Macrophage CD40 signaling: A pivotal regulator of disease protection and pathogenesis

Seminars in Immunology 21 (2009) 257–264

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Seminars in Immunology journal homepage: www.elsevier.com/locate/ysmim

Review

Macrophage CD40 signaling: A pivotal regulator of disease protection and pathogenesis Jill Suttles ∗ , Robert D. Stout Department of Microbiology and Immunology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Louisville, KY 40292, USA

a r t i c l e Keywords: CD40 CD154 Macrophage Monocyte Inflammation Cell signaling

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a b s t r a c t Macrophages reside in all tissues as resident populations and as immigrants recruited in response to tissue injury, inflammation or pathogen invasion. Under normal conditions, macrophages contribute to tissue homeostasis and provide innate immune surveillance. Both macrophages and their progenitors, bone marrow-derived monocytes, constitutively express the tumor necrosis factor receptor superfamily member, CD40, and are capable of a robust response to CD40 ligation resulting in the induction or enhancement of expression of genes with a predominantly pro-inflammatory function. CD40 signaling in macrophages in the context of host responses to pathogens plays a crucial role in host defense. However, macrophage responses to CD40 ligation in the context of autoimmune and cardiovascular disease contribute to disease pathogenesis. In this review, we discuss the role of CD40 in both protective and destructive processes, including the signaling pathways engaged and the factors capable of modulating CD40 signal transduction. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Macrophages reside in all tissues of the body where they provide innate immune surveillance and contribute to tissue homeostasis. Macrophages originate from bone marrow-derived mature monocytes that migrate to tissues in response to chemokine signaling, either for replenishment of the tissue resident population, or in response to tissue insult [1–4]. Upon entry into tissues monocytes/macrophages acquire unique functional profiles in response to the tissue environment. Even within a tissue, macrophages display heterogeneity dependent on the microanatomical location in that tissue [5–7]. This feature of macrophages is representative of their remarkable plasticity and range of activities that can be initiated and maintained by environmental signals [8]. Macrophages are capable of a highly diverse repertoire of functional activities, including phagocytosis, generation of microbicidal oxygen and nitrogen radicals, production of metalloproteinases, and production of both inflammatory and anti-inflammatory cytokines as well as factors promoting tissue growth and angiogenesis [8]. Both monocytes in circulation and macrophages in tissues are exposed to a wide variety of potential stimuli, including cytokines, chemokines, hormones, lipid mediators, and immunoglobulins, all of which have the capacity to modulate macrophage behavior. In addition to these various soluble factors, it was recognized over two decades ago that

∗ Corresponding author. Tel.: +1 502 852 5144; fax: +1 502 852 7531. E-mail address: [email protected] (J. Suttles). 1044-5323/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2009.05.011

monocytes and macrophages can be activated solely via cell contact with activated T cells [9,10]. A number of receptor–ligand pairs were reported to contribute to this activity, including membrane TNF␣ [11]. However, it was subsequently found that T cells that do not express membrane TNF␣ are equally effective in contact-dependent stimulation of macrophages [12]. Although previously considered as primarily a B cell surface receptor, CD40 was then viewed as a likely candidate receptor on monocytes/macrophages for the signal received by activated T cells. The expression of CD40 on human monocytes and murine macrophages and its functional ligation via CD154 expressed on activated CD4+ T cells was revealed [13–15], and followed by the conclusive finding that CD4+ T cells isolated from mice deficient for expression of CD154 were greatly impaired in ability to activate macrophage TNF␣ expression and generation of nitric oxide [16]. The discovery of the CD40:CD154 interaction as a mechanism of macrophage activation greatly expanded the view of the contribution of CD40 signaling to infectious and autoimmune inflammatory diseases. The subsequent identification of CD154 on platelets, mast cells and basophils [17,18], and the discovery of a soluble form of CD154 [19], further extend the possible scenarios in which ligation of CD40 expressed by monocytes and macrophages may play a role in disease progression or management. 2. Functional outcomes of macrophage CD40 stimulation The first studies of monocyte and macrophage CD40 responses revealed a primarily pro-inflammatory outcome of CD40 signal transduction. Monocytes and macrophages for the most part

