responses, potentiates inflammatory cytokine and chemokine production and induces neutrophil recruitment to the intestine and other organs (12, 13, 14). Studies from our laboratory suggest that acute EtOH intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell proliferation as well as interleukin-2 (IL-2) and interferon γ (IFN-γ) production. This effect is accompanied by an increase in bacterial translocation to MLN. We have also demonstrated a role for p38 and extracellular signal-regulated kinase (ERK) activation in T-cell suppression following EtOH and burn injury (15, 16, 17).
Toll-like receptors (TLRs) are known to play a critical role in host immunity. Classically, TLRs were believed to be expressed only on cells of the innate immune system, including neutrophils, dendritic cells and macrophages, where they function as first-line sensors of invading pathogens (18). TLRs recognize highly conserved molecules derived from microbes. Upon activation, TLRs induce the release of inflammatory mediators and cytokines to initiate adaptive immune responses against the invading pathogens. To date, at least 13 TLRs (TLR1 to TLR13) have been identified in mice and humans (19,20), each recognizing a distinct conserved pathogen-associated molecular pattern (PAMP) (19). Pathogenic microorganisms contain multiple PAMPs that act as TLR agonists: peptidoglycan (TLR2), lipopolysaccharide (LPS) (TLR4), flagellin (TLR5) and single-stranded RNA (ssRNA) (TLR7). The TLR signaling pathway has been widely investigated in the innate immune system. TLR signal transduction associates with Toll/IL-1 receptor (TIR) domain-containing adaptor molecules, such as myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), Toll-receptor-associated activator of interferon (TRIF) and Toll-receptor-associated molecule (TRAM). Except for TLR3, all the TLR proteins use the MyD88 adaptor protein to activate the mitogen activated protein kinase (MAPK) pathway, and subsequently, the translocation of nuclear factor-κB (NF-κB) to nucleus (21,22).
Although most TLR studies have focused on cells of the innate immune system, recent studies have indicated that TLRs directly or indirectly activate the adaptive immune system (23, 24, 25, 26, 27, 28, 29). Some of these studies further demonstrated that T cells express certain TLRs and that activation of these proteins can directly promote T-cell survival and proliferation, as well as IL-2 and IFN-γ production, independently of antigen presenting cells (APCs) (23, 24, 25). Yet, the mechanism of TLR intracellular signaling within T cells remains unclear. Moreover, the majority of data describing TLR expression and function within T cells is limited to cell lines or T cells from healthy animals. In this study, we used a mouse model of acute EtOH intoxication and burn injury to determine the effect of TLR2, 4, 5 and 7 agonists on T-cell effector functions in healthy and injury conditions. Among these, only the TLR2 agonist was found to directly modulate T-cell cytokine production. Furthermore, activation of TLR2 on T cells prevents suppression of IFN-γ production following EtOH and burn injury. Lastly, our findings suggest a role for p38 and ERK in TLR2-mediated modulation of T-cell IFN-γ production.
Materials and Methods
Animals and Reagents
Male C57BL/6 mice (22–25 g) were obtained from Harlan Laboratories (Indianapolis, IN, USA). IL-2 and IFN-γ enzyme-linked immunosorbent assay (ELISA) kits were obtained from BD Biosciences (San Diego, CA, USA). A mouse TLR 1–9 agonist kit was obtained from InvivoGen (San Diego, CA, USA), Alexa Fluor-647 conjugated anti-TLR2 and phycoerythrin (PE)-Cy5-conjugated anti-CD3e antibodies were obtained from eBioscience (San Diego, CA, USA). A MyD88 homodimerization inhibitory peptide set and TIRAP inhibitory peptide set were obtained from IMGENEX (San Diego, CA, USA). p38 inhibitor SB 203580 and ERK inhibitor PD 98059 were obtained from EMD Chemicals (Gibbstown, NJ, USA). Antibodies to p38, phospho-p38 (Thr180/Tyr182), ERK 1/2 and phospho-ERK 1/2 (Thr202/Tyr204) were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibody to β-actin was obtained from Abcam (Cambridge, MA, USA).
