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Alcohol and inflammation and immune responses: Summary of the 2005 Alcohol and Immunology Research Interest Group (AIRIG) meeting
Alcohol 38 (2006) 121e125
Review
Alcohol and inflammation and immune responses: Summary of the 2005 Alcohol and Immunology Research Interest Group (AIRIG) meeting Thomas J. Waldschmidta,*, Robert T. Cooka, Elizabeth J. Kovacsb a Department of Pathology, The University of Iowa, Carver College of Medicine, 1038 ML, Iowa City, IA 52242, USA Department of Surgery, Burn and Shock Trauma Institute, Loyola University Medical Center, Maywood, IL 20153, USA Received 17 April 2006; received in revised form 4 May 2006; accepted 5 May 2006
b
Abstract The 10th annual meeting of the Alcohol and Immunology Research Interest Group (AIRIG) was held at Loyola University Medical Center, Maywood, Illinois on November 18, 2005. The AIRIG meeting was held to exchange new findings and ideas regarding the profound suppressive effects of alcohol exposure on the immune system. The event consisted of five sessions, two of which featured plenary talks from invited speakers, two with oral presentations from selected abstracts, and a final poster session. Participants presented a range of novel information focused on ethanol-induced effects on innate and adaptive immunity after either acute or chronic exposure. In particular, participants offered insights into the negative effects of ethanol on the innate processes of adhesion, migration, inflammation, wound repair, and bone remodeling. Presentations also focused on the means by which ethanol disrupts activation of macrophages and dendritic cells (DC), especially stimulation mediated by Toll-like receptor ligands. Additional talks provided new data on the means by which ethanol suppresses adaptive immunity, with an emphasis on DC-mediated activation of T cells, effector T cell activity, and T cell-driven B cell responses. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Alcohol; Inflammation; Immunology; Dendritic cells; Macrophages; Cytokines
1. Introduction It is increasingly clear that alcohol abuse has a major impact on both the innate and adaptive arms of the immune response. The innate immune system is designed to prevent colonization of tissues by pathogens and consists of granulocytes, monocytes/macrophages, and natural killer cells. Adaptive immunity is focused on eliminating organisms that have penetrated sterile portions of the body, and uses B cells and T cells that have been alerted to the presence of pathogens by dendritic cells (DC). Ethanol-induced dysfunction within the immune system has a range of deleterious effects on human health, including major elevations in the rates of infectious disease (Cook, 1998; Happel & Nelson, 2005; MacGregor & Louria, 1997; Nelson & Kolls, 2002; Szabo, 1999). Investigators are not only making progress documenting the nature and range of immune lesions, but are also beginning to identify the means by which ethanol exposure leads to impairment. This is particularly true with innate immunity, where current research is leading to a better understanding of alcohol-induced * Corresponding author. Tel.: þ1-319-335-8223; fax: þ1-319-3358453. E-mail address: [email protected] (T.J. Waldschmidt). 0741-8329/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2006.05.001
dysfunction in granulocytes, macrophages, and DC as well as innate processes such as leukocyteeendothelial interactions, inflammation, and tissue remodeling (Happel & Nelson, 2005; Messingham et al., 2002; Nagy, 2003). Although it is well understood that long-term alcohol abuse severely damages immune function, the field of alcohol research has gained an appreciation that acute ethanol exposure in the form of binge drinking can also compromise protective immunity (Bagby et al., 1998; Boe et al, 2003; D’Souza et al., 1995; Faunce et al., 2003). However, the means by which acute and chronic ethanol exposure disrupt the immune system are likely to differ, with acute or binge drinking having a prominent effect on innate immunity and long-term intake leading to alterations in both the innate and adaptive systems. As such, greater attention is being given to the development and characterization of animal models that mimic acute versus chronic alcohol abuse. Using these models, researchers in the field are becoming better equipped to approach mechanistic questions. To further explore these issues, the 10th meeting of the Alcohol and Immunology Research Interest Group (AIRIG) was held at Loyola University Medical Center on November 18, 2005. The meeting was supported by an R13 grant from the National Institute on Alcohol Abuse and Alcoholism
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(AA016057) and by funds from the Department of Surgery at Loyola University Medical Center. The meeting was organized by Dr. Elizabeth J. Kovacs and Dr. Luisa A. DiPietro (Loyola University Medical Center), Dr. Lou Ann S. Brown (Emory University School of Medicine), Dr. Robert T. Cook (University of Iowa College of Medicine), and Dr. Thomas R. Jerrells (University of Nebraska Medical Center). The day consisted of five sessions, two of which featured invited speakers and two structured around short presentations of selected abstracts, as well as a poster session. Abstracts for all oral and poster presentations were previously published in this journal (Alcohol 36, 127e135).