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display similar functional outcomes to CD40 ligation, although the level of responsiveness may differ. Differences in responses based on the tissue source of cells are also apparent and likely reflect the priming effect of the tissue environment. Even within tissues or organs, macrophage responses may vary based on location within that tissue or organ. For example, T cell contact-dependent activation of lung tissue macrophages, but not alveolar macrophages, induces expression of matrix metalloproteinases [20]. Ligation of CD40 on monocytes and macrophages results in the efficient induction of synthesis of proinflammatory cytokines including IL-1␣ and IL-1␤ [14], TNF␣ [16,21], IL-6 [13,21,22], IL-12 [23,24], as well as chemokines, including IL-8 [21], CCL2, CCL3, CCL4 and CCL5 [25]. Ligation of CD40 on monocytes/macrophages also upregulates expression of MHC class II and co-stimulatory molecules CD80 and CD86, as well as CD40, itself [21]. Other effector functions of monocytes/macrophages induced via CD40 stimulation include the expression of matrix metalloproteinases (as mentioned above) [20,26–28] and production of nitric oxide (NO) [15,16]. In addition, CD40 stimulation of monocytes has also been shown to rescue them from apoptosis induced via serum deprivation [29]. Thus, upon exposure to CD154, monocytes and macrophages can be directed towards a functional phenotype that promotes antigen presentation and proinflammatory activity. However, many of the studies examining the functional outcomes of CD40 stimulation of macrophages have been performed in vitro, therefore not accounting for the contribution of the tissue environment. Studies using soluble forms of CD154, T cell membranes or fixed T cells do not take into consideration the cytokine milieu. For example, both Th1 and Th2 clones have been shown to activate macrophages [10,12,30], yet the outcome is quite different due to the presence or absence of IFN␥ or IL-4/IL-10. IFN␥ provides a second stimulus for the production of NO, thus, typically, in its absence CD40 ligation will not result in NO production [10]. The presence of IL-4 and IL-10, however, will downregulate the proinflammatory influence of CD40 ligation [31]. Stimulus of CD40 on macrophages in tissues with a high TGF␤ content will also have unique consequences. For example, many tumor types express CD154 as well as secrete TGF␤. Tumor-associated macrophages (TAMs) are maintained in a tumor-supportive state and there is evidence that CD40 signaling may contribute to this unique state of activation of TAMs (Stout and Suttles, unpublished observations). In contrast, treatment of tumor-bearing mice with an agonistic anti-CD40 mAb activates macrophage cytostatic activity and suppresses tumor cell growth [32,33]. Thus, the nature of the delivery of CD40 stimulus, and the tissue environment, result in variable consequences that can be either protective from disease or contribute to disease progression. As we discuss later, the CD40:CD154 interaction involving T cells is reciprocal and contributes to the functional plasticity of both cell types, thus directing, or re-directing, immune responses. 3. Monocyte/macrophage CD40 signal transduction Despite the heterogeneity of monocytes and macrophages, analysis of expression of CD40 on circulating monocytes (human) or in vitro generated murine macrophages by flow cytometry typically reveals homogeneous expression of CD40 that can be upregulated by cytokine stimulus [13,14]. Despite the fairly low levels of CD40 expression exhibited by resting monocytes/macrophages, these cells are readily activated by CD40 ligation. However, the requirements for activation of monocytes/macrophages through CD40 appear to be more stringent than reported for B cells, in that typically agonistic anti-CD40 IgG antibodies, alone, are not efficient activators of signal transduction in monocytes/macrophages, nor are monomeric forms of soluble CD154. More extensive crosslinking is required, such as can be achieved by use of IgM anti-CD154 antibody, cross-linking of anti-CD40 antibodies with

