PTX cruiser: driving autoimmunity via TLR4

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TRENDS in Immunology

Vol.26 No.6 June 2005

Research Focus

PTX cruiser: driving autoimmunity via TLR4 Michael K. Racke1,2, Wei Hu1 and Amy E. Lovett-Racke1 1 2

Department of Neurology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9036, USA The Center for Immunology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9036, USA

Although the cause of autoimmune diseases is unknown, it has long been speculated that an infectious agent might have a role in their initiation and progression. Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), has been used to study factors in disease pathogenesis. A recent study shows that pertussis toxin, which is used as an adjuvant in EAE, uses Toll-like receptor 4 signaling to mediate its disease-inducing effect. Introduction The role of infectious agents in precipitating or preventing autoimmune diseases remains unresolved [1]. Many have argued that an infection could act as a nonspecific trigger for the immune system, thereby initiating a disease relapse or the onset of autoimmunity [2]. The animal model for multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), has long been studied because it has many features of the human disease, particularly central nervous system (CNS) inflammation and demyelination. Interestingly, most models of EAE require the use of pertussis toxin (PTX) as part of the induction regimen. In a recent study by Kerfoot and colleagues, some of the molecular mechanisms have been elucidated in terms of the role that PTX has in EAE induction [3]. Previous studies on the role of PTX in EAE The prevailing dogma has been that PTX is required for EAE induction because it helps ‘open up’ the blood–brain barrier. Early studies showed that mice could be immunized with mouse spinal cord homogenate in complete Freund’s adjuvant (CFA) but that Bordetella organisms were also required to be administered for animals to develop the disease [4]. Subsequent studies by Kamradt et al. suggested that PTX was important in EAE induction because it helps prevent anergy induction of autoreactive T cells [5]. A recent study by Waldner et al. showed that PTX, lipopolysaccharide (LPS) and CpG DNA can break T-cell tolerance [6]. Other effects attributed to PTX include irreversible inhibition of second messengers, such as G proteins, which might subsequently affect signaling pathways that regulate T-cell differentiation in the antigen-presenting cells (APCs) or in the T cells themselves [7]. Work by Shive and colleagues showed that PTX stimulates APCs to foster Th1 differentiation [8]. However, none of these studies specifically addressed effects at Corresponding author: Racke, M.K. ([email protected]). Available online 8 April 2005 www.sciencedirect.com

the blood–brain barrier and the exact mechanism of how these microbial products exert their effects remained unknown. Several studies have examined the contribution of PTX in the EAE model. One of the most significant studies was by Goverman and colleagues [9], who used PTX when inducing EAE in myelin basic protein (MBP) T-cell receptor (TCR) transgenic mice. Interestingly, these mice have provided important insights into the role of the environment in the initiation of autoimmunity [9]. Mice that are bred in a pathogen-free environment and free from nonspecific immune stimulation are much less likely to develop autoimmunity compared to mice kept in a conventional animal colony. Studies on these mice also showed that the crucial factor in the development of EAE is that the TCR transgenic T cells need to cross the blood– brain barrier to gain access to the CNS and that administration of PTX results in the accumulation of T cells into the CNS and the onset of EAE [10]. These data are consistent with the concept that PTX ‘breaks down’ the blood–brain barrier but do not address specific mechanisms for how this occurs. Interestingly, Hofstetter et al. showed that co-injection with PTX, when immunizing SJL/J mice with either MBP or proteolipid protein 139–151 emulsified in incomplete Freund’s adjuvant (IFA), results in the mice developing EAE, demonstrating that PTX also activates APCs in the peripheral lymphoid organs and the CNS [11]. However, the effects observed were felt to be a result of the ability of PTX to inhibit signaling through G proteins, rather than signaling through Toll-like receptor 4 (TLR4), and do not address effects on the vascular endothelium itself.

PTX upregulates P-selectin by signaling through TLR4 The recent study by Kerfoot and colleagues sheds new light on the role of PTX in the EAE model and the role of the environment in the development of autoimmune disease [3]. This group used intravital microscopy of the murine microvasculature to examine the effects of PTX on lymphocyte rolling and adhesion. Intravenous injection of 250 ng of PTX, a dose commonly used in EAE studies, resulted in increased lymphocyte rolling from 5–48 h after the injection as well as increased adhesion. The authors then showed that this rolling and subsequent adhesion is due to P-selectin upregulation on the endothelial surface. They went on to show that it is the interaction of activated lymphocytes with the P-selectin that subsequently results in the increased permeability of the blood–brain barrier, providing the

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mechanism for the ‘breakdown’ of the blood–brain barrier. Perhaps the most exciting observation made in this study is that PTX effects are dependent on signaling through TLR4. The initial aspects of the immune response are mediated by TLRs and they are activated by the recognition of pathogen-associated molecular patterns [12]. TLRs are expressed on several cell types, including cells of the immune system. The authors showed that lymphocyte rolling and adhesion does not occur when PTX is injected into mice deficient in TLR4 [3]. Further studies showed that signaling events that occur in response to PTX are similar to those that occur with the TLR4 agonist LPS [3]. Using peritoneal macrophages from TLR4deficient mice, they also showed that PTX is no longer able to induce signaling events associated with TLR4 binding, suggesting that PTX-induced signaling is dependent on TLR4. TLR4 signaling ultimately results in translocation of NF-kB, which induces transcription of a variety of genes, including genes for proinflammatory cytokines and P-selectin (Figure 1). P-selectin expression on the endothelium might result from increased P-selectin gene expression or cytokine-induced release of P-selectin from Weibel-Palade bodies [13].

