A hypothetical role for Notch signaling pathway in ...

A hypothetical role for Notch signaling pathway in immunopathogenesis of leprosy

Héctor Serrano-Colla, MD. MSc, Liliana Acevedo-Saenza, PhD Nora Cardona-Castro a,b,c, *, MD, PhD

a

Basic Science Research Group, School of Graduate Studies, CES University, Medellín,

Colombia. b

School of Medicine, CES University, Medellín, Colombia.

c

Colombian Tropical Medicine Institute (ICMT), Medellín, Colombia.

*Corresponding author Nora Cardona-Castro, MD. MSc. PhD (Address) Carrera 43A#52 Sur-99 Telephone: 3053500 ext 2280 E-mail: [email protected].

Financial support: This work was conducted with financial support to Héctor Serrano Coll from Colciencias [grant 727-2015].

Abstract Leprosy is a chronic infectious disease caused by Mycobacterium leprae mainly affecting skin and peripheral nerves. Leprosy has a broad range of clinical manifestations that range from mild (tuberculoid leprosy) to severe (lepromatous leprosy) forms, and are highly dependent on the host’s immune response. Among the immune response elements involved in the pathogenesis of leprosy are the Toll-like receptors (TLRs), vitamin D receptor (VDR), natural killer cells (NK), and T cells. These innate and adaptive immune response elements may be related to the Notch signaling pathway, which is involved in immune cell growth, differentiation, and proliferation. We hypothesize that failure in Notch signaling in leprosy patients may be associated to: 1) compromising NK cell maturation, lysing of infected cells, and CD4+ Th1 differentiation. 2) VDR alterations and TLR polymorphisms may affect expression of Notch Delta-like ligands (DLL) in antigen presenting cells (APCs). 3) altered DLL expression by APCs could compromise CD4+ T cell differentiation towards the Th1 and Th17 effector phenotypes; and finally 4) expression of Notch Jagged ligands would induce CD4+ T cell differentiation towards Th2 effector phenotype and alternative activation of macrophages. Altogether, these signaling failures could favor proliferation of M. leprae in the host. Therefore, evidence of the proposed immunologic failures in leprosy patients would be essential for the better understanding of immunopathogenesis of this disease, and would ultimately enable detection of susceptible individuals, providing a valuable tool for prevention of this debilitating disease.

Key words: Mycobacterium leprae, immunity, T lymphocytes, Toll-like receptors, natural killer cells, vitamin D receptor, Notch signaling pathway.

INTRODUCTION Leprosy is chronic granulomatous infectious disease produced by Mycobacterium leprae, mainly affecting skin and peripheral nervous system, and is associated to deformity, mutilation, and social isolation (1,2). The natural course of this disease is manifested by disabilities in eyes, hands, and feet, which affect more than 4 million people worldwide (3). Additionally, clinical manifestations of leprosy are broad since they are highly dependent on the host’s immune system, and can range from a mild form or tuberculoid to a severe form or lepromatous leprosy (4,5).

Upon contacting the host, the first barrier encountered by M. leprae is the innate immune system, described by Metchnikoff in 1884 (6). This basic defense mechanism is dependent on epithelium integrity, natural killer (NK) cell activation, and receptors that recognize microbeassociated molecular patterns (MAMPs), including Toll-like receptors (TLRs) (7–9). TLRs recognize M. leprae MAMPs and initiate signaling pathways that lead to synthesis of proinflammatory cytokines, chemokines, and endothelial adhesion molecules. These proteins are key factors in the activation of cell-mediated adaptive immune response, which is fundamental against M. leprae infection (10,11).