2. Alcohol and inflammation The first plenary session, chaired by Dr. Lou Ann Brown, focused on the effects of ethanol in innate immunity and inflammation. Dr. Luisa DiPietro detailed the effects of prior ethanol exposure on dermal wound healing using both in vivo and in vitro systems. Using animal models, previous work from Dr. DiPietro and colleagues demonstrated a delay in wound healing after ethanol exposure, with a decrease in both wound collagen content and vascularity (Radek et al., 2005). Dr. DiPietro presented new data showing that endothelial cells within the wound beds of ethanol-treated mice expand poorly compared to those of the controls, thus leading to inefficient formation of new vasculature and a hypoxic environment. Studies examining levels of proangiogenic factors found normal to elevated levels of vascular endothelial growth factor (VEGF). Although the latter finding was surprising, in vitro experiments testing the effects of alcohol on endothelial cells demonstrated suboptimal responses to VEGF due to lower expression of VEGF receptor-2. Taken together, these findings demonstrate a range of ethanol-induced effects on cells and growth factors within wound beds that lead to diminished angiogenesis, inefficient vessel formation, localized hypoxia, and delayed tissue remodeling. Dr. Christine Metz from The Feinstein Institute for Medical Research discussed the ability of acute alcohol exposure to disrupt endothelial cell activation and leukocyte recruitment in response to inflammatory stimuli. Using the dorsal air pouch model in mice, Dr. Metz and colleagues demonstrated a significant reduction in the accumulation of inflammatory cells in the air pouches following alcohol injection (compared with control animals) in response to Toll-like receptor (TLR) ligands (Saeed et al., 2004). This result was also observed using the Schwartzman reaction model, in which the ears of ethanol-treated mice exhibited poor leukocyte infiltration with reduced expression of activation markers by endothelial cells. Similar to the findings observed with whole animals, acute alcohol treatment of human microvascular endothelial cells in vitro inhibited endothelial cell activation following exposure to proinflammatory cytokines or TLR ligand. Finally, using
an Escherichia coli-induced murine sepsis model, significantly lower survival rates were observed in mice following acute ethanol treatment, a result associated with increased bacterial counts in target organs. Preliminary data suggest the role of alcohol in modifying signal transducer and activator of transcription 3 (STAT3) activation in endothelial cells during inflammation. These data clearly underscore the serious nature of acute alcohol exposure (possibly through binge drinking) on recruitment of innate immunity to sites of infection and subsequent inflammatory responses. The last presentation in this session was given by Dr. Joanna Goral of the Chicago College of Osteopathic Medicine and focused on the effects of acute ethanol on TLR signaling in macrophages. Using a mouse model, activation of freshly derived macrophages was altered after a single injection of ethanol (Goral & Kovacs, 2005). Specifically, macrophages were harvested from mice 3 h after ethanol exposure and activated with ligands for TLR 2, 4, or 9 in vitro. Compared to macrophages from control mice, macrophages from ethanol-exposed mice produced lower levels of interleukin (IL)-6 and tumor necrosis factor (TNF) after TLR engagement, a result accompanied by compromised p38 and ERK1/2 activation. Further studies demonstrated that pharmacologic inhibitors of ser/thr protein phosphatases type 1 and type 2A, enzymes capable of deactivating MAP kinases, elevated p38 and ERK1/2 activation, as well as cytokine production, in ethanol-exposed macrophages after TLR 4 and 9 but not TLR 2 stimulation. These findings contribute toward our understanding of not only the mechanisms that ethanol uses to disrupt signal transduction pathways, but also the complex nature of TLR signaling on cells of the innate immune system. 3. Alcohol and immunity The second plenary session was chaired by Dr. DiPietro, and centered on the effects of ethanol on induction and maintenance of adaptive immunity. Dr. Pranoti Mandrekar of the University of Massachusetts Medical Center discussed how acute ethanol exposure affects the ability of myeloid-derived DC to activate T cells (Mandrekar et al., 2004). In this study, the investigators used two methods to obtain ethanol-exposed myeloid-derived DC. In the first (in vitro exposure), peripheral blood monocytes were harvested from normal donors and cultured for 1 week with granulocyte/macrophage colony-stimulating factor (GMCSF) and IL-4 in the presence of ethanol. In the second (in vivo exposure), monocytes from subjects who consumed moderate levels of alcohol were cultured with cytokine cocktail. Whether exposed to ethanol in vitro or in vivo, myeloid-derived DC induced suboptimal T cell activation in combination with either allogeneic or recall antigen stimulation. The poor functional activity of ethanol-exposed DC was accounted for by lower expression of CD80 and CD86 and decreased IL-12 production. Of interest, these investigators found that T cells stimulated with ethanol-treated
