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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Semin Immunol. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2758065
NIHMSID: NIHMS128238

CD40 AND THE IMUNE RESPONSE TO PARASITIC INFECTIONS

Abstract

The interaction between CD40 and CD154 regulates many aspects of cellular and humoral immunity. The CD40 — CD154 pathway is important for resistance against a variety of parasites. Studies done with these pathogens have provided important insight into the various mechanisms by which this pathway enhances host protection, mechanisms by which pathogens subvert CD40 signaling, conditions in which the CD40 — CD154 pathway promotes disease and on modulation of this pathway for immunotherapy.

Keywords: Protozoa, helminth, cytokine, autophagy

Introduction

CD40 is a member of the TNF receptor superfamily that is expressed on antigen presenting cells (APC) and various non-hematopoietic cells [1-4]. Its counter receptor CD154 (CD40 ligand) is expressed primarily on activated CD4+ T cells [5, 6]. The interaction between CD40 and CD154 controls many aspects of cellular and humoral immunity including T cell-mediated activation of dendritic cells (DC) and monocyte/macrophages, T cell priming, proliferation of B cells, Ig synthesis, isotype switch, and germinal center formation [2, 7-12].

The relevance of CD40 in humans was established by the discovery that the congenital immunodeficiency called X-linked hyper IgM syndrome (X-HIM) is caused by the lack of functional CD154 [13]. Although patients with this syndrome exhibit defects in humoral immunity, the most important clinical feature of this immunodeficiency is an increased incidence of infections with opportunistic pathogens that include parasites such as Cryptosporidium parvum and Toxoplasma gondii [14-17]. Studies using parasites provided important insight into the role of CD40 in regulation of cell-mediated immunity as well as likely explanations for the susceptibility to opportunistic infections observed in patients with X-HIM [17].

Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan of worldwide distribution. The parasite exists in three forms during its life cycle: 1) the tachyzoite, the form of the parasite that infects almost any nucleated cell; 2) the tissue cyst that appears to persist in tissues of the infected host for life; and 3) the oocyst that forms during the sexual cycle that takes place exclusively in the intestine of felines. Infection in humans follows ingestion of poorly coked meat that contains tissue cysts, or ingesting food or water contaminated with oocysts.

Acute infection with T. gondii is characterized by dissemination of tachyzoites throughout the body. T cell-mediated immunity and the production of cytokines, notably IFN-γ and IL-12, are critical for control of the infection [18, 19]. However, the organism successfully evades eradication. The chronic phase of infection that ensues is characterized by the disappearance of tachyzoites and the formation of tissue cysts (primarily in the central nervous system and skeletal muscle).

T. gondii infection is usually asymptomatic in immunocompetent humans. The development of disorders of cell-mediated immunity results in reactivation of the chronic infection that typically manifests as toxoplasmic encephalitis [20, 21]. The other scenario where T. gondii infection is of clinical relevance is when the infection is acquired congenitally.

Role of CD40 — CD154 signaling in regulation of type 1 cytokine response during T. gondii infection

The fact that patients with X-HIM are susceptible to toxoplasmic encephalitis and disseminated toxoplasmosis [16, 17, 22, 23] plus the demonstration that CD154-/- mice infected with T. gondii develop encephalitis provide clear evidence of the relevance of the CD40 — CD154 pathway in resistance against T. gondii. Studies in humans revealed that this pathway regulates IL-12 and IFN-γ production during the interaction between human T cells and T. gondii-infected APC [17, 24]. Interestingly, T. gondii induces profound phenotypic changes in APC that influence type 1 cytokine production. Infection with viable T. gondii but not phagocytosis of killed parasites or incubation with T. gondii lysate antigens induces activation of purified human monocytes and immature monocyte-derived DC characterized by up-regulation of MHC class II, CD40, CD80, and CD86 [17, 25-27] (Figure 1). Human monocyte-derived DC that contain intracellular tachyzoites do not produce IL-12 p70 unless they receive CD40 stimulation from T cells [26] (Figure 1). Interestingly, IL-12 p70 production is only observed if monocyte-derived DC are infected with viable tachyzoites and not if these cells phagocytose killed parasites or if they are exposed to T. gondii lysates [26, 28]. Studies using mouse DC revealed that production of IL-12 p70 appears to depend on T. gondii-induced IL-12 p40 production and CD40 upregulation, while CD40 stimulation of DC results in balanced production of IL-12 p35 [29]. The studies using human and mouse cells indicate that the response of DC to T. gondii together with CD40 stimulation of these cells allow the immune system to induce IL-12 p70 production in situations where such a response would be appropriate [26, 29, 30].

Figure 1
Role of CD40 — CD154 interaction in the induction of a type 1 cytokine response against T. gondii. Infection of either human immature dendritic cells (iDC) or monocytes (Mo) with viable tachyzoites of T. gondii induces up-regulation of CD40, CD80, ...