anti-Ig antibody, use of activated T cell membrane preparations, CD154 transfectants, or multimeric soluble forms of the ligand [14,22,34]. Possibly due to the limited availability of reagents that effectively activate macrophage-expressed CD40, studies of CD40 signaling in monocytes/macrophages are not extensive. Two independent early studies of monocyte/macrophage CD40 signaling revealed a tyrosine kinase-dependent pathway leading to activation of the mitogen-activated protein kinases (MAPKs) ERK1/2 which proved critical to the induction of proinflammatory cytokine and chemokine expression (i.e., IL-1, TNF␣, IL-6, and IL-8) [31,35]. Low-level CD40-mediated activation of the MAPK Jun-N-terminal kinase (JNK) was found in one of these studies [35], whereas both reports found no enhancement of MAPK p38 phosphorylation. Later studies reported p38 activation in monocytes in response to soluble CD154 stimulation and demonstrated a role of p38 in the induction of iNOS, COX-2 and IL-12 expression [36,37]. The discrepancies in the signaling outcomes observed in studies of CD40 ligation of monocytes and macrophages is likely due to the variation in the stimuli used. For example, a report by Mathur et al. [37] suggested that the strength of CD40 signaling dictated signaling outcome, with weak signals inducing ERK1/2 phosphorylation and IL-10 production, with stronger signals resulting in p38 activation and induction of IL-12. The tyrosine kinase activity rapidly induced by CD40 ligation was later attributed to Src kinase, which serves as an upstream initiator of pathways leading to both ERK1/2 and NF-␬B activation [22]. CD40 ligation on macrophages has also been shown to activate a phosphoinositide 3-kinase/Akt pathway that contributes to induction of IL-10 [38], which may occur via similar mechanisms as proposed for signaling via Toll-like receptor 4 (TLR4) [39]. There is also evidence for a role of the serine/threonine kinase Tpl2 (tumor progression locus 2), also known as Cot/MAP3K8, as an upstream initiator of the activation of ERK1/2, but not of JNK, p38 in response to macrophage CD40 ligation [40]. CD40 has no intrinsic catalytic activity and relies on the recruitment of adaptor proteins of the TNFR-associated factor (TRAF) family for signal transduction. Ligation of CD40 causes its oligomerization, which leads to recruitment of specific TRAF proteins, of which six have been identified. TRAF family members share a common stretch of amino acids at the carboxy terminus designated as the TRAF domain, which is further divided into two sub-regions. The carboxy terminus of the TRAF domain, TRAF-C, mediates homoand hetero-dimerization with other TRAF proteins and also to the receptor that recruits them. With the exception of TRAF1, all TRAFs contain a ring finger and 5–6 zinc finger-like motifs N-terminal to the TRAF domain, which presumably play a role in NF-␬B and c-Jun N-terminal kinase (JNK) activation [41]. The cytoplasmic domain of human CD40 has a proximal TRAF6 binding site and a more distal TRAF2/3/5 binding site. Although TRAF:CD40 interactions and the roles of TRAFs in B cell CD40 signaling has been evaluated in substantial detail [42,43], the specific functions of TRAFs in CD40 signal transduction in monocytes/macrophages have not been fully explored. Human monocytes express all TRAF family members constitutively [35]. Studies to date suggest that TRAF6 is likely the major player in mediating a wide range of proinflammatory functions initiated by CD40 ligation on monocytes and macrophages, as well as myeloid DC [22,44]. The role of TRAF family members in mediating CD40 signaling in macrophages was evaluated by transfection of a CD40-deficient macrophage cell line with wild-type human CD40, or with mutant forms of CD40 that contained disrupted TRAF binding sites [22]. It was shown that ligation of either wild-type CD40, or a CD40 mutant unable to bind TRAF2/3/5, resulted in effective stimulation of inflammatory cytokine production by macrophages, whereas ligation of a CD40 mutant lacking a functional TRAF6 binding site did not. An intact TRAF6 bindings site was found to be required for CD40-mediated activation of ERK1/2, as well as