Current advancements in genomics have allowed a better understanding of alterations in the host’s immune system that favor intracellular proliferation of M. leprae (12). To date, TLR polymorphisms, altered vitamin D receptor (VDR) expression levels, unbalanced activating and inhibitory signals in NK cells, and reduced CD4+ T cell differentiation towards Th1 or Th17 effector phenotypes have been reported to play a role in the host’s response to M. leprae infection (13). Recently, Kibbie et al (14) reported that Notch signaling pathway is involved in macrophage differentiation in response to infection with M. leprae. Taking into account that

this is a key signaling pathway in host’s activation of both the innate and adaptive immune responses, and these responses can be modulated by Notch ligands as Jagged inducing a humoral response , or Delta inducing a cellular response (15), it is thus relevant to understand the role of Notch signaling pathway in immunopathogenesis of leprosy.

HYPOTHESIS Different levels of Notch signaling pathway are involved in immunopathogenesis of leprosy. This hypothesis is based on the following assumptions: 1. Late NK maturation is enhanced by Notch signaling, therefore alterations in this pathway would affect expression of IFN- by NK cells, compromising macrophage activation and infected cell lysing, as well as CD4+ T cell differentiation towards Th1 phenotype. 2. VDR is involved in Notch ligand expression upon recognition of M. leprae MAMPs by TLRs in antigen presenting cells (APC), thus, alterations or polymorphisms in TLRs would compromise VDR expression, reducing Notch ligand expression in APCs, affecting CD4+ T cells differentiation towards Th1 and Th17 effector phenotypes. 3. Changes in expression or function of Notch Delta-like ligands (DLL) in APCs would compromise CD4+T cell differentiation towards Th1 and Th17 phenotypes, which would difficult classical macrophage activation that is key for lysis of infected cells. 4. Expression of Jagged –another Notch ligand- instead of DLLs by APCs, would enhance CD4+T cell differentiation towards a Th2 phenotype. This would induce alternative activation of macrophages that express IL-10 and TGF-, which are cytokines that facilitate intracellular proliferation of M. leprae in the host.

Here we discuss Notch signaling pathway and its relationship with TLRs, VDR, NK cells, as well as its role in CD4+T cell differentiation. Furthermore, we postulate hypothesis related to alterations in Notch signaling pathway that could be associated to immunopathogenesis of leprosy.

Notch signaling pathway The Notch signaling pathway is an evolutionary conserved cell signaling pathway, involved in regulation of diverse events during eukaryotic cell development, such as proliferation, cell growth, migration, and programmed cell death (16). To date, four Notch receptors have been described in mammalian cells (Notch1-4), and five specific ligands have been identified in mammals: delta-like ligand (DLL) 1, DLL3, DLL4, Jagged1, and Jagged2 (17,18). Notch receptors have three domains: an extracellular domain (NECD) and an intracellular domain (NICD) that are bound to the cell membrane by the transmembrane (TM) domain (Figure 1) (19,20).

Specific Notch ligands are proteins that are membrane bound or expressed by adjacent cells. Upon ligand binding, Notch receptors undergo proteolytic cleavage that is mediated by the ADAM (A disintegrin and metalloproteases) family of metalloproteases (21), which cleave the NICD to activate Notch signaling. Cleaved NICD translocates to the nucleus and binds to transcription factors such as Recombining binding protein suppressor of hairless (RBPJ) complex, inducing expression of Hes transcription factors, which in turn induce differentiation and proliferation of multiple cell types, including cells of the innate and adaptive immune system (Figure 1) (22,23).

Toll-like receptors and their role in Notch signaling and leprosy

TLRs are expressed by APCs (dendritic cells, Langerhans cells), by phagocytes, by epithelial cells in mucosa, and by some endothelial cells (11). In humans, ten TLR receptors have been described as recognizing MAMPs, while in other mammals (mice) up to thirteen different TLR receptors have been described (24,25). TLRs involved in recognition of M. leprae MAMPs include TLR1/2, TLR2, TLR4, and TLR6 (26,27). In host cells, TLR1/2 heterodimer is the first one to recognize M. leprae lipoproteins, stimulating secretion of cytokines such as interferon gamma (IFN-γ), IL-6, and IL-12, which are key cytokines for activation and differentiation of naïve T cells towards Th1 effector phenotype (13).