CD40 - CD154 interaction between human T cells and T. gondii-infected APC results in IL-12 production that in turn, drives secretion of IFN-γ [17, 26] (Figure 1). However, experiments using neutralizing anti-IL-12 mAb indicated that T. gondii-infected APC also induce IFN-γ production independently of IL-12 [26]. This IL-12-indepedent production of IFN-γ may be caused by direct co-stimulation to T cells since it is ablated by simultaneous neutralization of the CD40-CD154 and CD80/CD86-CD28 pathways [26] (Figure 1). Of relevance, in vivo studies in mice demonstrated that pathogen-specific CD4+ T cells of the Th1 phenotype develop in animals deficient in IL-12 [31]. In addition, blockade of both CD154 and CD28 pathways is required for effective inhibition of IFN-γ production in a mouse model of toxoplasmosis [32].

While the role of CD40 - CD154 signaling for control of IL-12 production has also been reported in T. gondii—infected CD154-/- mice [33], other animal studies revealed the existence of CD40-independent IL-12 secretion in response to T. gondii [34]. T. gondii lysates induce IL-12 production by mouse CD8α+ DC in a CD40-independentt manner [34]. IL-12 production in response to soluble antigens was explained by T. gondii profilin-like protein that binds to TLR11 [35] and in part by T. gondii cyclophilin 18, a molecule released by tachyzoites that binds to CCR5 [36, 37] (Figure 2). While this pathway induces IL-12 production in mice, it is not known whether the same can be said for humans. T. gondii lysates antigens do not appear to cause IL-12 production by human dendritic cells [28]. Moreover, TLR11 is represented in humans only by a pseudogene [38] that contains a stop codon that would prevent protein expression [39].

Figure 2
CD40-indepedent production of IL-12 in response to T. gondii. Profiling and cyclophilin-18 (C-18) secreted by T. gondii or present in parasite lysates bind to TLR11 and CCR5 respectively expressed on mouse CD8α+ dendritic cells (DC). These two ...

Studies in patients with defective CD40 - CD154 signaling support the relevance of this pathway in the regulation of type 1 cytokine response in humans. Toxoplasmic encephalitis and disseminated toxoplasmosis have been reported in patients with X-HIM [16, 17, 22, 23]. Peripheral blood mononuclear cells (PBMC) and T cells from X-HIM patients secrete markedly decreased amounts of IFN-γ in response to T. gondii compared to healthy controls [17]. Similarly, PBMC from patients with X-HIM fail to secrete or secrete low amounts of IL-12 after incubation with T. gondii [17]. In contrast, IL-12 production in response to Staphylococcus aureus Cowan I strain plus IFN-γ is similar to that in control subjects [17]. Studies using recombinant CD154 trimer further confirmed the relevance of CD154 signaling for regulation of cytokine synthesis. CD154 trimer restores IL-12 secretion in response to T. gondii by PBMC from patients with X-HIM, and through this mechanism, it normalizes IFN-γ production in response to the parasite [17]. In addition to regulation of type 1 cytokine production, studies in patients with X-HIM indicate that CD154 appears to be crucial for in vivo priming of human T cells against T. gondii [17]. Taken together, the studies sing T. gondii identified defective type 1 cytokine response and impaired T cell priming as likely explanations for susceptibility to opportunistic infections in X-HIM patients.

CD40 — CD154 signaling is also relevant to HIV-1+ patients. Studies using antigenic stimulation by pathogens including T. gondii as well as polyclonal T cell stimulation revealed that CD4+ T cells from these individuals exhibit lower levels of CD154 [40-43]. This defect appears to contribute to impaired type 1 cytokine response associated with HIV-1 infection [40, 41]. The explanation for diminished CD154 expression in HIV-1 infection likely lies on the fact that the CD40 — CD154 interaction induces bi-directional signaling: CD40 not only induces activation of APC but CD40 also regulates CD154 expression. Many studies reported that CD40 decreases CD154 expression [44-46]. Enhanced susceptibility to CD40-mediated regulation likely explains why expression of CD154 during T cell — APC interaction is diminished in CD4+ T cells from HIV-1+ patients [42]. Studies that used T. gondii support a model whereby CD4+ T cells from these patients have an altered set point that controls the level of CD154 expression and thus, the strength of CD40-induced activation of APC [42]. Diminished CD40 stimulation would particularly affect IL-12 production since secretion of this cytokine appears to require strong CD40 stimulation [47].

CD40 is a novel regulator of autophagy

While CD40 regulates type 1 cytokine production in response to T. gondii, studies in CD154-/- mice pointed to additional mechanisms of resistance controlled by the CD40-CD154 pathway [33]. Indeed, CD40 stimulation of humans and mouse macrophages results in killing of the parasite [48-52]. This effect does not require the presence of IFN-γ and occurs independently of NOS2, the immune related GTPase (IRG) LRG-47, IGTP and IRG-47, the oxidative pathway and tryptophan degradation [48, 49, 52]. These findings raised the possibility that CD40 stimulation might be inducing killing of T. gondii by inducing lysosomal degradation.

