Costimulation in Allergic Asthma: The Roles of B7 and Semaphorin Molecules

Abstract

It is well established that allergic asthma is T cell-driven disease where CD4+ T cells of Th2 phenotype play a critical role in disease initiation and maintenance. There are several critical steps in the induction of Th2 type immune response to the allergen. The first critical step is the antigen processing and presentation of allergen-derived peptides in the context of specific major histocompatibility Class II (MHCII) molecules by antigen-presenting cells (APC). Recognition of this complex by T cell receptor (TCR) and interaction of costimulatory ligands with corresponding receptors represents the second step in T cell activation. As the third part of optimal T cell differentiation, proliferation, and expansion, several cytokines, integrins, and chemokines get involved in the fine-tuning of DC-T cell interaction and activation. Multiple recent evidences point to the selected members of B7 and semaphorin families as important checkpoints providing a fine-tuning regulation of immune response. In this book chapter, we discuss the properties of costimulatory molecules and address their roles in allergic asthma.

1. Introduction

Allergic asthma is a Th2-driven, immunological chronic disease [1]. CD4+ T cells of Th2 phenotype secreting Th2 cytokines such as IL-4, IL-5, and IL-13 play a critical role in asthma initiation and propagation [2]. In this book chapter, we address the question of how different costimulatory molecules influence the allergic immune response which is central to asthma pathogenesis.

The initial step in the immune response is the antigen capture and processing by APC. APC subdivide into “professional” such as dendritic cells (DC), B cells, and macrophages, and “unprofessional” such as epithelial cells, fibroblasts, basophils, eosinophils, ILC2 (type 2 innate lymphoid cells), which normally have other functions in tissues and do not act as APC [3, 4, 5]. Antigenic epitopes derived from a captured allergen are presented to T cells in the context of specific MHC (human leukocyte antigen, HLA, for human cells) molecules [1]. This is the first signal for T cell activation, whereas a second signal is derived from costimulation where specific costimulatory molecules on APC interact with their receptors on T cells (Figure 1) [6]. The first signal alone does not lead to the immune response to allergen (Figure 1), it rather induces T cell unresponsiveness or “anergy” [6, 7].

Figure 1.

The two-signal model of the T-cell activation. (a) Functions of the immune checkpoint molecules (IChMs) are completely dependent on the first signal because the interaction of the receptor (Co-R) on T-cells with the ligand (Co-L) on APCs (the second signal) do not result in an activation of T-cells without the first signal. (b) T-cell activation has not occurred in the absence of the second signal. In several cases, the absence of the second signal leads to T-cell tolerance and anergy. (c) the correct activation of T-lymphocytes occurs after the TCR interaction with the MHC-presented peptide (Ag) (the first signal) and after the interaction of a ligand of the B7 family (Co-L) with its receptor (Co-R) (the second signal). A synergism of the two signals results in an optimal activation of T-cells.

The members of the B7 family are the most characterized immunomodulatory ligands that bind to receptors on lymphocytes. They can act as costimulators or inhibitors/checkpoints. Currently, there are eleven known representatives of the B7 family, namely: B7–1 (CD80), B7–2 (CD86), B7-H1 (PD-L1, CD274), B7-DC (PDCD1LG2, PD-L2, CD273), B7-H2 (B7RP1, ICOS-L, CD275), B7-H3 (CD276), B7-H4 (B7x, B7S1, Vtcn1), B7-H5 (VISTA, Platelet receptor Gi24, SISP1), B7-H6 (NCR3LG1), B7-H7 (HHLA2), and ILDR2 (the synonyms of IChM names of the B7 family are given in parentheses) [7, 8]. Two molecules of B7 family proteins [9], B7–1 and B7–2, are the best characterized costimulators [7, 8]. Their ligation of CD28 expressed on T cells leads to T cell activation whereas interaction with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) functions as an inhibitory signal.