An antimicrobial response is triggered upon TLR2 homodimer recognition of peptidoglycans present in M. leprae wall (6,11). Activation of this pathway regulates VDR and 1α-hydroxylase (CYP27B) expression, leading to transformation of vitamin D to its active form 1,25 dihydroxyvitamin D [1,25(OH)2D] and its subsequent binding to VDR, inducing synthesis of cathelicidin, an antimicrobial peptide that plays a critical role in against mycobacteria (28–30). Immune mechanisms activated by TLRs upon recognition of M. leprae MAMPs and its interplay with the Notch pathway are shown in table 1.

Previous studies have proposed that unidentified M. leprae ligands are recognized by TLR4 homodimers (ref). Additionally, TLR4 is the only member of the TLR family that is able to activate signaling dependently and independently of MyD88 via its TIR (Toll/IL-1 receptor) intracellular domain, leading to expression of proinflammatory (TNF-α, IL-6, IL-12) and type I IFN cytokines. It is worth pointing out that these signaling pathways induce type I IFN overexpression, which favors clinical manifestations of generalized forms of leprosy

(lepromatous leprosy) since these cytokines inhibit IFN- and vitamin D bactericidal activity (table 1)(31,32).

In the study by Mattos et al (27), susceptibility to develop generalized and anergic forms of leprosy is associated to recognition of M. leprae polypeptides by TLR6 heterodimers in fibroblasts and Schwann cells. This fact induces lipid biogenesis that leads to expression of prostaglandin E2, IL-10, and inhibits IL-12 and NO, providing an appropriate environment for proliferation of M. leprae (Table 1) (27,33)

Single nucleotide polymorphisms (SNPs) in TLRs have been described as susceptibility or resistance factors for M. leprae (34,35). Among the TLR SNPs associated to susceptibility to leprosy are TLR1/2 N248s (rs4833095) and TLR2 C2029T (rs121917864) SNPs. The former has been reported in Bengalese and Brazilian populations to reduce expression of TNF-α (13), while the latter has been shown to reduce expression of nuclear factor (NF-) in Asian and African population samples (34). Regarding SNPs associated to resistance to M. leprae, a study from India reported TLR1/2 I602S (rs5743618) SNP to lead to increased IFN-γ expression (13,34,36).

TLR signaling pathway interacts with other pathways such as the Notch signaling pathway. Shang et al (37) described that upon recognition of mycobacteria by TLRs, a yet to be described intracellular signaling events induce expression of Notch ligands at the cell surface. Furthermore, studies by Ito and colleagues (38) reported that modifications in TLRs lead to alterations in expression and recognition of DLL in APCs, thus compromising CD4+ T cell differentiation towards a Th17 phenotype, which play an essential role in defense against mycobacteria (38).

The notion that TLR polymorphisms may be associated to altered DLL expression in APC cells leads us to hypothesize that those APCs may be non-functional, and would hinder CD4+ T cell differentiation towards Th1 and Th17effector phenotypes, which are active against M. leprae. Therefore, the cross-talk between these two pathways may play a key role in physiopathogenesis of leposy (Table 1 and figure 2).

Alterations in VDR and its possible cross-talk with Notch signaling pathway Vitamin D receptor is a member of the nuclear receptor superfamily and plays an essential role in biological actions related to vitamin D (39). VDR regulates expression of several genes involved in calcium/phosphate homeostasis, proliferation, cell differentiation, and immune response (40). Furthermore, vitamin D is an essential stimulant for innate immunity against mycobacteria, since binding of active vitamin D to VDR induces antimicrobial peptide synthesis such as cathelicin and β-defensins, which destroy bacterial cellular membrane (table 2) (41,42).