Tachyzoites of T. gondii reside within a parasitophorous vacuole that allows parasite survival by preventing the release of the contents of endosomes/lysosomes into the parasitophorous vacuole. [53-55]. Until recently, the dogma was that the lack of fusion between the parasitophorous vacuole and the late endosomes/lysosomes was irreversible and was established at the time of active invasion [54, 55]. However, through CD40, cell-mediated immunity changes the non-fusogenicity of the parasitophorous vacuole and targets it for lysosomal degradation. CD40 causes fusion of parasite-containing vacuoles with late endosomes and lysosomes [49, 56]. Pharmacologic and genetic approaches that block different components of the endosomal/lysosomal pathway demonstrated that CD40 induces killing of T. gondii through vacuole-lysosome fusion [49]. These studies uncovered a new paradigm where interaction between CD154 on T cells and CD40 expressed on macrophages leads to the killing of an intracellular pathogen via the induction of vacuole-lysosomal fusion [49, 56]. The fact that in vivo CD40 stimulation induces macrophage toxoplasmacidal activity and reduces the parasite load [52] strongly suggests that vacuole-lysosome fusion induced by CD40 contributes to host protection.

CD40 re-directs the parasitophorous vacuole to the endosomal/lysosomal compartment through macroautophagy, a pathway to lysosomal degradation that is different from the classical phago-lysosomal fusion [49]. Macroautophagy, usually referred to as autophagy, is a process that directs cytoplasmic material and organelles to lysosomes [57, 58]. During autophagy an isolation membrane encircles portions of cytosol and organelles leading to the formation of autophagosomes. This structure fuses with late endosomes and lysosomes resulting in lysosomal degradation of its contents. Autophagy is a homeostatic mechanism in response to cellular stress such as nutrient deprivation or the accumulation of damaged organelles [57-60]. However, autophagy can also function as an innate mechanism that leads to lysosomal degradation of pathogens such as Mycobacterium tuberculosis, Streptococcus pyogenes, Listeria monocytogenes and Salmonella enterica [61-64]. Adaptive immunity also activates autophagy as anti-microbial strategy. IFN-γ appears to kill M. tuberculosis via autophagy [61]. Studies using T. gondii demonstrated that autophagy can be activated by adaptive immunity to induce pathogen killing. CD40 stimulation of macrophages causes recruitment of the highly specific autophagy marker LC3 (Atg8) around the parasitophorous vacuole [49]. This is followed by fusion with late endosomes/lysosomes [49]. Moreover, knockdown of the autophagy molecule Beclin 1 revealed that autophagy is required for fusion of the parasitophorous vacuole with late endosomes/lysosomes and for killing of T. gondii by CD40-activated macrophages [49].

Autophagic killing of T. gondii requires TRAF6-dependent synergy between CD40 and TNF-α. The TRAF6 binding site plays a dual role in the autophagic killing of T. gondii: it enhances autocrine production of TNF-α [65] and TRAF6 signals downstream of CD40 cooperate with TNF-α to mediate activate autophagy and lysosomal degradation of the parasite [49, 56] (Figure 3A).

Figure 3
Autophagy—dependent killing of T. gondii induced by CD40. A, T. gondii—specific CD4+ T cell acquires expression of CD154 after interaction with infected macrophages. CD40—CD154 binding results in recruitment of TRAF6 to the intracytoplasmic ...

The CD40 - CD154 pathway is key for control of toxoplasmic encephalitis likely through autophagy. CD154-/- mice develop this disease despite upregulation of IFN-γ [33] indicating that the CD40 - CD154 pathway likely promotes protection against encephalitis by activating mechanisms of host resistance that act independently of IFN-γ and its downstream effector molecules. Indeed, autophagic killing induced by CD40 occurs independently of IFN-γ, NOS2 and IRG. A model can be proposed whereby the IFN-γ/TNF-α-induced NOS2 pathway is essential but not sufficient resistance against cerebral toxoplasmosis [33, 66], and full control of toxoplasmosis would require CD40-induced autophagic killing of T. gondii [67] (Figure 3B).

The immune response against T. gondii in mice does not fully mimic that in humans. In addition to differences described above (TLR11 expression), the relative importance of IFN-γ and downstream effector molecules is likely different in humans and mice. Mice infected with T. gondii require STAT-1, IRGs (LRG-47, IGTP and IIGP1) and NOS2 for survival during different phases of infection [68-75]. In marked contrast, children with an autosomal dominant defect in IFN-γR1 that causes a deletion in the STAT-1 binding site are not susceptible to toxoplasmosis [76], IRG in humans have been reduced to a truncated gene IRGM and IRGC that lack an IFN-inducible element [77], and NOS2 in humans is more tightly regulated than in rodents and the production of nitric oxide appears to be weaker in humans than in mice [78]. CD40-induced autophagic killing of T. gondii may promote resistance against T. gondii in humans because CD40 induces killing of T. gondii independently of IFN-γ, STAT1, NOS2 and IRG [48, 49, 52, 56]. In addition to patients with X-HIGM, defects in the CD40 pathway may explain susceptibility to cerebral and/or ocular toxoplasmosis in newborns and HIV-1+ patients. Several groups reported impaired expression of CD154 on neonatal CD4+ T cells [79-83] and reduced levels of CD40 on DC [83]. Defective CD154 induction is particularly more pronounced in preterm babies [83], a finding relevant to toxoplasmosis in newborns since this is an infection acquired prior to birth. As described above, HIV-1+ patients can have defective CD154 induction on their CD4+ T cells.