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Figure 1. PTX signaling through TLR4 induces P-selectin expression on the cerebrovascular endothelium. PTX binds TLR4, which signals through myeloid differentiation factor 88 (MyD88) and interleukin-1-associated kinases (IRAKs). The Ik kinase (IKK) complex becomes phosphorylated, which subsequently phosphorylates IkB, releasing NF-kB for translocation to the nucleus. NF-kB binds to kB sites of genes of proinflammatory cytokines, such as tumor necrosis factor-a (TNF-a) and possibly P-selectin, and induces expression of these genes. P-selectin expression on the cell surface might result from increased P-selectin gene expression or release of P-selectin from Weibel-Palade bodies, where it is stored. www.sciencedirect.com

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Because TLR4 appears to be responsible for PTX-induced lymphocyte rolling and adhesion, it follows that TLR4 should be necessary for the induction of EAE. However, here is where the picture gets murky. Although some experiments show a dependence on TLR4 for the induction of EAE, others do not [3]. The authors attribute the result to the fact that TLR4-independent mechanisms could be sufficient to induce EAE with PTX plus ‘environmental factors.’ The observation that PTX can signal through TLR4, upregulate P-selectin and increase lymphocyte rolling and adhesion enables a model for how PTX enhances blood– brain barrier breakdown to be established (Figure 2). However, one does not need to necessarily invoke ‘environmental factors’ for EAE induction in the absence of TLR4 because adoptive transfer models of EAE often do not use PTX. Perhaps under the conditions of active immunization, the role of PTX is more important because there are not as many activated myelin-reactive T cells trafficking through the CNS vasculature. Because PTX is known to enhance adoptively transferred EAE, it is likely that upregulation of P-selectin on the CNS endothelium results in enhanced lymphocyte accumulation in the CNS. In addition, because PTX can signal through TLR4, APC activation and the subsequent differentiation of T cells down the Th1 pathway can now also be explained through effects other than G-protein inhibition. Future studies on TLRs in EAE and MS Importantly, these findings might provide a molecular explanation for the observations that infections often precede MS exacerbations. TLRs are expressed on cerebrovascular endothelium in rodents [14,15] and TLR2 and TLR4 are expressed on human endothelium, although it remains to be shown whether TLRs are expressed on human cerebrovascular endothelial cells [16]. Thus, infections in which agonists for these receptors might be released into the circulation could result in the upregulation of P-selectin and therefore enhanced lymphocyte rolling and adhesion in the CNS vasculature. For example, in studies in which CpG DNA and LPS induce EAE [6], is P-selectin upregulated on the cerebrovascular endothelium? In addition, because TLR agonists can also activate APCs, the conditions might be ripe for the activation of myelin-reactive T cells that are recruited into the CNS in this manner (Figure 2). Thus, one could envision a scenario in which an infection could participate in the initiation of an autoimmune event, such as an MS exacerbation. Further studies will need to examine whether agonists for TLR2 and TLR4 can function similarly to PTX in the EAE model and could perhaps begin to explain the diversity of infectious agents, both viral and bacterial, that have been implicated in MS and other autoimmune diseases [17]. However, mechanisms to upregulate P-selectin might differ in murine and human systems [18]. In addition, if these TLR agonists can get through the blood–brain barrier and activate the local microglia, one could also envision these activated microglia participating in CNS injury, which has been shown in a model of hypoxia–ischemia [19]. Thus, the presence of infection

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Supported by grants from the National Institutes of Health (MKR) and National Multiple Sclerosis Society (MKR, AELR).

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Figure 2. Mechanism of PTX effects in EAE. (a) The resting CNS endothelium does not express P-selectin but it does have tight junctions to help form the blood–brain barrier. T cells do not interact with this resting endothelium. (b) Injection of PTX results in stimulation of the endothelium through TLR4. The endothelium upregulates P-selectin, which results in increased rolling of activated T cells on the endothelial cell surface. (c). Rolling T cells are subsequently captured by adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1). (d).Activated T cells then cross the CNS endothelium. With the breakdown in the blood–brain barrier, PTX can also activate the perivascular microglia cells, which are located next to the endothelium and participate in the inflammatory response.

might not only initiate an episode of autoimmunity but might also participate in regulating the severity of injury that could subsequently occur. The demonstration that PTX signals through TLR4 is an important step that will lead to further studies on how other infectious agents can participate in ‘driving autoimmunity’ in several autoimmune diseases, in addition to those of the CNS.

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