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I␬B kinase (IKK) and NF-␬B. Introduction of a dominant negative TRAF6 into a wild-type (CD40+ ) macrophage cell line resulted in abrogation of CD40-mediated induction of inflammatory cytokine synthesis. The crucial role of TRAF6 in these pathways was corroborated by the use of a cell permeable peptide corresponding to the TRAF6 binding motif of CD40. This peptide, capable of blocking the TRAF6:CD40 interaction, inhibited CD40 activation of ERK1/2, IKK and inflammatory cytokine production [22]. Blockade of the TRAF6:CD40 interaction did not inhibit the initial phosphorylation of Src kinase induced through CD40, suggesting that the requirement for TRAF6 is downstream of Src in the signaling pathways leading to ERK1/2 and NF-␬B activation. As mentioned above, the serine/threonine kinase Tpl2 has also been implicated in CD40-induction of ERK1/2 phosphorylation in macrophages. Macrophages deficient for Tpl2 expression showed impaired ERK1/2 activation [40]. In this study, recruitment of Tpl2 in a complex with CD40 and TRAF6 was demonstrated in fibroblasts, which may be the case as well in macrophages, although this has not yet been directly shown. TRAF6 has also been shown to be critical to CD40-induced autophagy in a model of Toxoplasma gondii infection of macrophages [45,46]. This is due, in part, to the TRAF6-dependency of TNF␣ production induced in response to CD40 ligation [22]. CD40 and TNF␣ work synergistically to promote vacuole–lysosome fusion through autophagy, resulting killing of this intracellular pathogen [46].

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Surface plasmon resonance studies revealed that TRAF6 has a low affinity for CD40 and requires extensive multimerization of CD40 to mediate signaling events [47]. Thus, the identification of TRAF6 as an important signal transducer for CD40 in monocytes/macrophages may explain the requirement for extensive cross-linking to initiate CD40 signaling in these cell types. Although TRAF6 clearly plays a key role in CD40 signaling in monocytes and macrophages, the disruption of the TRAF2,3,5 reduces selected signaling events, as well, indicating that TRAF interactions with region contribute to CD40-mediated signaling. For example, the absence of an intact TRAF2,3,5 binding site results in reduced TNF␣ production in response to CD40 ligation, suggesting that TRAFs interacting with this binding region, though not essential for TNF␣ expression, contribute to this outcome [22]. Treatment of macrophages with a cell-permeable TRAF2,3,5 binding peptide also resulted in reduced expression of TNF␣, (J. Suttles, unpublished data). The signaling pathways engaged by CD40 ligation on monocytes/macrophages, and their functional outcomes are shown in Fig. 1. The common use of TRAF6 as adapter for signaling by both CD40 as well as TLR-mediated proinflammatory signaling pathways presents interesting possibilities for signaling cross-talk or cross-inhibition. It was found that the phenomenon of LPS tolerance, whereby macrophages stimulated by LPS are refractory to a secondary stimulus with LPS, also applies to CD40 signaling, as well. As discussed below, this phenomenon could be a major contributor

Fig. 1. Signaling pathways and functional outcomes of CD40 stimulation of monocytes/macrophages. TRAF6 is required for, and TRAF2 contributes to NF-␬B activation. ERK1/2 are activated via a Src, TRAF6-mediated pathway. Both the NF-␬B and ERK1/2 pathways contribute to a large number of primarily pro-inflammatory activities of monocytes and macrophages. Activation of ERK1/2 is also responsible for monocyte survival. Activation of p38 plays a role in activation of iNOS expression and PI3K activity contributes to induction of IL-10 synthesis.