Role of CD40 signaling in immunopathology during T. gondii infection

Although type 1 cytokine response is central to resistance against T. gondii, uncontrolled cytokine production leads to immunopathology. Indeed, IL-10-/- mice control the parasite and yet exhibit mortality associated with high production of IL-12, TNF-α, and IFN-γ production and liver damage [84]. Similarly, C57BL/6 mice develop acute inflammatory ileitis after oral infection with T. gondii, a process that is dependent on CD4+ T cells and IFN-γ [85]. Immunopathology in this model of infection as well as in IL-10-/- mice is inhibited by blockade of CD154 [32, 86], especially in the setting of simultaneous blockade of CD28 [32]. Taken together, like other mediators of host resistance, the CD40 - CD154 pathway plays a role in protection against T. gondii and in situations where the infection causes immunopathology.

Cryptosporidium parvum

Cryptosporidium is a protozoan that can cause diarrhea. Humans become infected by ingestion of oocysts that release sporozoites in the intestinal lumen. Sporozoites then infect epithelial cells. Infected individuals shed oocyst in their feces. Thus, humans can become infected by person-to-person contact. Waterborne outbreaks of cryptosporidiosis can also occur. Infection can be asymptomatic or can cause self-limited diarrhea. However, patients with AIDS or other immunodeficiencies can develop severe disease that may involve the biliary tract.

Animal studies revealed that cell-mediated immunity confers protection against C. parvum. CD4+ T cells and MHC class II are required to control C. parvum [87, 88]. In addition, IFN-γ and IL-12 mediate protection against the pathogen [89, 90]. Although infection leads to antibody production, it appears that humoral immunity is of secondary importance compared to cell-mediated immunity.

The identification of C. parvum as an important pathogen in patients wit X-HIM syndrome provided clear evidence for the relevance of the CD40-CD154 pathway for resistance against C. parvum [91]. Indeed, both CD40-/- and CD154-/- mice are unable to control C. parvum [92]. While parasite shedding in the stools disappears by 4 weeks in control mice, CD40-/- and CD154-/- mice show unabated parasite shedding for at least 8 weeks [92]. Moreover, C. parvum is detected in the gut, gallbladder and biliary tree of CD40-/- and CD154-/- mice and causes cholangitis, a feature of cryptosporidiosis in immunodeficient patients [92]. Further evidence of the importance of CD154 came from studies in SCID mice [92]. These animals are susceptible to C. parvum. While administration of spleen cells from wild type mice induces control of the parasite, spleen cells from CD154-/- are unable to confer protection [92].

Studies in HepG2 cells, a hepatocellular carcinoma cell line with features of biliary epithelial cells, revealed that CD40 stimulation induces anti-microbial activity against C. parvum [91]. This effect may be explained by induction of apoptosis of infected cells [92]. However, the in vivo relevance of this mechanism is unclear. Experiments with bone marrow chimeras showed that expression of CD40 on bone marrow cells is sufficient to induce resistance against C. parvum while CD40 expression on non-hematopoietic cells is unable to confer protection [93].

Leishmania

Leishmania is an obligate intracellular protozoan that causes disease that is endemic in the tropics, subtropics and southern Europe. Female phebotomine sandflies release promastigotes into the skin while probing for a blood meal. The promastigote infects primarily macrophages and DC. The amastiogote form of Leishmania replicates within phagolysosomes. The host cell is eventually destroyed and the parasites that are released infect other host cells.

Leishmania can cause cutaneous, mucosal and visceral disease. There are a variety of species of Leishmania that are of clinical relevance in humans. Some examples include: Leishmania major, a cause of cutaneous disease; Leishmania amazoniesis and Leishmania tropica, both causes of cutaneous leishmaniasis (less commonly visceral disease); and Leishmania donovani, a cause of visceral leishmaniasis (less commonly cutaneous disease).

Leishmania provides a classical model system to study the role of cytokines in determining resistance or susceptibility to a pathogen. A mouse model of cutaneous leishmaniasis after infection with L. major allowed to demonstrate that strains of mice that develop preferential production of IL-12 and activation of IFN-γ -producing Th1 cells are able to control the infection [94]. In contrast, progressive disease occurs in mice that respond by preferential activation of Th2 cells that produce IL-4 [94].