Mouse Model of Acute EtOH Intoxication and Burn Injury
As described previously (14), mice were randomly divided into four groups: sham vehicle, sham EtOH, burn vehicle and burn EtOH. In the EtOH-treated groups, mice were gavaged with 0.4 mL of 25% EtOH in water (2.9 gm/kg). In the vehicle groups, mice were gavaged with 0.4 mL of water. At 4 h after gavage, mice were anesthetized with a mixture of ketamine and xylazine at a dose of 79.7 mg/kg and 1.18 mg/kg, respectively, by intraperitoneal injection. The dorsal surface was shaved and mice were transferred into a template fabricated to expose ~12.5% of the total body surface area (TBSA). TBSA was calculated by using Meeh’s formula as described by Walker and Mason (30). For burn injury, mice were immersed into hot water maintained at 85°C-90°C for 7s. For sham injury, mice received identical anesthesia and were shaved, but were immersed in lukewarm water for 7 s. All animals were dried immediately and resuscitated with 1.0 mL physiological saline by intraperitoneal injection. All the animal procedures were carried out in adherence to the National Institutes of Health 2011 Guide for the Care and Use of Laboratory Animals, 8th edition (31) and were approved by the Loyola University Chicago Health Sciences Division Institutional Animal Care and Use Committee.
Measurement of T-Cell IL-2 and IFN-γ Levels
To determine whether TLRs modulate T-cell responses following EtOH and burn injury, we designed the following series of experiments.
Experiment 1: To confirm whether EtOH intoxication combined with burn injury suppresses T-cell responses, MicroBeads-purified T cells were resuspended at a density of 5 × 106 cells/mL in RPMI 1640 supplemented with 2mmol/L l-glutamine, 10 mmol/L HEPES, 50 µg/mL gentamicin, 100 U/mL penicillin with 100 µg/mL streptomycin and 10% FCS. As previously described (16,17), T cells (5 × 105 cells/well) were cultured in 96-well plates in the presence of plate-bound anti-CD3 (2 µg/mL) at 37°C and 5% CO2 for 48 h. Following culture, supernatants were harvested and tested for IL-2 and IFN-γ levels, by use of respective ELISA kits.
Experiment 2: To determine whether TLR agonists directly modulate T-cell responses following EtOH and burn injury, T cells were cultured with/without plate-bound anti-CD3 antibody in the presence or absence of TLR agonists. Specific agonists include: TLR2, heat-killed preparation of Listeria monocytogenes (HKLM; 108 cells/mL); TLR4, LPS (1 µg/mL); TLR5, flagellin from Salmonella typhimurium (ST-FLA; 1 µg/mL) and TLR7, ssRNA40 (1 µg/mL). After 48 h of culture, supernatants were tested for IL-2 and IFN-γ production by ELISA.
Experiment 3: To assess whether TLR2 agonist modulates T-cell responses after EtOH intoxication and burn injury through the MyD88 and TIRAP pathway, T cells were cultured with plate-bound anti-CD3 in the presence or absence of TLR2 agonist (HKLM, 108 cells/mL), MyD88 homodimerization inhibitory peptide (25), 50 and 100 µmol/L) and/or TIRAP inhibitory peptide (25), 50 and 100 µmol/L). After 48 h, cell supernatants were harvested and analyzed for IFN-γ by ELISA.
Experiment 4: To further determine whether p38 and ERK1/2 play any role in the TLR2-mediated T-cell responses following EtOH and burn injury, T cells were cultured with/without plate-bound anti-CD3 and TLR2 agonist (HKLM, 108 cells/mL) in the presence or absence of p38 inhibitor (SB 203580 10 µmol/L) and ERK inhibitor (PD 98059 50 µmol/L). p38 and ERK inhibitor doses were selected from our previous studies (17). After 48 h, IFN-γ was tested in cell supernatant.
T-Cell Staining and Flow Cytometry
Spleen T cells were cultured in the presence and absence of plate-bound anti-CD3 (2 µg/mL) in RPMI 1640 supplemented with 2 mmol/L l-glutamine, 10 mmol/L HEPES, 50 µg/mL gentamicin and 100 U/mL penicillin with 100 µg/mL streptomycin for 16 h. Fresh T cells or cultured T cells (106 cells/100 µL) were incubated with PE-Cy5-conjugated anti-mouse CD3e and Alexa Fluor-647 conjugated anti-mouse TLR2 in PBS containing 5% FCS on ice for 30 min in the dark. The cell suspensions were washed two times and resuspended in 0.5 mL PBS. Expression of CD3 and TLR2 were determined at the Loyola University Chicago Health Sciences Division FACS Core Facility using a six-color flow cytometer (BD FACSCanto); data were analyzed with the flow cytometry analysis software FlowJo 7.5 (Tree Star, Inc., Ashland, OR, USA).