Multiple recent reports pointed to selected semaphorin family [10, 11] members acting as checkpoints in the immune response regulating optimal T cell activation and cytokine production [10, 12]. Semaphorins alone are unable to induce or suppress T cell activation regulated by a combination of signals 1 and 2 but can significantly potentiate or downregulate it [10, 12]. Moreover, their involvement in asthmatic disease development has been supported by several recent publications (reviewed in [13, 14, 15] establishing them as potential immunomodulatory targets.

The goal of this book chapter is to discuss the roles of these molecules in asthma and provide the ground for their therapeutic use in disease prevention, management, or treatment.

2. B7 family members in asthma

2.1 B7: 1 and B7: 2

Asthma is Th2 cell-driven disease with Th2 type cytokines such as IL-4, IL-5, and IL-13 driving the disease pathology [2]. The effect of costimulation in asthma has been a subject of several decades’ of research. The differential role of two B7 family members in allergic response has been extensively studied and described in multiple articles published in the late 1990-s (16–19, reviewed in 20, 21). The work by Freeman et al. [16] questioned the functional necessity of two known at that time B7 family members. Using the in vitro cell cultures and distinct transfectants, they reported that both B7–1 and B7–2 effectively and equally costimulate T cells to produce IL-2 and IFNγ, however, B7–2 was more efficient in costimulation of IL-4 production with cell priming and especially with a repetitive cell stimulation, whereas B7–1 was efficient for GM-CSF production. Similarly, anti-B7–2 mAb significantly reduced the induction of IL-4 mRNA in a primary human allogeneic MLR whereas anti-B7–1 mAb failed to do so. The work by Van Neerven [17] addressed the same question as the discussed above research but in different experimental settings, namely the stimulation of human PBMC obtained from allergic and non-allergic persons in vitro with house dust mite allergen (HDM) in the presence or absence of B7–1 or B7–2 blocking Abs, or CTLA-Ig. CTLA4-Ig was efficient in inhibiting allergen-induced cell proliferation and cytokine production. The proliferation of CTLA4-Ig-treated cells was partially restored by stimulating them with anti-CD28 mAb which indicated that CTLA4-Ig inhibits the interaction of CD28 with both, CD80 and CD86. Interestingly, anti-CD86 mAb inhibited the HDM-induced cell proliferation similarly to CTLA4-Ig but with less degree of inhibition. However, the addition of anti-CD80 blocking mAb to the anti- CD86 mAb treated cells resulted in identical inhibition as with CTLA4-Ig. This report suggested that the costimulation inactivation could be efficient in the downregulation of allergen-dependent Th2 responses in asthmatic patients (Figure 2). The research by Larche et al. [18] used allergen stimulation of human PBMC and cells obtained by alveolar lavages to examine B7–1 and B7–2 dependence of T cell immune response. While allergen-induced PBMC proliferation and cytokine production were inhibited by the use of CTLA4-Ig and anti-B7–2 Ab in cell cultures, anti-B7–1 Ab showed no effect. Moreover, HDM-induced broncho-alveolar lavage (BAL) T cell proliferation was also B7–2 but not B7–1 dependent. This study further supported the notion that T cell costimulation-targeted therapy could be beneficial in asthma management. The study by Jaffar et al. [19] stimulated with HDM allergen the explants from endobronchial mucosal biopsies obtained from asthmatic patients. Although this study did not address the requirement of individual B7–1 or B7–2 molecules in anti-allergic T cell response, it clearly demonstrated the requirement of B7/CD28 costimulation in IL-5 and IL-13 production using a novel tool for asthma research, the bronchial explant system. Moreover, they were the first to demonstrate a significant difference in cytokine profile in bronchial explants between asthmatic and non-asthmatic lungs.

Figure 2.