Previous studies have reported VDR polymorphisms to be associated to leprosy. In India, Neela et al (43) showed that –in their study population- VDR SNPs (Taq-I rs731236, Fok-I rs2228570, and Apa-I rs7975232) were associated to risk of leprosy, being Fok-I rs2228570 and Apa-I rs7975232 associated to increased risk of leprosy. In addition, Goulart et al (44) reported in a Brazilian population VDR Taq-I polymorphism to be associated to increased risk of leprosy, since it may affect T cell differentiation and maturation, thus compromising host immune response.

VDR and vitamin D may be involved in Notch signaling through the expression of Notch ligands in APCs and the role these cells play in CD4+ Tcell differentiation towards Th1, Th2, or Th17 effector phenotypes. Using vitamin D deficient wistar rats, Domingues-Faria et al (45) reported down-regulation of Notch receptors and its ligands, compromising the effector function of this signaling pathway. Taking into account the above-mentioned studies, we hypothesize that upon M. leprae MAMPs recognition by TLR2, a yet to be described intracellular signaling cascade that induces expression of Notch ligands in the macrophages may be related to vitamin D and its receptor. Therefore, this alteration would compromise expression of Notch ligands in APCs and hence CD4+ T cell differentiation (Table 2 and figure 2).

NK cell differentiation may be compromised by alterations in Notch signaling pathway

NK cells are a type of lymphocyte stemming from CD34+ progenitors in the bone marrow. These cells recognize and eliminate neoplastic and infected cells by secreting perforins, granzymes, and IFN-γ (46,47). Additionally, cytotoxicity and late activation of NF cells is regulated by the balance between cell-death activating and inhibitory signals, which are mediated by receptors such as killer-cell immunoglobulin-like receptors (KIRs) (48,49). Described in table 2 are the immune mechanisms of some cellular components against M. leprae infection, and their possible relationship to the Notch pathway.

An imbalance between KIR mediated cell-death activating and inhibitory genes has been reported for leprosy patients. The study by Franceschi and colleagues (50) showed that, compared to patients with tuberculoid form of leprosy and healthy individuals, expression of KIR activating genes was down-regulated in patients with lepromatous leprosy (LL). In

agreement with those findings, the study by Jarduli et al (51) compared expression of KIR receptors in 408 leprosy patients and 412 healthy controls, and reported an imbalance in the expression of KIR activating and inhibitory genes in patients with the anergic form of the disease (LL), compared to those with tuberculoid leprosy or healthy controls. On the other hand, the imbalance between KIR activating and inhibitory genes is associated to late activation of NK cells, thus favoring intracellular proliferation of M. leprae.

Expression of KIR receptors on NK cells may also be associated to Notch signaling. Using NK cell cultures, Felices and colleagues (52) showed that inhibition of Notch signaling led to downregulation of expression of KIR activating genes, suggesting that Notch pathway directly regulates expression of these receptors. Therefore, it is plausible that in leprosy, the reported imbalances between activating and inhibiting KIR genes may be related to alterations in Notch signaling (table 2).

An additional hypothesis that may explain alterations in Notch signaling in the context of leprosy, relates to the expression on non-functional KIR activating genes by NK cells. If the later maturation stage of NK cells is compromised, the cell would be unable to secrete IFN-γ, and hence CD4+ T cells would not differentiate towards the Th1 effector phenotype, favoring development of LL, the anergic form of the disease (figure 3).

CD4+ T cell effector phenotypes and their potential association with Notch signaling pathway T cells originate from hematopoietic stem cells in the bone marrow that migrate to the thymus, as immature T cells, where they undergo differentiation and selection processes to become naïve CD4+ or CD8+ T cells (53,54). During these stages, the Notch signaling pathway plays a key

role in promoting pre-T cell survival during β-selection (19), and in preventing immature T cells from maturing into other lymphoid cell types including dendritic cells (DCs), B lymphocytes, and myeloid cells (19,55).