CD40 — CD154 interaction and the regulation of type 1 cytokine response in Leishmania infection

Three simultaneous studies revealed the central role of the CD40 — CD154 pathway for protection against Leishmania. CD154-/- or CD40-/- mice on a resistant background (C57BL/6) developed ulcerating cutaneous lesions after infection with promastigotes of L .major or L. amazoniensis [95-97]. The central role of CD154 in host resistance was supported by the demonstration that treatment of infected CD154-/- mice with recombinant CD154 afforded partial protection against L. major [95]. CD154-/- and CD40-/- mice had a profound defect in the induction of a Th1 type response [95-97]. T cells from CD154-/- mice were unable to induce of IL-12 production when stimulated with Leishmania-infected macrophages [95]. Moreover, this defect in IL-12 production appears to be central to enhanced susceptibility to infection because in vivo treatment with IL-12 increased IFN-γ production and prevented disease progression [95]. CD40 appears to require NF-κB2 to induce IL-12 production [98]. CD154 is also required not only for protection against cutaneous leishmaniasis but also against visceral leishmaniasis. CD154-/- mice are susceptible to L. donovani [99], and the lack of CD154 promotes replication of residual L. donovani that survive after chemotherapy [100].

The importance of CD40 signaling for resistance against Leishmania was confirmed by studies using wild-type mice. Administration of anti-CD154 mAb to C57BL/6 mice infected with L. major resulted in impaired IL-12 production and worsened cutaneous lesions [101, 102]. These studies indicate that CD40 signaling is required to maintain control of L. major. The CD40 — CD154 pathway controls type 1 cytokine response not only in mice but also in humans. Incubation of human peripheral blood lymphocytes with autologous macrophages infected with L. major resulted in IFN-γ and IL-12 that were inhibited by addition of an anti-CD154 mAb [103].

The fact that Leishmania reside within macrophages suggested that these cells might be the source of IL-12 required to control the infection. However, Leishmania inhibits IL-12 production by macrophages [104]. Thus, investigators turned their attention to DC. Indeed, these cells produce IL-12 p40 in response to L. donovani and L. major [105, 106]. Importantly, production of bioactive IL-12 p70 is dependent on the CD40 — CD154 pathway. Human monocyte-derived DC infected with L. major produce high levels of Il-12 p70 when stimulated with CD154 [107]. Moreover, L. major-infected monocyte-derived DC from patients with cutaneous leishmaniasis induced IFN-γ production by autologous T cells in a CD154-dependent manner [107]. These results suggest that CD40 signaling in skin DC at the site of infection may contribute to the development of a protective immune response [107]. CD40 stimulation also induces IL-12 production by DC infected with L. amazoniensis [108]. However, this response was detected only in DC from a resistant strain of mice (C3H/HeJ). In contrast, DC from a susceptible strain (BALB/c) produced IL-4 in response to CD40 stimulation [108].

The ability of Leishmania-infected dendritic cells to produce IL-12 in response to CD40 stimulation may provide an explanation for the different clinical presentation of leishmaniasis: localized versus disseminated disease and healing versus non-healing skin lesions. In this regard, while monocyte-derived DC infected with L. major produce IL-12 p70 in response to CD40 stimulation, DC infected with L. donovani and L. tropica fail to do so [109]. However, it appears that the inability of L. donovani to prime DC for IL-12 p70 production may be dependent on the form of parasite used. In contrast to human monocyte-derived DC infected with promastigotes of L. donovani, DC infected with L. donovani amastigotes upregulated IL-12 p70 production in response to CD40 stimulation [110].

While the CD40 — CD154 pathway is important for induction of type 1 cytokine response and resistance against Leishmania, this pathway may not always be required for protection. CD154-/- mice that are infected with a low dose L. major promastigotes or that are also CD28-/- can acquire and maintain resistance against the pathogen [102, 111]. Treatment of infected CD154-/- mice with TRANCE receptor fusion protein revealed that IL-12 production and the induction of a Th1-type response is mediated by the TRANCE — RANK pathway in the absence of CD154 [112]. These findings likely represent an example of the development of compensatory mechanisms of host protection in knockout mice.

The CD40 — CD154 pathway and the induction of macrophage anti-Leishmania activity

In addition to conferring host resistance by promoting induction of a type 1 cytokine response, the CD40 — CD154 pathway likely enhances protection by also activating anti-Leishmania activity in macrophages. Draining lymph nodes from CD154-/- mice infected with L. amazoniensis have an impaired ability to induce nitric oxide (an important mediator of anti-Leishmania activity) by infected macrophages [96]. In addition, macrophages that receive CD40 stimulation plus IFN-γ acquire leishmanicidal activity [97]. CD40 also cooperates with other molecules to trigger killing of Leishmania by macrophages. Activated T cells induce anti-Leishmania activity in macrophages through cooperation between CD40 and CD11a (LFA-1) [113]. These two molecules stimulate nitric oxide production and thus, induce killing of L. major [113]. This process occurs independently of TNF receptors providing a potential explanation for the ability of TNFR1/2-/- mice to control L. major [113].