Role of costimulation in T cell immune response and asthma. a. B7–1 and B7–2 interaction with CTLA-4 contributes to suppressive activity of allergen-specific Treg cells whereas their interaction with CD28 costimulates Th1 and Th2 responses. b. ICOS-L – ICOS interaction regulates Th2 effector cell function; it is efficient in stimulation of IL-4 and IL-10 production but not IFNγ. It regulates Th2 cell infiltration into lungs, promotes B cell differentiation and IgE production, contributes to AHR. This pathway also regulates IL-10 production in Treg cells. c. PD-L1 interaction with PD-1 receptor play a protective role in allergic asthma as it was reported to drive a differentiation of Treg cells and to downregulate contact hypersensitivity reaction. On the other hand, the use of neutralizing anti-PD-1 ab in vivo decreased eosinophilic lung infiltration but increased AHR and lung neutrophilia. d. PD-L2 interaction with PD-1 receptor downregulates allergic asthmatic response by suppressing Th2 cell activation, AHR, eosinophil infiltration, and IgE production. e. B7-H3 interaction with unknown receptor promotes Th2 and Th17 cell differentiation, lung infiltration by eosinophils, AHR, IL-4 and IL-17 production.

2.2 B7-H1 (PD-L1) and B7-DC (PD-L2)

The B7 homolog 1 (B7-H1) shares the same inducible PD-1 receptor on T cells with B7-DC (reviewed in 20, 21). While B7-H1 is constitutively expressed on monocytes and is downregulated with cell activation, B7-DC expression is induced by cell activation (reviewed in 7, 8, 20). Functionally, it was speculated that PDL-1 may suppress Th1-mediated inflammation and PDL-2 may suppress Th2-mediated inflammation (Figure 2) [20, 21]. The expression and regulation of PD-L1 and PD-L2 in asthma were analyzed using a segmental challenge of human lungs with allergen followed by BAL [22]. This study was initiated to clarify the importance of these costimulators in human asthma as previous reports using mouse models of the disease gave conflicting results [23, 24, 25]. The mouse lung expression levels of PD-L1 and PD-L2 were significantly upregulated by the OVA challenge [25]. However, the treatment of DC with CPG DNA, CD40L, GM-CSF, LPS, and IFN-γ led to the increased expression of PD-L2 on the cell surface whereas IL-4 and IL-13 induced the highest PD-L2 expression on DC among all mentioned above stimuli [25]. Interestingly, Th2 cytokines induce PD-L2 expression on DC but not B7–1 or B7–2 expression suggesting a regulatory role of this costimulatory in Th2 cell activation. In vivo treatment of mice with recombinant PD-L2-Fc resulted in a Th2-like inflammation in murine lungs. Such effect of PD-L2-Fc could be explained by a potential PD-L2-Fc blocking of the inhibitory interaction of cell-associated PD-L2 or PD-L1 with PD-1 or by a potential PD-L2-Fc interaction with a second receptor and enhancement of T cell activation via a PD-1-independent mechanism. This alternative yet unidentified receptor may dominate the inhibitory PD-1 receptor in vivo. The observed negative correlation between PD-1 expression by circulating CD4+ T cells and IgE concentrations in serum in asthma patients suggested a protective role of PD-L1 in allergic asthma [22]. However, there was an increase in blood and a decrease in BAL of PD-1+ CD4+ T cells after segmental bronchial challenge with the allergen [22]. The authors concluded that the up-regulation of PD-L1 and downregulation of PD-L2 on endobronchial DC subsets favor a Th2 inflammation in their human asthma model based on a segmental lung allergen challenge. They propose that modulating PD-1 ligand-mediated pathways by blocking PD-L1 or activating PD-L2 signaling could be a promising immunomodulatory approach in allergic asthma management. A more recent study has shown that in the in vivo experimental model of HDM-induced asthma anti-PD-L1 mAb completely abrogated eosinophil recruitment and PD-1/PD-L1 blockade by the use of neutralizing mAb to either PD-1 or PD-L1 led to the enhanced airway hyperreactivity (AHR) due to activation of Th17 cells and resulting increase of airway neutrophilia [26]. The authors identified the increased frequency of CD4 + IFNγ + and CD4 + IL-17A+ cells in PD-1-deficient (Pdcd1−/−) mice which directly correlated with higher levels of circulating IFNγ and IL-17A. This study goes in accord with previous research establishing allergic asthma as a mix Th2/Th17 response. Clinical observation in patients with chronic occupational asthma showed a persistent PD-L2 expressing mDC-mediated Th2 response that was partially PD-L2-dependent [27]. This suggests that other costimulators participate in Th2 activation in the asthmatic setting.