For the analysis of p38 and ERK1/2 protein and phosphorylation, T cells were cultured in RPMI 1640 supplemented with 2 mmol/L l-glutamine, 10 mmol/L HEPES, 50 µg/mL gentamicin, 100 U/mL penicillin and 100 µg/mL streptomycin in the presence or absence of MyD88 inhibitor peptide (25 µmol/L) and TIRAP inhibitor peptide (25 µmol/L) for 3 h. T cells were subsequently stimulated with TLR2 agonist, HKLM (108 cells/mL), for 5 min followed by anti-CD3 (1 µg/mL) for 10 min. Following stimulation, T cells were lysed in lysis buffer containing 50 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L EDTA, 100 mmol/L NaF, 1 mmol/L MgCl2, 10 mmol/L Na4P2O7, 200 µmol/L Na3VO4, 0.5% Triton X-100 and 10% glycerol on ice for 45 min to 1 h, as previously described (16,17). Lysates were centrifuged and supernatants were harvested and stored at −70°C until analysis. For the analysis of p38 and ERK protein and phosphorylation, an equal amount of protein from each T-cell lysate preparation was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon polyvinylidine fluoride membranes by using a semidry Trans-Blot system (Bio-Rad, Hercules, CA, USA), as previously described (16,17). The membranes were saturated with blocking buffer (10 mmol/L Tris and 150 mmol/L NaCl [TBS], 0.05% Tween 20 supplemented with 5% dry milk) for 2 h at room temperature and incubated with the desired primary antibody (1/1000 dilution) at 4°C overnight. The membranes were washed five times with TBS supplemented with 0.05% Tween 20 (TBST) and then incubated with a secondary anti-body conjugated with horseradish peroxidase (1/2000 dilution) for 1 h at room temperature. The membranes were washed five times with TBST, probed using Western Lightning Plus-ECL (PerkinElmer Inc., Waltham, MA, USA), and autoradiographed (16,17). Membranes were stripped with Western blot Stripping Buffer (Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. After five washes with TBST, the membranes were reprobed with desired antibodies or β-actin antibody for loading control. Representative blots shown in the Results section come from the same membranes, which may have multiple samples from the various experimental groups.
The data, wherever applicable, are presented as means ± standard error of the mean (SEM) and were analyzed with analysis of variance (ANOVA) with Tukey post hoc test or Student t test (In-Stat; GraphPad Software Inc., La Jolla, CA, USA). P < 0.05 was considered statistically significant.
TLR Agonists Modulate T-Cell IFN-γ Production, but Not IL-2, following EtOH Intoxication Combined with Burn Injury
Activation of TLR2 Directly Prevents Suppression of T-Cell IFN-γ Production following EtOH Intoxication and Burn Injury
Expression of TLR2 on T Cells following EtOH Intoxication and Burn Injury
MyD88 and TIRAP Are Required for TLR2-Mediated T-Cell IFN-γ Production following EtOH Intoxication and Burn Injury
TLR2 Modulates T-Cell p38/ERK Signaling following EtOH Intoxication and Burn Injury
TLR2-Mediated T-Cell IFN-γ Production Utilizes p38/ERK Signaling following EtOH Intoxication and Burn Injury
Recent studies demonstrate that certain TLRs are expressed on various T-cell subsets, including memory T cells, natural killer T cells (NKTs) and regulatory T cells (Treg) (33, 34, 35). In addition, TLR ligands function as costimulatory factors to induce T-cell activation (36,37). In this study, we determined whether stimulation of TLR2, 4, 5 and 7, with specific TLR agonists, can modulate the suppression of T-cell responses after EtOH intoxication and burn injury. To clearly delineate the role of TLR expression and function on T cells, we isolated T cells from MLN and spleen by using immunomagnetic beads and confirmed T-cell purity by flow cytometry. We observed that T cells cultured in the presence of plate-bound anti-CD3 and TLR2, 4 or 5-specific agonists prevented the decrease in T-cell IFN-γ production in MLN, as well as in splenic T cells obtained from mice receiving EtOH intoxication and burn injury. TLR2 agonist was also found to significantly increase IFN-γ production in the sham vehicle group. Moreover, in the absence of CD3 stimulation, only TLR2 agonist prevented the suppression of IFN-γ production in splenic T cells following EtOH intoxication and burn injury. In contrast to IFN-γ, the effect of TLR ligands on T-cell IL-2 release was found to be organ specific. In MLN T cells, agonists for TLR2, 4, 5 and 7 prevented EtOH-plus-burn-induced suppression of IL-2 secretion in MLN. Similar treatment of splenic T cells with TLR agonists (except TLR7 agonist) did not significantly influence the IL-2 secretion following EtOH and burn injury. Moreover, these agonists caused a significant decrease in IL-2 release in the sham vehicle group. TLR7, on the other hand, increased IL-2 secretion by splenic T cells following EtOH and burn injury. The exact mechanism of such dichotomy in IL-2 release by two different lymphoid organs (MLN versus spleen) in healthy and injured conditions remains unknown at this time and is a limitation of this study.