Manipulation of CD40 signaling by Leishmania

The evidence that strains of mice susceptible to L. major have decreased production of IL-12 plus the central role of the CD40 - CD154 pathway in induction of this cytokine led to the proposal that defects in this pathway may be important in explaining susceptibility to infection [101]. CD154 expression was similar in susceptible and resistant strains of mice infected with L. major[101]. Thus, it was proposed that there might be a defect in CD40 signaling in Leishmania infection [101]. Indeed, macrophages from BALB/c mice infected with L. major exhibit defective induction of NOS2 in response to CD40 stimulation [114]. This defect appeared to be due to diminished p38 MAPK signaling [114]. Anisomycin, a p38 MAPK stimulator, restored NOS2 expression and anti-Leishmania activity of macrophages [114]. Moreover, in vivo treatment of BALB/c mice with anisomycin led to reduced parasite burden and induction of a Th1-type memory response [114].

The effects of Leishmania on CD40 signaling vary in macrophages from resistant (C57BL/6) versus susceptible (BALB/c) mice [115]. While CD40 stimulation induced anti-L. major activity in macrophages from C57BL/6 mice, macrophages from BALB/c exhibited increased parasite replication [115]. This discrepant outcome appears to be explained by the induction of IL-10 in CD40-stimulated macrophages from BALB/c mice [115]. Relevant to these findings, L. major has been reported to skew intracellular signaling in CD40-stimulated macrophages from BALB/c mice [47]. Weak CD40 signals induce ERK-1/2 signaling and IL-10 expression, whereas stronger signals induce p38 MAPK signaling and IL-12 p40 expression [47]. L. major skews CD40 signaling toward ERK-1/2 and IL-10 production in macrophages from BALB/c mice [47]. As a result, there is inhibition of p38 MAPK activation, of expression of NOS2 and of IL-12 production [47] (Figure 4). ERK-1/2 inhibition or IL-10 neutralization restores CD40-induced p38MAPK activation and parasite killing in macrophages [47]. Whether Leishmania fails to skew intracellular signaling in macrophages from C57BL/6 mice remains to be determined.

Figure 4
L. major skews CD40 signaling. In response to CD40 stimulation of macrophages from BALB/c mice, L. major amastigotes promote ERK1/2 and IL-10 production. This pathway inhibits p38 MAPK activation, the expression of NOS2 and the production of IL-12.

A recent study suggests that L. major influences signaling downstream of CD40 through its ability to deplete cholesterol [116]. Pharmacologic depletion of cholesterol or infection with L. major impairs the assembly of CD40 signalosomes that contain TRAF2, TRAF3 TRAF6 and Lyn, and as a result, cause inhibition in IL-12 production [116]. In contrast, cholesterol depletion or L. major infection promote the assembly of CD40 signalosomes that contain TRAF6 and Syk, leading to production of IL-10 [116]. However, in an apparent paradox, it has previously been demonstrated that CD40 triggers IL-12 production though the TRAF6 binding site and not the TRAF2,3 binding site [117].

Manipulation of CD40 signaling as immunotherapy

Stimulation of CD40 — CD154 signaling enhances resistance against Leishmania. Mice of a susceptible background (BALB/c) infected with L. major developed a type 1 cytokine response and acquire protection against the infection if they are treated with an agonistic anti-CD40 mAb [118]. BALB/c and C57BL/6 infected with L. donovani exhibited a reduction in parasite burden in the liver, increased IFN-γ production and enhanced mononuclear cell recruitment as well as granuloma formation after treatment with agonistic anti-CD40 mAb [99]. Anti-CD40 mAb potentiated the effect of pentavalent antimony, a chemotherapeutic agent for visceral leishmaniasis [99]. These results suggest that approaches to stimulate CD40 signaling may nave therapeutic application for the treatment of leishmaniasis. However, the induction of an undesirable pro-inflammatory response raises concern about utilizing generalized stimulation of CD40 signaling.

The IL-12-inducing activity of CD154 suggested that this molecule could be used as an adjuvant to vaccination to preferentially induce a type 1 cytokine response. Indeed, vaccination of susceptible BALB/c mice with soluble Leishmania antigen plus a plasmid that encodes CD154 enhanced antigen-specific IFN-γ production and induced protection against L. major [119]. Likewise, vaccination of susceptible BALB/c mice with soluble Leishmania lysate antigens plus L929 cells that express CD154 induced a type 1 cytokine response and protected mice against challenge infection with L. major [120]. Similar effects were observed when mice were immunized with L292 cells that express both CD154 and the Leishmania antigen gp63, or with immunization with L. major promastigotes adsorbed with recombinant CD154 [120]. Another approach to stimulate CD40 signaling during immunization has been to utilize transgenic parasites that express CD154. Compared to wild-type parasites, infection with L. major promastigotes that express the extracellular domain of CD154 caused mild disease in susceptible BALB/c mice and afforded resistance against challenge infection to C57BL/6 mice [121].