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to the immunosuppression associated with sepsis. Sinistro and colleagues found that stimulation of monocytes with LPS resulted in a reduced response to a subsequent stimulation via CD40 ligation, including reduced IL-12 and TNF␣ production [48]. Importantly, similar results were seen when monocytes from patients undergoing sepsis were evaluated. Monocytes from septic patients showed similar defects in TNF␣, IL-12, and IL-1␤ production, as well reduced expression of CD80 and CD86 [49]. We found that pre-incubation with CD154 results in strong suppression of a secondary LPS stimulus, indicating that the tolerance effect is bidirectional. CD40 simulation resulted in robust induction of SOCS3, which may be responsible for this inhibitory effect (J. Suttles, unpublished data). Not unexpectedly, CD40 stimulation of macrophages also results in a greatly reduced response to a subsequent stimulation of CD40. This could be due to a combination of receptor endocytosis, TRAF degradation, and induction SOCS3 expression. CD40 signal transduction in monocytes/macrophages can be modulated by a variety of cytokines, chemokines and hormones. The anti-inflammatory cytokines IL-4 and IL-10 have been shown to inhibit CD40-mediated activation of ERK1/2 and induction of IL1␤ synthesis, and were synergistic in this regard, suggesting that they may act at different points in the CD40 signaling pathway [31]. This was also evident by the finding that IL-4, but not IL-10 inhibited CD40-mediated rescue of monocytes from apoptosis [50]. Both IL-4 and IL-10 were also found to inhibit CD40-mediated induction of iNOS expression and PGE2 production by monocytes, but, again, this appeared to be due to different mechanisms. IL-10 significantly inhibited CD40-induced activation of the ERK1/2, p38, and NF-␬B pathways, whereas inhibition by IL-4 was limited to the ERK1/2 pathway [36]. Interestingly, although treatment of monocytes with IL-l0 strongly downregulated IL-12 production, treatment with IL-4 or IL-13 was found to enhance expression of IL-12 p40, and production of IL-12 p70 [51]. Thus, these two typically anti-inflammatory cytokines appear to differ in their ability to modulate CD40 signaling in monocytes/macrophages and act via differing mechanisms that have not been fully explored. Mechanisms of action likely include roles of suppressors of cytokine synthesis (SOCS) [52,53], activation of phosphatases [54] and activation of AMP-activated protein kinase, AMPK, recently identified as a potent suppressor of macrophage inflammatory signaling pathways that is activated by IL-4 and IL-10 [55]. Monocyte/macrophage inflammatory function has shown to be inhibited by the nuclear receptor superfamily members, peroxisome-proliferator activated receptors (PPARs), in particular PPAR␥ [56]. Although studies to date have focused on the ability of PPAR␥ to suppress a subset of TLR-regulated genes, it has been shown that macrophages that display elevated PPAR␥ activity, show greatly reduced NF-␬B activity and proinflammatory cytokine production induced by CD40 ligation [57] and PPAR␥ agonists inhibit CD40-induced activation of IL-12 production in DC [58]. Factors that enhance CD40 signaling include CCL2/MCP-1, which was shown to act synergistically with CD40 ligation in the induction of COX2 expression by macrophages [59]. Of course, the regulation of CD40 expression is a critical control point for reducing or enhancing the contribution of this receptor to monocyte/macrophage function. CD40 is an NF-␬B-regulated gene and, not surprisingly, it appears that proinflammatory stimuli upregulate CD40 expression, whereas typically anti-inflammatory stimuli downregulate CD40 expression [60]. CD40 expression on macrophages, as well as on numerous other cell types, is positively influenced by IFN␥. Thus the infiltration of IFN␥-producing cells serves to recruit cells of the tissue environment (e.g., resident tissue macrophages, endothelial cells, vascular smooth muscle cells, and fibroblasts) to become active participants in the inflammatory response [61]. Monocyte/macrophage expression of CD40 was shown to be enhanced by treatment with GM-CSF and IL-3, as well as via stim-

ulation with LPS [13,62–64]. IFN␥ and LPS stimulated expression of CD40 on macrophages and microglial cells can be negatively regulated by TGF␤, IL-4, and IL-10 [65–67]. IFN␥ enhancement of CD40 expression on monocytes/macrophages can also be inhibited by statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) [68–70], which may contribute to their antiinflammatory action [69].