We further explored the relationship between CD3 stimulation and TLR2 activation in the modulation of IFN-γ production. We observed that in the absence of CD3 stimulation, T cells cultured with TLR2 ligand demonstrated increased IFN-γ production compared with T cells cultured with media alone, in both sham and injured mice. Moreover, CD3 stimulation greatly augmented this response, increasing IFN-γ levels more than 13fold compared with T cells cultured with TLR2 ligand alone, suggesting that TLR2 activation directly modulates T-cell IFN-γ production and that activation of CD3 has a synergic effect on this response. Furthermore, in the absence of CD3 stimulation, TLR 4, 5 and 7 agonists fail to induce IFN-γ production. Consistent with our findings, Xu et al. observed that activated CD4+ T cells express similar levels of TLR2 and TLR4. Yet, when cultured with anti-CD3 with/without Pam3Cys-SK (TLR2 ligand) or LPS (TLR4 ligand), only Pam3Cys-SK increased T-cell proliferation and IFN-γ production (20). Similarly, recent studies have indicated that CD3/CD28-activated CD8+ T cells are functionally responsive to direct stimulation by TLR2 ligand Pam3CYs (38). In the absence of specific antigen, TLR2 acts as a costimulatory receptor on CD8+ T cells to directly control memory CD8+ T-cell proliferation and IFN-γ production (39). Moreover, T cells isolated from TLR2-deficient mice failed to respond to Lip-Ospa, a prototypic lipoprotein known to induce T-cell proliferation and IFN-γ production (40). Anti-CD3-activated T cells from TLR2−/− mice also did not respond to Pam3CYs, even in the presence of 10% of wild-type APCs, emphasizing the role of TLR2 signaling on T-cell immunity (20). TLR2 activation on T cells was further validated when Imanishi et al. indicated that TLR2 directly induces T-helper 1 (Th1) IFN-γ production in the absence of TCR stimulation. Treatment of Th1 cells with cyclosporine A (CsA), an inhibitor of TCR-mediated calcineurin activation, did not affect the TLR2-induced IFN-γ production (41). Together, these findings suggest that TLR2 modulates T-cell IFN-γ production independent of TCR activation. Our data fall in line with these findings, as we demonstrate that TLR2 activation not only modulates, but also prevents suppression of T-cell IFN-γ production following EtOH and burn injury. Our findings further demonstrate a role for TCR activation, because CD3 stimulation acted in synergy with TLR2 to modulate T-cell IFN-γ release.
In determining the mechanism by which TLR2 activation modulates IFN-γ production, we determined the levels of TLR2 expression on isolated T cells, and analyzed whether this expression was altered following EtOH and burn injury. We found that only ~1% of freshly isolated CD3+ T cells express TLR2. Interestingly, TLR2 expression increases to ~30% of CD3+ T cells following 16 h in culture conditions, irrespective of CD3 stimulation or injury. Lee et al. previously reported that TLR2 expression on Listeriaspecific CD8+ memory T cells did not require CD3 prestimulation (42). Because EtOH and burn injury did not influence TLR2 expression on T cells, we analyzed the effect of combined insult on the TLR2 signaling pathway.