Trypanosoma

Trypanosmes are protozoa that can cause Chagas’ disease in the Americas (Trypanosoma cruzi) or african trypanosomiasis (Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense). Infection in humans is transmitted by hematophagous insects. These insects release infective trypomastigotes that enter through disruptions of the skin or mucosa. Trypomastigotes disseminate throughout the blood and infect a variety of cells leading to the intracellular replicative form called the amastigote. Infection with T. cruzi in BALB/c mice mimics disease as it occurs in humans: an acute phase characterized by parasitemia followed by a chronic where the parasite is not detectable in the blood but is present in peripheral tissues. IL-12, IFN-γ, TNF-α and nitric oxide have been implicated in resistance against T. cruzi [122-125].

CD40 induces anti-microbial activity against T. cruzi. Peritoneal macrophages treated with supernatants of spleen cells stimulated with CD154-expressing fibroblasts prevent infection with T. cruzi [126]. This effect is mediated by nitric oxide and the production of this radical requires IL-12, IFN-γ and TNF-α [126]. Moreover, CD40 stimulation of IFN-γ-primed macrophages also induces anti-microbial activity against T. cruzi [126]. The relevance of the cascade CD40 - IL-12 - IFN-γ is supported by in vivo experiments were mice that received CD154-expressing fibroblast exhibited reduced parasitemia and increased survival [126].

Two additional approaches of in vivo modulation of CD40 — CD154 signaling support the relevance of this pathway for induction of protection against T. cruzi. In one of them, parasites were genetically engineered to express host CD154 [127]. Infection of mice with CD154-expressing T. cruzi trypomastigotes led to low parasitemia, survival and resistance to challenge infection [127]. These effects were associated by evidence of preserved cell mediated immunity as assessed by lymphocyte proliferation and production of IFN-γ [127]. In the second approach, mice were treated with small trivalent molecules that bind CD40 and mimic the effects of soluble CD154 (miniCD40L) [128]. Mice treated with miniCD40L exhibited a decrease in parasitemia and in mortality that were accompanied by prevention of the immunosuppression that typically follows T. cruzi infection [128]. MiniCD40L-treted mice were protected from depletion of CD8+ T cells as well as impairment in lymphocyte proliferation and IFN-γ production [128]. These studies demonstrated that rationally designed CD154 mimetics can be effective in vivo [128].

CD40 appears to also promote resistance against trypanosoma also through stimulation of humoral immunity. African trypanosomes such as T. congolese are an important cause of disease in livestock. The extracellular nature of this pathogen likely explains the important role of humoral immunity in host protection. Experiments were SCID mice were reconstituted with bovine fetal liver/thymus/lymph nodes (SCID-bo) examined the role of CD40 in resistance against T. congolese [129]. SCID-bo mice infected with T. congolese were treated with a suboptimal dose of the trypanocidal agent Berenil [129]. Co-administration of a stimulatory anti- CD40 mAb led to a modest increase in survival, increased in the population of CD5+ B cells and appeared to also increase parasite-specific antibody response [129].

Plasmodium

Malaria is a major health problem in developing tropical countries. Malaria in humans is caused by 4 species of Plasmodium (Plasmodium vivax, Plasmodium falciparum, Plasmodium ovale and Plasmodium ovale). Infection is transmitted by an anopheles mosquito that releases sporozoites into the bloodstream. This form of the parasite infects hepatocytes where intracellular replication of merozoites takes place. Merozoites are released into the bloodstream and infect red cells where they become trophozoites. Malaria is a common cause of fever and anemia in tropical countries. P. falciparum can cause severe malaria characterized by decreased mental status (cerebral malaria) and multi-organ failure.

P. berghei provides an animal model of severe malaria. Infection in susceptible strains of mice is characterized by breakdown of the blood-brain barrier, hemorrhages and sequestration of macrophages and platelets in cortical venules, neurological abnormalities and death [130]. TNF-α is a major mediator of severe malaria in mice [131]. Studies using CD40-/- and CD154-/- mice reported that the CD40 - CD154 pathway contributes to death in malaria. While parasitemia is unaffected in CD40-/- and CD154-/- infected with P. berghei, these animals exhibited decreased mortality [130]. Genetic disruption of the CD40 - CD154 pathway also protected mice against blood-brain barrier breakdown and against sequestration of macrophages in brain venules [130]. These beneficial effects are not due to diminished TNF-α levels. Although CD40-/- mice exhibited diminished CD54 mRNA levels, this decrease is modest and it is uncertain if it can explain diminished macrophage sequestration [130]. Taken together, these findings indicate that the CD40 — CD154 pathway plays an essential role in the pathogenesis of severe malaria.

Susceptibility to severe P. falciparum malaria has been associated with polymorphisms of genes such as glucose 6 phosphate dehydrogenase, β-globin, Duffy and HLA-B53 [132]. An allele of CD154 which has a mutation in the promoter region has been linked to susceptibility to severe malaria [133]. Gambian males that are hemizigous for CD154-726C exhibit significant protection against cerebral malaria and severe malarial anemia [133]. These results suggest that future studies should further characterize the role of CD154 polymorphisms is susceptibility to infections.