4. Role of macrophage CD40 function in autoimmune inflammatory disease The most well-studied role of CD40 expression on myeloid cells is its function as a critical co-stimulatory molecule in myeloid DC:T cell interactions during the process of antigen presentation. Blockade of the CD40:CD154 interaction has been shown to inhibit onset of inflammatory disease in numerous animal models, including experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA), thyroiditis, uveitis, inflammatory bowel disease, and diabetes [71–77]. Although blockade of CD40:CD154 interactions can disrupt DC:T cell interactions and inhibit induction of disease, following activation of autoreactive T cells, T cell interactions with monocytes and macrophages at the sites of inflammation are a substantive contributor to the pathogenesis of autoimmune inflammatory disease. There are a number of examples in which blockade of CD40:CD154 interactions ameliorates ongoing disease. For example, in the relapsing EAE (SJL) model, treatment of mice with anti-CD154 antibody either during peak disease, or during remission was found to inhibit disease progression [78]. Anti-CD154 antibody treatment also inhibited disease in adoptive transfer recipients of encephalitogenic T cells, suggesting that blockade of CD40 activation of microglial cells (the resident macrophages of the brain) and infiltrating peripheral macrophages, contributes to the therapeutic effect of this treatment on ongoing disease [78]. It is cautioned that the abrogation of disease using this strategy in the relapsing EAE model may be transient if there is continued inflammatory stimulus, e.g., via epitope spreading [79]. Studies employing CD40 bone marrow chimeric mice have also revealed the importance of the CD40:CD154 interaction occurring between T cells and macrophages/microglial cells in the CNS. Using the myelin oligodendrocyte glycoprotein (MOG)-induced model of EAE, Becher et al. found that development of CNS inflammation was reduced in CD40+/+ → CD40−/− chimeric mice, even though T cell activation appeared normal [80]. Interestingly, infiltration of activated T cells into CNS was also impaired, suggesting that CD40 expression in CNS is required for production of inflammatory mediators/chemokines necessary for recruitment of T cells. These influences of CD40 expression in CNS are most likely attributable to the microglial compartment. In a similar study conducted by Ponomarev and colleagues, induction of EAE by adoptive transfer of encephalitogenic T cells resulted in reduced diseases in the absence of CD40 expression in CNS, which was accompanied by reduced infiltration of macrophages into CNS. The authors concluded that during the course of disease, microglial cells are induced to express MHC class II, CD40 and CD86 in a CD40-independent manner. A second, CD40-dependent activation step was required for disease progression, including expansion of encephalitogenic T cells and efficient recruitment of macrophages from the periphery [81]. Macrophages are also key contributors to the pathogenesis of rheumatoid arthritis (RA) [82] and models of collagen-induced arthritis also have shown dependency on the CD40:CD154 interaction for disease [72]. A recent report demonstrated that delivery of liposome carrier encapsulated CD40-directed antisense oligonucleotides reduced clinical severity of ongoing disease in murine CIA [83]. The efficacy of this approach, specifically with regard to the treatment of ongoing disease, was attributed to the tropism

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of the reagent for macrophages and monocytes, in addition to DC. It is important to consider that the T cell macrophage interaction is reciprocal. In autoimmune inflammatory disease such as EAE/multiple sclerosis and CIA/rheumatoid arthritis, as well as inflammatory bowel disease, a role of both Th1 and Th17 Th subsets has been established [84] and, in the case of EAE, T cells expressing both IFN␥ and IL-17 are present during disease [85]. There is new appreciation for the potential plasticity of T cells and evidence suggests that interactions of T cells with monocytes/macrophages at the sites of inflammation can direct, or re-direct T cell responses. In a recent report, it was shown that monocytes derived from inflamed joints of patients with active RA, unlike monocytes from peripheral blood, specifically drove Th17 responses in a cell contact-dependent manner [86]. The cell contact-dependency suggests a possible role of CD40:CD154 interactions. Although not shown in this study, it is possible that the cytokine milieu of inflamed tissue may modify the response of monocytes/macrophages to CD40 ligation resulting in a specialized reciprocal stimulation of the CD154-expressing T cell. Overall, the functional significance of CD40 expression on monocytes and macrophages in the context of inflammatory autoimmune disease is very broad-reaching, including not only the wide range of activities of monocytes/macrophages induced through CD40 ligation, but also the reciprocal effects on T cell function.