Naïve T cells can differentiate to several effectors patterns as are Th1, Th2, Th3, Th9, Th17, Th22, and in these processes of differentiation is involved Notch (11,19). Regarding the role of Notch signaling pathway in naïve T cell differentiation towards Th1 and Th17 effector subtypes, previous studies have shown that binding of DLL1 ligands in APCs cells to Notch1 or Notch2 receptors in naïve T cells activates this pathway. This interaction induces expression of IFN- γ, favoring CD4+ T cell differentiation towards a Th1 phenotype (19, 56). On the other hand, interaction of Notch1 or Notch2 receptors with DLL4 ligands promotes T cell differentiation into a Th17 phenotype (Figure 4). These two T cell effector phenotypes are key in the adaptive immune response to M. leprae, and are commonly found in tuberculoid leprosy patients (57).

Additionally, Notch signaling pathway is also involved in Th2 differentiation by binding of Jagged ligands in APC cells to Notch1 or Notch2 receptors in naïve T cells, which leads to expression of IL-4 in T cells that favors Th2 differentiation. This effector phenotype is usually found in LL patients (Figure 4) (58,59).

Throughout this discussion we have emphasized the essential role of Notch signaling pathway in CD4+ T cell differentiation towards different effector phenotypes. Therefore, the type of Notch ligands that are expressed by APC cells in hosts of M. leprae, or even in their coinhabitants, is key to determine which of them are susceptible of developing anergic forms of leprosy.

Hypothesis on the role of Notch signaling pathway in adaptive and innate response to M. leprae

In this section, we will discuss the possible relationship of TLRs and NK cells with Notch signaling pathway in the context of the innate immune response to M. leprae. Upon infection with M. leprae, TLRs recognize mycobacterial MAMPs and initiate a signaling cascade that leads to expression of cytokines and chemokines such as IL-12 and IL-15 (60, 63), which are crucial for NK activation. Upon activation, NK cells must undergo affinity maturation in order to acquire cytolytic activity, which requires up-regulation of KIR activating genes that allow NK cells to secrete IFN-γ, perforins, and granzymes, needed to lyse M. leprae infected cells (43).

Previous studies have reported a role for Notch signaling pathway in later stages of maturation of NK cells (52). In the event of pathway failure, differentiation of CD4+ T cell towards Th1 phenotype would be compromised, and in turn, differentiation towards Th2 would be favored, facilitating intracellular growth of mycobacteria (Figure 3).

Regarding the relationship of adaptive immune response to M. leprae and the Notch signaling pathway, it is important to point out that during antigen presentation by APCs to naïve T cells, in addition to expression of cytokines such as IL-12, IL-18, and IFN-γ, differentiation of CD4+ T cells towards Th1 phenotype is favored by the interaction of DLLs in APCs with Notch1 and Notch2 receptors in CD4+ T cells (19, 60). Th1 T cells are characterized by expression of cytokines such as IL-2, TNF-α, and IFN-γ. Importantly, IL-2 promotes type 1 cytotoxic CD8+ T cell (Tc1) proliferation, TNF-α induces recruitment of inflammatory cells such as neutrophils and macrophages, and IFN-γ promotes classical activation of macrophages and induction of their microbicidal activity (11).

In order for this adaptive immune response to be fully effective against M. leprae, activation of Th17 cells is also required. Notch signaling pathway plays an essential role in CD4+ T cell differentiation towards Th17 phenotype. These cells are characterized by expression of IL-17, a cytokine that promotes recruitment of inflammatory cells. Thus, the interplay between these two immune cell lineages, Th1 and Th17, is essential for successful elimination of M. leprae (figure 4) (23,57,60).

Altogether,

alterations

in

Notch

signaling

pathway

and

their

consequences

in

immunopathogenesis of leprosy lead us to hypothesize that modifications in expression or functionality of DLLs in APCs would compromise CD4+ T cell differentiation towards Th1 and Th17 effector phenotypes, subsequently affecting classical activation of macrophages, and finally favoring proliferation of M. leprae. We also propose that in anergic forms of the disase, namely LL, APCs express the Notch ligand Jagged, which upon interacting with naïve CD4+ T cells induce expression of IL-4, IL-5, and IL-13. These conditions favor alternative activation of macrophages, which is characterized by expression of anti-inflammatory cytokines, IL-10 and TGF-β, generating immunologic tolerance and facilitating intracellular proliferation of M. leprae (Figure 5).