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DC not only exhibited poor expansion and cytokine production, but also remained hyporesponsive when restimulated with control DC. Further studies examined the functional capacity of plasmacytoid DC after ethanol exposure and demonstrated this population to also exhibit lower cytokine secretion (IFN-a) after stimulation. The second and third papers in this session were presented by Dr. Thomas Waldschmidt and Dr. Annette Schlueter, respectively, from The University of Iowa. These investigators used a chronic model of ethanol intake originally described by Meadows and coworkers (Meadows et al., 1989; Spitzer & Meadows, 1999; Zhang & Meadows, 2005) and expanded upon by Cook and colleagues (Cook et al., 2004; Song et al., 2002; Zhu et al., 2004) in which mice are acclimated to 20% ethanol in drinking water (as the sole source of fluid) while having full access to conventional rodent chow. After acclimation, it was reported that mice can be kept on 20% ethanol for a year or more allowing for examination of immune status after chronic, as opposed to acute, exposure. Dr. Waldschmidt presented data demonstrating that mice tolerate extended ethanol exposure without losing body mass or exhibiting overt signs of systemic stress. Dr. Waldschmidt and colleagues further examined the B cell compartment of mice after extended periods of ethanol exposure (up to 32 weeks) and found progressive loss of mature B cells. This loss was not due to a defect in B cell lymphopoiesis because pro- and pre-B cells in the bone marrow were normal as was B cell output. As expected, loss of peripheral B cells was accompanied by diminished antibody production after either T cell-dependent or -independent antigen challenge. Final studies examining the microanatomy of peripheral lymphoid organs revealed disrupted morphology of B cell and T cell zones, suggesting a possible mechanism for peripheral lymphocyte attrition. Using the same chronic model of ethanol intake, Dr. Schlueter tested the functional capacity of various DC populations. Although freshly isolated splenic DC from ethanol-fed mice expressed normal baseline levels of co-stimulatory molecules, upregulation of these proteins proved to be erratic after stimulation with either TLR ligands or TNFa. Consistent with these findings, DC from chronically exposed mice inefficiently stimulated naı¨ve T cells compared to those of the control mice, whether using an allogeneic or peptide activation system. Further experiments examined Langerhans cells in mice after long-term ethanol intake, and revealed a decreased number of these cells in the skin. Of interest, the kinetics of the migration of Langerhans cells to regional lymph nodes was altered in ethanol mice after topical application of antigen. Thus, in addition to suboptimal T cell stimulatory ability, DC in mice exposed to long-term ethanol may also exhibit poor migration properties. 4. Short presentations As a complement to the plenary speakers, two sessions were organized to feature podium presentations selected
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from a number of abstracts submitted by meeting participants. As with the plenary sessions, these presentations covered a range of topics and focused on both innate and adaptive immunity. Dr. Pratibha Joshi from Emory University led off a number of talks on innate immunity and discussed work in the rat showing a negative effect of chronic ethanol intake on GM-CSF receptor expression on alveolar macrophages. This was accompanied by decreased levels of PU.1, a key transcription factor that drives a number of essential GM-CSF-dependent macrophage functions (Joshi et al., 2005). Importantly, they found that delivery of GM-CSF into the upper airway of ethanol-fed animals overcame the defect and restored both PU.1 levels and macrophage function. Qun Dai of the Louisiana State University Health Science Center presented research exploring the effect of acute ethanol exposure on macrophage TLR 4 cluster formation. This group had previously demonstrated that ethanol altered lipopolysaccharide (LPS)mediated partition of CD14 into lipid rafts on macrophage membranes (Dai et al., 2005). Using confocal microscopic analysis, new experiments showed LPS to direct colocalization of TLR4 and CD14 on the membrane along with cytoplasmic actin reorganization. Of interest, ethanol exposure diminished TLR4 and CD14 cluster formation, suggesting that alcohol may alter TLR-mediated activation by disabling receptor complex remodeling within lipid domains of the plasma membrane. Dr. John Callaci from Loyola University Medical Center reported the effects of ethanol administration on bone formation and resorption. Using an acute injection model in the rat, previous studies revealed that 3 weeks of in vivo alcohol exposure resulted in significantly decreased bone mineral density, and correspondingly, deceased bone compressive strength (Callaci et al., 2004). Based on these findings, RNA was isolated from bone of treated rats and microarray screens were performed to search for bone-specific genes that contribute to the loss of density and strength. Alcohol was found to temporally alter genes involved in bone remodeling with upregulation of bone resorption-related genes after acute binge exposure and downregulation of bone formation-related genes after more prolonged binge exposure. Concurrent antiresorptive therapy blocked alcohol-related bone damage and normalized alcohol-modulated gene expression profiles suggesting both a mechanism of alcohol-induced bone damage and a target for therapeutic intervention. A second group of short presentations focused on the ability of ethanol to alter adaptive immunity at both the inductive and effector stages. Audrey Lau from the University of Pittsburgh presented recent studies examining the effects of ethanol on murine DC function after both in vitro and in vivo exposure (Lau et al., 2006). The DC were obtained for in vitro experiments by culturing bone marrow cell suspensions with Flt3 ligand for 8 days. Ethanol was added to the cultures during this outgrowth period. DC derived from ethanol-containing cultures exhibited lower expression levels of classic co-stimulatory molecules, reduced titers