Previous studies have demonstrated that T cells can be activated by PAMPs, and that TLR2 recognizes the largest spectrum of PAMPs among the TLRs (18,43,44). TLR-mediated responses are controlled mainly by the MyD88-dependent signaling pathway, which has been conserved by all described TLR members, except TLR3. The stimulation of cells with TLR ligands recruits adaptor proteins containing a TIR domain, such as MyD88 and TIRAP, to cell surface receptors. MyD88 contains a death domain, which allows it to recruit IL-1R-associated kinase 4 (IRAK4) and IRAK1 through a homophilic interaction of the death domains. The interaction between the two death domains results in phosphorylation of IRAKs, and formation of a complex with TNFR-associated factor 6 (TRAF 6). TRAF 6 then recruits TGF-β-activated kinase 1 (TAK1) and TAK1 binding proteins (TAB1, TAB2 and TAB3 complex). Activated TAK1 induces the activation of Iκ kinase α (IKK-α), IKK-β, IKK-γ and NF-κB, which subsequently results in the translocation of NF-κB into nucleus to induce target gene expression. In addition, activated TAK1 also induces activation of MAPK kinase 6 (MKK6), which modulates activation of the MAPK pathway (18,44). Thus, MyD88 and TIRAP are key adaptor molecules in the classically described TLR pathway. However, the majority of TLR signaling studies have focused on the role of this pathway in innate immune cell responses. In this study, we determined whether similar pathways are involved in adaptive T-cell responses following EtOH intoxication and burn injury. We observed that purified T cells cultured with anti-CD3 and TLR2 ligand in the presence of high doses (100 µmol/L) of MyD88 or TIRAP inhibitory peptides completely diminished TLR2-mediated IFN-γ production both in sham and burn-injured mice. Culturing T cells in the presence of anti-CD3, TLR2 ligand and a low dose of MyD88 (25 µmol/L) or TIRAP (25 µmol/L) inhibitory peptides was not sufficient to diminish TLR2-mediated IFN-γ production in sham mice, but did diminish TLR2-mediated IFN-γ production in EtOH and burn-injured mice. This finding suggests that T cells from the EtOH burn group are more sensitive to inhibition of MyD88 and TIRAP than cells from the sham vehicle group because the effect of MyD88/TIRAP inhibitors at 25 µmol/L is significant in the EtOH burn group compared with the sham vehicle group. Moreover, higher concentrations of MyD88/TIRAP inhibitors are required to inhibit cells from the sham vehicle group. Although the exact cause for increased sensitivity of T cells from the EtOH burn group to MyD88/TIRAP inhibitors remains to be established, it is likely that the combined insult perturbs TLR2 signaling. Consistent with our finding, Tomita et al. reported that CD4+ T cells from MyD88−/− animals showed lower proliferation and less IFN-γ production compared with wild-type CD4+ T cells in a murine model of chronic colitis (45).
Several lines of evidence indicate that MAPKs (for example, p38 and ERK) play a central role in T-cell activation, proliferation and subsequent differentiation into Th cells. A recent study from our laboratory indicated that acute EtOH intoxication combined with burn injury suppresses T-cell IL-2 and IFN-γ production by inhibiting p38 and ERK phosphorylation. Moreover, our study demonstrates that IL-12 modulates T-cell IL-2 and IFN-γ production by utilizing the p38 and ERK pathways, following EtOH and burn injury. In this current study, we observed that treatment of T cells with MyD88 or TIRAP inhibitor, in the presence of TLR2 agonist and anti-CD3, significantly decreased p38 and ERK activation, compared with T cells stimulated with TLR2 agonist and anti-CD3, following EtOH and burn injury. Moreover, treatment of T cells with p38 or ERK inhibitor, and subsequent stimulation with TLR2 agonist, abolished TLR2-mediated IFN-γ production in both sham and burn-injured mice, irrespective of CD3 activation. Consistent with our findings, Imanishi et al. demonstrated that IFN-γ production was severely impaired in MyD88−/− and IRAK4−/− Th1 cells stimulated with TLR2 agonist Pam3, even in the presence of IL-2. IL-2 and IL-12 enhance TLR2-mediated IFN-γ production in Th1 cells by activation of MAPKs. Yet, Pam3-induced activation of p38 and ERK was also impaired in MyD88−/− and IRAK4−/− Th1 cells (41). Because TLR2 and CD3-induced TCR activation in T cells are p38/ERK-dependent, there may be crosstalk between the TLR2 and TCR signaling pathways. TLR2 has a synergistic effect with CD3 activation and may play a critical role in T-cell IFN-γ production following EtOH and burn injury.
In this study, our results indicate that in conjunction with CD3 stimulation, several TLR ligands enhance T-cell IFN-γ production following EtOH intoxication and burn injury, a response that does not require the presence of APCs. Our results also show that the TLR2 agonist directly prevents the suppression of T-cell IFN-γ production following injury, irrespective of CD3 stimulation. However, the TLR2 agonist has a synergistic effect with CD3 activation on T-cell IFN-γ release. It might be that the activated T cells, such as those from EtOH and burn-injured mice, are more sensitive to PAMPs. Furthermore, we demonstrate that MyD88/TIRAP-dependent pathways play a critical role in TLR2-mediated T-cell activation, through their modulation of p38 and ERK, following EtOH and burn injury. Our findings provide a novel role for TLR2 in T-cell activation and function, and provide an understanding of TLR2-mediated T-cell responses following EtOH intoxication combined with burn injury.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This study was supported by National Institutes of Health (NIH) grants R01AA015731, R01AA015731-04S1 and in part by the Dr. Ralph and Marian C. Falk Medical Research Trust. JL Rendon was supported by NIH grants F30AA020167, T32AA013527 and the Loyola University Chicago Stritch School of Medicine Combined MD/PhD Program.
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