Helminths

Helminths are multicellular organisms that are the cause of some of the most prevalent infections in the world. They include trematodes (schistosomes), nematodes (roundworms) and cestodes (tape worms). Chronic infection caused by the trematode Schistosoma mansoni can cause intestinal and liver disease. Humans become infected by cercaria released from infected snails. The parasite migrates through veins and lymphatics as a schistosomula. The adult female worm reaches veins of the intestine where it releases eggs. These eggs can be excreted via the feces, or can remain within the host either in the intestine or in the liver. Eggs trigger formation of granulomas and are responsible for most disease manifestations.

Chronic disease cause by helminths such as schistosomes typically induces a Th2-type immune responses associated with the production of IL-4, IL-13, IL-5, IgE and eosinophila [134]. Antigens secreted by the eggs are responsible for inducing a Th2 response. This cytokine response is required for host survival but is also responsible for the induction of complications of the infection such as hepatic fibrosis [135, 136].

Studies in a mouse model of schistosomiasis revealed that CD40 - CD154 interaction controls Th2 responses against a pathogen. CD154-/- mice infected with S. mansoni exhibit a defect in production of Th2-associated cytokines [137]. This defect is of functional relevance because CD154-/- mice have increased mortality as well as worsened histopathology in the lung after infection with S. mansoni [137]. Thus, the CD40 - CD154 pathway is required for the development of an appropriate immune response [137]. Studies with DC further support the relevance of CD40 in the immune response against S. mansoni. In vivo administration of bone marrow derived DC pulsed with schistosome egg antigen (SEA) results in induction of a Th2 response [138]. This response is blunted when mice receive DC that lack CD40 [138]. The likely explanation for the importance of this receptor for induction of an immune response against schistosoma is that the CD40 - CD154 pathway is required for in vivo activation of dendritic cells. Dendritic cells incubated with SEA do not upregulate costimulatory ligands or MHC class II molecules [138]. However, animals infected with S. mansoni exhibit evidence of in vivo activation of DC, a process that requires CD154 [139].

Administration of radiation-attenuated S. mansoni larvae induces protective immunity associated with a Th1 type response [140]. The CD40 - CD154 pathway is also important for the generation of this immune response. In mice immunized with radiation-attenuated larvae, production of IL-12 p40 by DC, T cell priming and production of IFN-γ are dependent on the presence of CD154 [141]. Moreover, the CD40 - CD154 pathway is essential for the development of protective immunity since CD154-/- mice vaccinated with radiation-attenuated larvae are not resistant to challenge infection [142]. Interestingly, the protective effect of CD154 was not mediated exclusively by the induction of a Th1 response since administration of IL-12 to vaccinated CD154-/- mice restored Th1 response but failed to confer protection [142]. It is not clear whether the protective role of CD154 is also mediated by antibody production.

Increased intestinal muscle contractility leads to expulsion of nematodes [143]. This response is dependent on STAT6, IL-4 and IL-13 [144]. Moreover. T cell infiltration of intestinal muscle layers and well as expression of IL4 and IL-13 at this site has been reported [144, 145]. Studies in mice infected with the nematode Trichinella spiralis revealed that production of IL-4, IL-13, IgG1, IgE, mucosal MCP-1 and goblet cell response are impaired in mice that lack CD154 [146]. As a result, there is diminished intestinal muscle contractility and impaired parasite expulsion [146]. Thus, the CD40 - CD154 pathway acts as an upstream regulator of the induction of Th2 response and expulsion of a nematode. CD40 has also been found to be important for the induction of a Th2 response against the cestode Taenia crassiceps [147].

Conclusions

Studies done with parasites have helped establish the central role of the CD40 — CD154 pathway in the regulation of the immune response against infections. These studies revealed that CD40 — CD154 signaling activates anti-microbial mechanisms that are dependent not only on type 1 cytokine response but also on IFN-γ-independent autophagy as well as on stimulation of protective type 2 cytokine response in the case of helminths. Work using models of parasitic infections has provided likely explanations for the susceptibility to opportunistic infections in patients with defective CD40 — CD154 signaling. Recent studies are beginning to uncover how pathogens can skew CD40 signaling to impair protective cell-mediated immunity and on the likely role of polymorphisms of CD154 in determining susceptibility to infection. Finally, models of parasitic infections have contributed to the identification of modulation of CD40 — CD154 signaling as an approach to enhance the effectiveness of vaccines and, perhaps as an avenue to develop new treatment modalities against various infections.

Acknowledgments

The author was supported by the National Institutes of Health AI48406AI and EY018341, the American Heart Association (Ohio Valley Affiliate), the Juvenile Diabetes Research Foundation International, the Dietrich Diabetes Research Institute, the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation.

Footnotes

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