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of induced IL-12, and a suboptimal ability to stimulate na¨ıve T cells. Expression levels of the co-inhibitory molecule CD274/B7-H1 were unaffected. When testing splenic DC isolated from mice after 8 weeks of ethanol intake similar results were observed. Compared to control cells, splenic DC from alcohol-fed mice failed to display normal levels of co-stimulatory molecules when activated, and were less efficient in promoting proliferation in naı¨ve T cells. Of interest, hepatic DC from mice fed ethanol exhibited little change in co-stimulatory molecule expression and T cell stimulatory function after activation. These results thus contribute to a growing body of data demonstrating lesions in DC function after either acute or chronic ethanol exposure. Betty Young from the University of Iowa discussed the fate of activated antigen-specific CD4 and CD8 T cells in mice after long-term ethanol administration. Cook and colleagues previously demonstrated peripheral T cells from human alcoholics and chronic ethanol exposure to exhibit characteristics of nonspecific activation (Cook et al., 1995; Song et al., 2002). To specifically track and analyze antigen-activated T cells in a nontransgenic setting, Listeria monocytogenes infection was used. In this well-described system, Listeria-reactive CD4 and CD8 T cells can be enumerated at various time points postinfection by in vitro restimulation with defined MHC class I- and II-specific Listeria peptides followed by cytoplasmic staining of T cells for cytokine production. Infection of mice after 4 weeks on ethanol administration did not alter the overall frequencies and kinetics of Listeria-specific CD4 and CD8 T cells. Adoptive transfer experiments further demonstrated the Listeria-reactive memory cells obtained from mice subjected to chronic ethanol exposure to be functionally normal compared with control groups. These data suggest that even in the setting of increasing immune dysfunction, the T cell compartment retains some ability to provide protective immunity against intracellular pathogens. Dr. Martin Poenie of the University of Texas also presented work focused on aberrant activation of T cells in alcoholics. Using the Jurkat T cell line as a model system, acute in vitro ethanol exposure (in the absence of antigen) was found to activate T cells as measured by conjugate formation between Jurkat and Raji (B cell tumor) cells. These T celleB cell conjugates were CD11a dependent and required proximal signaling events in the T cell. Of interest, areas of B cell contact on the T cell membrane resembled immune synapses, similar to those observed after superantigendriven stimulation. These results offer the possibility that ethanol exposure may autonomously drive nonspecific activation of T cells leading to a state of T cell dysfunction. In addition to the scientific presentations, Dr. Denise Russo from the National Institute for Alcohol Abuse and Alcoholism (NIAAA) gave an overview of the various funding vehicles the NIH offers for trainees at all levels. Dr. Russo further summarized the NIH Roadmap initiatives supported by the NIAAA, and how investigators should seek to optimize funding opportunities through these initiatives.
Final discussion focused on the phased implementation of electronic submission of grant applications and the possibility of establishing core resources that can be shared by multiple investigators within the alcohol and immunity research community.
Acknowledgments The authors and participants gratefully acknowledge financial support for the 2005 AIRIG meeting from the NIAAA (AA016057) and Department of Surgery, Loyola Medical Center. The administrative and logistic support provided by Letta Kochalis is also greatly appreciated.
References Bagby, G. J., Zhang, P., Stoltz, D. A., & Nelson, S. (1998). Suppression of the granulocyte colony-stimulating factor response to Escherichia coli challenge by alcohol intoxication. Alcohol Clin Exp Res 22, 1740– 1745. Boe, D. M., Nelson, S., Zhang, P., Quinton, L., & Bagby, G. J. (2003). Alcohol-induced suppression of lung chemokine production and the host defense response to Streptococcus pneumoniae. Alcohol Clin Exp Res 27, 1838–1845. Callaci, J. J., Juknelis, D., Patwardhan, A., Sartori, M., Frost, N., & Wezeman, F. H. (2004). The effects of binge alcohol exposure on bone resorption and biomechanical and structural properties are offset by concurrent bisphosphonate treatment. Alcohol Clin Exp Res 28, 182–191. Cook, R. T. (1998). Alcohol abuse, alcoholism, and damage to the immune systemda review. Alcohol Clin Exp Res 22, 1927–1942. Cook, R. T., Ballas, Z. K., Waldschmidt, T. J., Vandersteen, D., LaBrecque, D. R., & Cook, B. L. (1995). Modulation of T-cell adhesion markers, and the CD45R and CD57 antigens in human alcoholics. Alcohol Clin Exp Res 19, 555–563. Cook, R. T., Zhu, X., Coleman, R. A., Ballas, Z. K., Waldschmidt, T. J., Ray, N. B., LaBrecque, D. R., & Cook, B. L. (2004). T-cell activation after chronic ethanol ingestion in mice. Alcohol 33, 175–181. Dai, Q., Zhang, J., & Pruett, S. B. (2005). Ethanol alters cellular activation and CD14 partitioning in lipid rafts. Biochem Biophys Res Commun 332, 37–42. D’Souza, N. B., Mandujano, J. F., Nelson, S., Summer, W. R., & Shellito, J. E. (1995). Alcohol ingestion impairs host defenses predisposing otherwise healthy mice to Pneumocystis carinii infection. Alcohol Clin Exp Res 19, 1219–1225. Faunce, D. E., Garner, J. L., Llanas, J. N., Patel, P. J., Gregory, M. S., Duffner, L. A., Gamelli, R. L., & Kovacs, E. J. (2003). Effect of acute ethanol exposure on the dermal inflammatory response after burn injury. Alcohol Clin Exp Res 27, 1199–1206. Goral, J., & Kovacs, E. J. (2005). In vivo ethanol exposure down-regulates TLR2-, TLR4-, and TLR9-mediated macrophage inflammatory response by limiting p38 and ERK1/2 activation. J Immunol 174, 456–463. Happel, K. I., & Nelson, S. (2005). Alcohol, immunosuppression, and the lung. Proc Am Thorac Soc 2, 428–432. Joshi, P. C., Applewhite, L., Ritzenthaler, J. D., Roman, J., Fernandez, A. L., Eaton, D. C., Brown, L. A., & Guidot, D. M. (2005). Chronic ethanol ingestion in rats decreases granulocyte-macrophage colony-stimulating factor receptor expression and downstream signaling in the alveolar macrophage. J Immunol 175, 6837–6845. Lau, A. H., Abe, M., & Thomson, A. W. (2006). Ethanol affects the generation, cosignaling molecule expression, and function of plasmacytoid and myeloid dendritic cell subsets in vitro and in vivo. J Leukoc Biol 14. (Epub ahead of print).