5. Contributions of macrophage CD40 signaling to atherosclerosis Atherosclerosis is an inflammatory disease of complex etiology that displays many common features with autoimmune inflammatory diseases, including infiltration of T cells and monocytes/macrophage to the target site, in this case the vessel wall, and the recruitment of non-hematopoietic cells into the inflammatory response. Mouse models of atherosclerosis, including mice deficient for apolipoprotein E (ApoE) critical for cholesterol metabolism, and mice lacking low-density lipoprotein receptor (LDLR) have provided useful tools for the evaluation of the processes underlying atherogenesis. Formation of atherosclerotic plaque is initiated by infiltration of monocytes and T cells, with monocyte differentiation to macrophages occurring in the tissue, accompanied by a Th1 type inflammatory response. The production of inflammatory cytokines such as IFN␥ and TNF␣ induce expression of CD40 on endothelial cells and vascular smooth muscle cells, rendering them targets for CD154-expressing cells [87–89]. Although co-expression of both CD40 and CD154 on macrophages, endothelial cells and vascular smooth muscle cells in atherosclerosis has been reported [90], other laboratories have found CD154 expression to be exclusive to T cells and platelets, and absent on resident cells of the vasculature [88,91]. The importance of CD154 to the pathogenesis of atherosclerosis was shown by several studies employing the mouse models mentioned above and using either an antibody blockade approach to disrupt CD154-induced signaling, or use of CD154 deficient mice backcrossed onto the appropriate background (i.e., ApoE-deficient, LDLR-deficient). For example, Mach et al. [92], found that treatment of LDLR-deficient mice (fed a high cholesterol diet) with antiCD154, reduced the lipid content and size of aortic atherosclerotic lesions, as well as inhibited infiltration of macrophages and T cells. Lutgens et al. used mice deficient for CD154, backcrossed onto an ApoE-deficient background and examined both early and advanced lesions, concluding that initial lesion formation was not impacted, but late stage atherosclerotic changes were affected, with advanced plaques displaying reduced lipid content and a more stable plaque phenotype [93]. The source of CD154 most relevant to atherosclerosis was questioned by reports that the atherosclerotic plaque phenotype was not affected when CD154-deficient bone marrow

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chimeras were generated on an LDLR-deficient background [94,95], which suggested that non-hematopoietic cells were the origin of CD154. Adding additional complexity are recent findings indicating that CD154 interacts with receptors other than CD40, including the integrins Mac-1 (CD11/CD18) and ␣5␤1 [96,97]. It has been reported that CD40-deficient/LDLR-deficient mice were not protected from development of atherosclerosis as compared to mice deficient for LDLR alone, suggesting to these authors that the contribution of CD154 to the disease is independent of CD40 and reliant on CD154:Mac-1 interactions [96]. However, this interpretation contrasts with numerous studies demonstrating that CD40-deficient mice display macrophage dysfunction, and that cross-linking of anti-CD40 antibodies results in macrophage signal transduction leading to a number of strongly pro-atherogenic outcomes. The elevated expression of CD40 on numerous cell types, including macrophages, in inflamed vascular tissue in atherosclerosis suggests a functional association with disease progression that requires further exploration. 6. Macrophage CD40 signaling in host defense CD40, in its role as a key co-stimulatory molecule in T cell:APC interactions, is required for the induction of effective immune responses against invading pathogens via directing Th1, Th2 and Th17 responses [98–100]. However, CD40 ligation of macrophages also contributes directly towards the induction of anti-microbial activities. As discussed above, CD40 ligation of macrophages effectively induces proinflammatory cytokine production [13,14,61], and CD40 ligation in the presence of IFN␥ induces full effector function of macrophages including production of NO [15,16]. These activities have been shown to contribute to responses against parasitic, fungal and bacterial infection. For example, mice deficient for CD154 infected with parasitic protozoan, Leishmania amazonensis, displayed a higher susceptibility to infection than wild-type mice, which was associated with impairment of both T cell and macrophage functions. In addition to the inability to generate a protective immune response, macrophage production of NO was impaired [101]. It has also been shown that infection by Trypanosoma cruzi is reduced by addition of exogenous CD154 [102], and this protection involved CD154 stimulation of macrophage NO production in addition to the induction of IL-12. In addition, infection of mice with T. cruzi transfected with CD154, resulted in greatly reduced parasitemia and mortality as compared with the control organism [103]. These authors also demonstrated that treatment of mice with small molecules that mimic trimeric CD154 (miniCD40Ls) can also successfully amplify host resistance to T cruzi. Importantly, this response was evident in wild-type, but not CD40-deficient mice, indicating that CD40 was the specific target of these CD154 mimetics [104]. Likewise, CD40:CD154 interactions were shown to contribute to restraint of growth of T. gondii, even in the absence of IFN␥ [105]. In this study, IFN␥-deficient mice infected with T. gondii were treated in vivo with agonistic anti-CD40 which was found to decrease parasite load. An investigation of the mechanism underlying this phenomenon revealed that in vivo ligation of CD40 increased TNF␣ production by T. gondii-infected macrophages, which appeared sufficient for anti-toxoplasmacidal activity [105]. This may be due to the described synergy between CD40 and TNF␣ in inducing macrophage effector function, which can bypass the need for IFN␥ [106]. T. gondii, as does many intracellular pathogens, survives in macrophages by residing in vacuoles and avoiding vacuole–lysosome fusion. Interestingly, an additional mechanism of CD40-mediated anti-microbial activity observed in this model was the ability of CD40 stimulation to induce autophagydependent vacuole–lysosome fusion [45], as mentioned previously. This TRAF6-dependent CD40-mediated mechanism may apply to