CONCLUSIONS Notch signaling pathway is an evolutionarily conserved pathway that may play an essential role in activation of innate and adaptive immune responses in leprosy patients, since proper activation of this pathway is directly related to successful NK cell and CD4+ T cells (Th1 and Th17) activation, which are essential cells for the immune response against M. leprae. Therefore, confirmation of these hypotheses –in the context of leprosy- would allow us to: 1)

identify Notch ligand-receptor interactions that promote expression of KIR activating genes in NK cells, which are key in later NK cell maturation stages, lysis of infected cells, and differentiation of CD4+ T cells into Th1 effector phenotype; 2) establish whether TLR polymorphisms affecting VDR expression compromise DLL expression in APCs in leprosy patients; 3) identify modifications in DLL expression in APCs in leprosy patients, as well as alterations in CD4+ T cell Th1 and Th17 differentiation; 4) establish if patients with anergic forms of leprosy (LL) exhibit an increased expression of Notch ligands Jagged that induce CD4+ T cell differentiation towards Th2 phenotype, facilitating M. leprae proliferation. Therefore, evidence of the proposed immunologic failures in leprosy patients would be essential for the better understanding of immunopathogenesis of this disease, and would ultimately enable detection of susceptible individuals, providing a valuable tool for prevention of this debilitating disease.

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FIGURE CAPTIONS Figure 1. Notch signaling pathway. Notch receptors interact with DLL and Jagged ligands, promote activation of ADAM proteases that cleave the NICD, and activate Notch signaling. Cleaved NICD translocates to the nucleus and binds to Recombining binding protein suppressor of hairless (RBPJ) transcription factor complex, inducing expression of Hes transcription factors, which in turn induce differentiation and proliferation of multiple cell types, including cells of the innate and adaptive immune system.

Figure 2. TLR polymorphisms, VDR, and Notch ligand expression. TLR polymorphisms affect VDR expression, which may compromise DLL expression by APCs, hindering CD4+ T cell differentiation towards Th1 or Th17 effector phenotypes.

Figure 3. Innate immune response to leprosy and its possible relationship to Notch signaling. TLRs recognize mycobacterial MAMPs and initiate a signaling cascade that leads to expression of IL-12 and IL-15, which are crucial cytokines for initial NK activation. NKs acquire cytolytic activity by activation of Notch signaling pathway, which would favor expression of activating KIR receptors, allowing NK cells to release granules and IFN-γ in order to lyse infected cells. Alterations in Notch signaling may lead to non-functional activating KIR receptors in NK cells, favoring proliferation of M. leprae.

Figure 4. Adaptive immune response to leprosy and its possible relationship to Notch signaling. During APC antigen presentation to naïve T cells, cytokines (IL-12, IL-18, and IFNγ) and Notch ligand DLL-1 binding to Notch receptors favor differentiation of CD4+ T cells to Th1 phenotype, characterized by expression of cytokines such as IL-2 and thus promote type 1 cytotoxic CD8+ T cell (Tc1) proliferation. Both cell types express TNF-α and IFN-γ to recruit

and promote classical activation of macrophages, favoring lysis of infected cells. Notchmediated differentiation of CD4+ T cells towards Th17 cells plays a key role in the adaptive immune response to M. leprae

Figure 5. Hypothesis on possible cross-talk between adaptive immune response to leprosy and Notch signaling. Alterations in Notch signaling pathway could be related to poor CD4+ T cell differentiation towards Th1 and Th17 effector phenotypes, favoring proliferation of M. leprae. APCs interact through Jagged ligands with Notch receptros in CD4+ T cells, favoring differentiation towards Th2 phenotype, thus proliferation and survival of M. leprae.