T.J. Waldschmidt et al. / Alcohol 38 (2006) 121e125 MacGregor, R. R., & Louria, D. B. (1997). Alcohol and infection. Curr Clin Top Infect Dis 17, 291–315. Mandrekar, P., Catalano, D., Dolganiuc, A., Kodys, K., & Szabo, G. (2004). Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production. J Immunol 173, 3398–33407. Meadows, G. G., Blank, S. E., & Duncan, D. D. (1989). Influence of ethanol consumption on natural killer cell activity in mice. Alcohol Clin Exp Res 13, 476–479. Messingham, K. A., Faunce, D. E., & Kovacs, E. J. (2002). Alcohol, injury, and cellular immunity. Alcohol 28, 137–149. Nagy, L. E. (2003). Recent insights into the role of the innate immune system in the development of alcoholic liver disease. Exp Biol Med 228, 882–890. Nelson, S., & Kolls, J. K. (2002). Alcohol, host defence and society. Nat Rev Immunol 2, 205–209. Radek, K. A., Matthies, A. M., Burns, A. L., Heinrich, S. A., Kovacs, E. J., & Dipietro, L. A. (2005). Acute ethanol exposure impairs angiogenesis and the proliferative phase of wound healing. Am J Physiol Heart Circ Physiol 289, 1084–1090.
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Saeed, R. W., Varma, S., Peng, T., Tracey, K. J., Sherry, B., & Metz, C. N. (2004). Ethanol blocks leukocyte recruitment and endothelial cell activation in vivo and in vitro. J Immunol 173, 6376–6383. Song, K., Coleman, R. A., Zhu, X., Alber, C., Ballas, Z. K., Waldschmidt, T. J., & Cook, R. T. (2002). Chronic ethanol consumption by mice results in activated splenic T cells. J Leukoc Biol 72, 1109–1116. Spitzer, J. H., & Meadows, G. G. (1999). Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption. Cell Immunol 194, 205–212. Szabo, G. (1999). Consequences of alcohol consumption on host defence. Alcohol Alcohol 34, 830–841. Zhang, H., & Meadows, G. G. (2005). Chronic alcohol consumption in mice increases the proportion of peripheral memory T cells by homeostatic proliferation. J Leukoc Biol 78, 1070–1080. Zhu, X., Coleman, R. A., Alber, C., Ballas, Z. K., Waldschmidt, T. J., Ray, N. B., Krieg, A. M., & Cook, R. T. (2004). Chronic ethanol ingestion by mice increases expression of CD80 and CD86 by activated macrophages. Alcohol 32, 91–100.
Review
Alcohol and inflammation and immune responses: Summary of the 2005 Alcohol and Immunology Research Interest Group (AIRIG) meeting Thomas J. Waldschmidta,*, Robert T. Cooka, Elizabeth J. Kovacsb a Department of Pathology, The University of Iowa, Carver College of Medicine, 1038 ML, Iowa City, IA 52242, USA Department of Surgery, Burn and Shock Trauma Institute, Loyola University Medical Center, Maywood, IL 20153, USA Received 17 April 2006; received in revised form 4 May 2006; accepted 5 May 2006
b
Abstract The 10th annual meeting of the Alcohol and Immunology Research Interest Group (AIRIG) was held at Loyola University Medical Center, Maywood, Illinois on November 18, 2005. The AIRIG meeting was held to exchange new findings and ideas regarding the profound suppressive effects of alcohol exposure on the immune system. The event consisted of five sessions, two of which featured plenary talks from invited speakers, two with oral presentations from selected abstracts, and a final poster session. Participants presented a range of novel information focused on ethanol-induced effects on innate and adaptive immunity after either acute or chronic exposure. In particular, participants offered insights into the negative effects of ethanol on the innate processes of adhesion, migration, inflammation, wound repair, and bone remodeling. Presentations also focused on the means by which ethanol disrupts activation of macrophages and dendritic cells (DC), especially stimulation mediated by Toll-like receptor ligands. Additional talks provided new data on the means by which ethanol suppresses adaptive immunity, with an emphasis on DC-mediated activation of T cells, effector T cell activity, and T cell-driven B cell responses. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Alcohol; Inflammation; Immunology; Dendritic cells; Macrophages; Cytokines
1. Introduction It is increasingly clear that alcohol abuse has a major impact on both the innate and adaptive arms of the immune response. The innate immune system is designed to prevent colonization of tissues by pathogens and consists of granulocytes, monocytes/macrophages, and natural killer cells. Adaptive immunity is focused on eliminating organisms that have penetrated sterile portions of the body, and uses B cells and T cells that have been alerted to the presence of pathogens by dendritic cells (DC). Ethanol-induced dysfunction within the immune system has a range of deleterious effects on human health, including major elevations in the rates of infectious disease (Cook, 1998; Happel & Nelson, 2005; MacGregor & Louria, 1997; Nelson & Kolls, 2002; Szabo, 1999). Investigators are not only making progress documenting the nature and range of immune lesions, but are also beginning to identify the means by which ethanol exposure leads to impairment. This is particularly true with innate immunity, where current research is leading to a better understanding of alcohol-induced * Corresponding author. Tel.: þ1-319-335-8223; fax: þ1-319-3358453. E-mail address: [email protected] (T.J. Waldschmidt). 0741-8329/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2006.05.001