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other organisms that use similar evasive strategies for survival in macrophages [46]. Macrophage CD40 signaling contributes similarly to defense against fungal infections. CD154-deficient mice infected via intratracheal inoculation with Cryptococcus neoformans displayed a greatly reduced inflammatory response in the lung, corresponding to an increased fungal burden, as compared to wild-type mice. These observations were accompanied by impaired macrophage NO production and impaired anti-microbial activity, as well as reduced IL-12 production [107]. Similarly, when CD154-deficient mice were subjected to Candida albicans infection, a lower fungal load in kidneys was observed as compared to wild-type mice, which could be mimicked by neutralizing anti-CD154 antibody. The CD154deficient mice showed lower plasma TNF␣ levels and reduced NO production by C. albicans-stimulated peritoneal macrophages was observed, which was discerned as the key mechanism for increased susceptibility to infection [108]. There is evidence that CD40 signaling also plays a role in bacterial sepsis, however, in this case it is exacerbating, rather than protective. Gold and colleagues subjected CD40-deficient mice to the cecal ligation and puncture model of polymicrobial sepsis and found that CD40-deficient mice displayed improved survival and reduced evidence of organ injury [109]. However, further studies by this group found that mice deficient for CD154 did not show this level of protection and evidence was presented indicating that CD40 stimulation in this model may be primarily via heat shock protein 70 (Hsp70) ligation of macrophage CD40 and subsequent induction of IL-12 production [110,111]. An outcome of sepsis is suppression of macrophage function such that responses to secondary stimuli are blunted. Although this may be a mechanism that could protect the host from uncontrolled inflammation and septic shock, it can result in a state of immunosuppression that renders the host unable to control a secondary infection. The cross-tolerance of TLR and CD40 creates a scenario by which attempts of CD154+ T cells to reactivate macrophages to combat infection would be impaired. Thus, this cross-tolerance effect could be viewed and an immunoevasive mechanism employed by bacteria. Interference of macrophage CD40 expression or interference with CD40 signaling machinery would favor pathogen survival. As an example, although CD40 ligation on macrophages typically results in IL-12 production, in Leishmania major infected macrophages, CD40 ligation preferentially induces IL-10. There is evidence that this may be a result of Leishmania’s propensity to deplete membrane cholesterol, which results in disruption of the assembly of IL-12-inducing CD40 signaling components in lipid rafts [112]. The net result is the creation of an environment that favors L. major survival.

7. Conclusions Macrophages display a remarkably diverse array of functions and adapt rapidly to changes in their microenvironment [8]. They are the most versatile of leukocyte populations and are present in nearly all tissues where they help maintain tissue homeostasis and provide defense against invading pathogens. However, their ubiquitous presence and their sensitivity to a broad range of stimuli also targets them as easy recruits into disease pathologies. Although macrophages are primarily considered in the context of innate responses, e.g., as responders to TLR stimulation, the functional expression of CD40 on macrophage confers upon them the ability to play a significant role in the outcome of adaptive immune responses. Antigen-activated CD154+ T cells can drive macrophage effector functions to provide antigen-driven protective effects in host defense. On the other hand, in the case of autoimmune inflammatory disease, autoantigen-activated CD154+ T cells can activate macrophage effector functions resulting in tis-

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