dysfunction in granulocytes, macrophages, and DC as well as innate processes such as leukocyteeendothelial interactions, inflammation, and tissue remodeling (Happel & Nelson, 2005; Messingham et al., 2002; Nagy, 2003). Although it is well understood that long-term alcohol abuse severely damages immune function, the field of alcohol research has gained an appreciation that acute ethanol exposure in the form of binge drinking can also compromise protective immunity (Bagby et al., 1998; Boe et al, 2003; D’Souza et al., 1995; Faunce et al., 2003). However, the means by which acute and chronic ethanol exposure disrupt the immune system are likely to differ, with acute or binge drinking having a prominent effect on innate immunity and long-term intake leading to alterations in both the innate and adaptive systems. As such, greater attention is being given to the development and characterization of animal models that mimic acute versus chronic alcohol abuse. Using these models, researchers in the field are becoming better equipped to approach mechanistic questions. To further explore these issues, the 10th meeting of the Alcohol and Immunology Research Interest Group (AIRIG) was held at Loyola University Medical Center on November 18, 2005. The meeting was supported by an R13 grant from the National Institute on Alcohol Abuse and Alcoholism
122
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(AA016057) and by funds from the Department of Surgery at Loyola University Medical Center. The meeting was organized by Dr. Elizabeth J. Kovacs and Dr. Luisa A. DiPietro (Loyola University Medical Center), Dr. Lou Ann S. Brown (Emory University School of Medicine), Dr. Robert T. Cook (University of Iowa College of Medicine), and Dr. Thomas R. Jerrells (University of Nebraska Medical Center). The day consisted of five sessions, two of which featured invited speakers and two structured around short presentations of selected abstracts, as well as a poster session. Abstracts for all oral and poster presentations were previously published in this journal (Alcohol 36, 127e135).
2. Alcohol and inflammation The first plenary session, chaired by Dr. Lou Ann Brown, focused on the effects of ethanol in innate immunity and inflammation. Dr. Luisa DiPietro detailed the effects of prior ethanol exposure on dermal wound healing using both in vivo and in vitro systems. Using animal models, previous work from Dr. DiPietro and colleagues demonstrated a delay in wound healing after ethanol exposure, with a decrease in both wound collagen content and vascularity (Radek et al., 2005). Dr. DiPietro presented new data showing that endothelial cells within the wound beds of ethanol-treated mice expand poorly compared to those of the controls, thus leading to inefficient formation of new vasculature and a hypoxic environment. Studies examining levels of proangiogenic factors found normal to elevated levels of vascular endothelial growth factor (VEGF). Although the latter finding was surprising, in vitro experiments testing the effects of alcohol on endothelial cells demonstrated suboptimal responses to VEGF due to lower expression of VEGF receptor-2. Taken together, these findings demonstrate a range of ethanol-induced effects on cells and growth factors within wound beds that lead to diminished angiogenesis, inefficient vessel formation, localized hypoxia, and delayed tissue remodeling. Dr. Christine Metz from The Feinstein Institute for Medical Research discussed the ability of acute alcohol exposure to disrupt endothelial cell activation and leukocyte recruitment in response to inflammatory stimuli. Using the dorsal air pouch model in mice, Dr. Metz and colleagues demonstrated a significant reduction in the accumulation of inflammatory cells in the air pouches following alcohol injection (compared with control animals) in response to Toll-like receptor (TLR) ligands (Saeed et al., 2004). This result was also observed using the Schwartzman reaction model, in which the ears of ethanol-treated mice exhibited poor leukocyte infiltration with reduced expression of activation markers by endothelial cells. Similar to the findings observed with whole animals, acute alcohol treatment of human microvascular endothelial cells in vitro inhibited endothelial cell activation following exposure to proinflammatory cytokines or TLR ligand. Finally, using
an Escherichia coli-induced murine sepsis model, significantly lower survival rates were observed in mice following acute ethanol treatment, a result associated with increased bacterial counts in target organs. Preliminary data suggest the role of alcohol in modifying signal transducer and activator of transcription 3 (STAT3) activation in endothelial cells during inflammation. These data clearly underscore the serious nature of acute alcohol exposure (possibly through binge drinking) on recruitment of innate immunity to sites of infection and subsequent inflammatory responses. The last presentation in this session was given by Dr. Joanna Goral of the Chicago College of Osteopathic Medicine and focused on the effects of acute ethanol on TLR signaling in macrophages. Using a mouse model, activation of freshly derived macrophages was altered after a single injection of ethanol (Goral & Kovacs, 2005). Specifically, macrophages were harvested from mice 3 h after ethanol exposure and activated with ligands for TLR 2, 4, or 9 in vitro. Compared to macrophages from control mice, macrophages from ethanol-exposed mice produced lower levels of interleukin (IL)-6 and tumor necrosis factor (TNF) after TLR engagement, a result accompanied by compromised p38 and ERK1/2 activation. Further studies demonstrated that pharmacologic inhibitors of ser/thr protein phosphatases type 1 and type 2A, enzymes capable of deactivating MAP kinases, elevated p38 and ERK1/2 activation, as well as cytokine production, in ethanol-exposed macrophages after TLR 4 and 9 but not TLR 2 stimulation. These findings contribute toward our understanding of not only the mechanisms that ethanol uses to disrupt signal transduction pathways, but also the complex nature of TLR signaling on cells of the innate immune system. 3. Alcohol and immunity The second plenary session was chaired by Dr. DiPietro, and centered on the effects of ethanol on induction and maintenance of adaptive immunity. Dr. Pranoti Mandrekar of the University of Massachusetts Medical Center discussed how acute ethanol exposure affects the ability of myeloid-derived DC to activate T cells (Mandrekar et al., 2004). In this study, the investigators used two methods to obtain ethanol-exposed myeloid-derived DC. In the first (in vitro exposure), peripheral blood monocytes were harvested from normal donors and cultured for 1 week with granulocyte/macrophage colony-stimulating factor (GMCSF) and IL-4 in the presence of ethanol. In the second (in vivo exposure), monocytes from subjects who consumed moderate levels of alcohol were cultured with cytokine cocktail. Whether exposed to ethanol in vitro or in vivo, myeloid-derived DC induced suboptimal T cell activation in combination with either allogeneic or recall antigen stimulation. The poor functional activity of ethanol-exposed DC was accounted for by lower expression of CD80 and CD86 and decreased IL-12 production. Of interest, these investigators found that T cells stimulated with ethanol-treated
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DC not only exhibited poor expansion and cytokine production, but also remained hyporesponsive when restimulated with control DC. Further studies examined the functional capacity of plasmacytoid DC after ethanol exposure and demonstrated this population to also exhibit lower cytokine secretion (IFN-a) after stimulation. The second and third papers in this session were presented by Dr. Thomas Waldschmidt and Dr. Annette Schlueter, respectively, from The University of Iowa. These investigators used a chronic model of ethanol intake originally described by Meadows and coworkers (Meadows et al., 1989; Spitzer & Meadows, 1999; Zhang & Meadows, 2005) and expanded upon by Cook and colleagues (Cook et al., 2004; Song et al., 2002; Zhu et al., 2004) in which mice are acclimated to 20% ethanol in drinking water (as the sole source of fluid) while having full access to conventional rodent chow. After acclimation, it was reported that mice can be kept on 20% ethanol for a year or more allowing for examination of immune status after chronic, as opposed to acute, exposure. Dr. Waldschmidt presented data demonstrating that mice tolerate extended ethanol exposure without losing body mass or exhibiting overt signs of systemic stress. Dr. Waldschmidt and colleagues further examined the B cell compartment of mice after extended periods of ethanol exposure (up to 32 weeks) and found progressive loss of mature B cells. This loss was not due to a defect in B cell lymphopoiesis because pro- and pre-B cells in the bone marrow were normal as was B cell output. As expected, loss of peripheral B cells was accompanied by diminished antibody production after either T cell-dependent or -independent antigen challenge. Final studies examining the microanatomy of peripheral lymphoid organs revealed disrupted morphology of B cell and T cell zones, suggesting a possible mechanism for peripheral lymphocyte attrition. Using the same chronic model of ethanol intake, Dr. Schlueter tested the functional capacity of various DC populations. Although freshly isolated splenic DC from ethanol-fed mice expressed normal baseline levels of co-stimulatory molecules, upregulation of these proteins proved to be erratic after stimulation with either TLR ligands or TNFa. Consistent with these findings, DC from chronically exposed mice inefficiently stimulated naı¨ve T cells compared to those of the control mice, whether using an allogeneic or peptide activation system. Further experiments examined Langerhans cells in mice after long-term ethanol intake, and revealed a decreased number of these cells in the skin. Of interest, the kinetics of the migration of Langerhans cells to regional lymph nodes was altered in ethanol mice after topical application of antigen. Thus, in addition to suboptimal T cell stimulatory ability, DC in mice exposed to long-term ethanol may also exhibit poor migration properties. 4. Short presentations As a complement to the plenary speakers, two sessions were organized to feature podium presentations selected
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from a number of abstracts submitted by meeting participants. As with the plenary sessions, these presentations covered a range of topics and focused on both innate and adaptive immunity. Dr. Pratibha Joshi from Emory University led off a number of talks on innate immunity and discussed work in the rat showing a negative effect of chronic ethanol intake on GM-CSF receptor expression on alveolar macrophages. This was accompanied by decreased levels of PU.1, a key transcription factor that drives a number of essential GM-CSF-dependent macrophage functions (Joshi et al., 2005). Importantly, they found that delivery of GM-CSF into the upper airway of ethanol-fed animals overcame the defect and restored both PU.1 levels and macrophage function. Qun Dai of the Louisiana State University Health Science Center presented research exploring the effect of acute ethanol exposure on macrophage TLR 4 cluster formation. This group had previously demonstrated that ethanol altered lipopolysaccharide (LPS)mediated partition of CD14 into lipid rafts on macrophage membranes (Dai et al., 2005). Using confocal microscopic analysis, new experiments showed LPS to direct colocalization of TLR4 and CD14 on the membrane along with cytoplasmic actin reorganization. Of interest, ethanol exposure diminished TLR4 and CD14 cluster formation, suggesting that alcohol may alter TLR-mediated activation by disabling receptor complex remodeling within lipid domains of the plasma membrane. Dr. John Callaci from Loyola University Medical Center reported the effects of ethanol administration on bone formation and resorption. Using an acute injection model in the rat, previous studies revealed that 3 weeks of in vivo alcohol exposure resulted in significantly decreased bone mineral density, and correspondingly, deceased bone compressive strength (Callaci et al., 2004). Based on these findings, RNA was isolated from bone of treated rats and microarray screens were performed to search for bone-specific genes that contribute to the loss of density and strength. Alcohol was found to temporally alter genes involved in bone remodeling with upregulation of bone resorption-related genes after acute binge exposure and downregulation of bone formation-related genes after more prolonged binge exposure. Concurrent antiresorptive therapy blocked alcohol-related bone damage and normalized alcohol-modulated gene expression profiles suggesting both a mechanism of alcohol-induced bone damage and a target for therapeutic intervention. A second group of short presentations focused on the ability of ethanol to alter adaptive immunity at both the inductive and effector stages. Audrey Lau from the University of Pittsburgh presented recent studies examining the effects of ethanol on murine DC function after both in vitro and in vivo exposure (Lau et al., 2006). The DC were obtained for in vitro experiments by culturing bone marrow cell suspensions with Flt3 ligand for 8 days. Ethanol was added to the cultures during this outgrowth period. DC derived from ethanol-containing cultures exhibited lower expression levels of classic co-stimulatory molecules, reduced titers
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of induced IL-12, and a suboptimal ability to stimulate na¨ıve T cells. Expression levels of the co-inhibitory molecule CD274/B7-H1 were unaffected. When testing splenic DC isolated from mice after 8 weeks of ethanol intake similar results were observed. Compared to control cells, splenic DC from alcohol-fed mice failed to display normal levels of co-stimulatory molecules when activated, and were less efficient in promoting proliferation in naı¨ve T cells. Of interest, hepatic DC from mice fed ethanol exhibited little change in co-stimulatory molecule expression and T cell stimulatory function after activation. These results thus contribute to a growing body of data demonstrating lesions in DC function after either acute or chronic ethanol exposure. Betty Young from the University of Iowa discussed the fate of activated antigen-specific CD4 and CD8 T cells in mice after long-term ethanol administration. Cook and colleagues previously demonstrated peripheral T cells from human alcoholics and chronic ethanol exposure to exhibit characteristics of nonspecific activation (Cook et al., 1995; Song et al., 2002). To specifically track and analyze antigen-activated T cells in a nontransgenic setting, Listeria monocytogenes infection was used. In this well-described system, Listeria-reactive CD4 and CD8 T cells can be enumerated at various time points postinfection by in vitro restimulation with defined MHC class I- and II-specific Listeria peptides followed by cytoplasmic staining of T cells for cytokine production. Infection of mice after 4 weeks on ethanol administration did not alter the overall frequencies and kinetics of Listeria-specific CD4 and CD8 T cells. Adoptive transfer experiments further demonstrated the Listeria-reactive memory cells obtained from mice subjected to chronic ethanol exposure to be functionally normal compared with control groups. These data suggest that even in the setting of increasing immune dysfunction, the T cell compartment retains some ability to provide protective immunity against intracellular pathogens. Dr. Martin Poenie of the University of Texas also presented work focused on aberrant activation of T cells in alcoholics. Using the Jurkat T cell line as a model system, acute in vitro ethanol exposure (in the absence of antigen) was found to activate T cells as measured by conjugate formation between Jurkat and Raji (B cell tumor) cells. These T celleB cell conjugates were CD11a dependent and required proximal signaling events in the T cell. Of interest, areas of B cell contact on the T cell membrane resembled immune synapses, similar to those observed after superantigendriven stimulation. These results offer the possibility that ethanol exposure may autonomously drive nonspecific activation of T cells leading to a state of T cell dysfunction. In addition to the scientific presentations, Dr. Denise Russo from the National Institute for Alcohol Abuse and Alcoholism (NIAAA) gave an overview of the various funding vehicles the NIH offers for trainees at all levels. Dr. Russo further summarized the NIH Roadmap initiatives supported by the NIAAA, and how investigators should seek to optimize funding opportunities through these initiatives.
Final discussion focused on the phased implementation of electronic submission of grant applications and the possibility of establishing core resources that can be shared by multiple investigators within the alcohol and immunity research community.
Acknowledgments The authors and participants gratefully acknowledge financial support for the 2005 AIRIG meeting from the NIAAA (AA016057) and Department of Surgery, Loyola Medical Center. The administrative and logistic support provided by Letta Kochalis is also greatly appreciated.
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