2.3 B7-H2 and ICOS

Another pair of the B7 family ligand and its receptor involved in the regulation of T cell activation comprises of B7-H2 and ICOS (Inducible CO-Stimulator) (Figure 2). It was originally shown that the engagement of ICOS by B7-H2 on CD4+ T cells increased the production of Th1 (IFN-γ and TNFα) and Th2 (IL-4, IL-5, and IL-10) cytokines [28, 29, 30]. ICOS-deficient mice were unable to induce the allergen-specific IgE responses when compared to WT mice which demonstrated an important role of ICOS:B7-H2 interaction in the induction of IgE production [31]. It was shown recently that the injection of anti-B7-H2 mAb resulted in the reduction of inflammation and Th2 cytokines production in the mouse model of allergic asthma [32]. Moreover, blocking the ICOS:B7-H2 interaction on human ILC2s reduced AHR and lung inflammation in the experimental asthma model [33]. In addition, it was demonstrated that in contrast to wild-type counterparts, B7-H2 deficient mice did not develop AHR after OVA sensitization and challenge [34].

2.4 B7-H3 and other B7-H molecules

To investigate the contribution of B7-H3 to the development of allergic asthma, mice were treated with antiB7-H3 blocking Ab during the course of OVA sensitization and challenges [35]. Anti-B7-H3 mAb treatment of mice at the experimental asthma induction phase (days 7–18 after allergen priming) suppressed allergic lung inflammation including eosinophilic infiltration, airway mucus hypersecretion, downregulated the number of B7-H3+ cells in the lung tissues as compared with the immunoglobulin G (IgG) treated control group. In addition, anti-B7-H3 mAb inhibited IL-4 and IL-17 levels and increased the expression IFN-γ in BALF of allergen-treated mice. However, anti-B7-H3 mAb treatment did not show an inhibitory effect on any measured asthma parameters at the effector phase (days 21–27 after priming). Nevertheless, B7-H3 blockage can provide a novel therapeutic approach for allergic asthma especially if used in a combination with immunotherapies that work in the effector phase. Two years later the same group of scientists reported an association of asthma exacerbation with increased levels of B7-H3 expression in the peripheral blood of asthmatic children which was significantly decreased by the use of steroids [36]. Their further studies in an animal model of asthma showed that recombinant B7-H3 administration to the mouse lungs in the time-frame of allergen priming (days 0 to 14), but before challenge (days 21, 27), significantly upregulate all parameters of allergic response such as inflammatory cell infiltration to the lung tissues, Th1 and Th2 cytokine levels in BAL and plasma, allergen-specific IgE production, and Th2/Th17 cell proliferation and cytokine levels [37].

The roles of other B7 family members such as B7-H4, B7-H5, and B7-H7 in asthma have never been investigated. Conflicting data on B7-H7 costimulation results led to a proposed concept of dual functionality as it is in the case of B7–1/B7–2 and CD28/CTLA-4. As an example, B7-H7 receptor CD28H could serve as an immunostimulatory receptor for T cell activation whereas KIR3DL3 could inhibit immune responses upon ligation of B7-H7 [38]. On the other hand, CD28H which is a CD28 homolog absent in mice but present in human serves as a functional receptor for B7-H5 [39]. B7-H5/CD28H interaction selectively costimulates human T-cell growth and cytokine production via an AKT-dependent signaling cascade. Interestingly, CD28H is constitutively expressed on all naïve T cells and its expression decreased with cell activation and is lost on terminally differentiated effector CD45RA + CCR7 − T cells [39]. Basically, the effector cytokine-producing CD4+ T helper cells and FoxP3+ CD4+ T reg cells lack CD28H expression. The authors associate such loss of expression for effector cells with repetitive cell stimulation. Moreover, the pattern on B7-H5 expression in peripheral tissue suggests that B7-H5/CD28H interaction is critical for the co-stimulation of newly generated effector or effector/memory T cells at the periphery. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells [40]. B7-H6 triggers NKp30-mediated activation of human NK cells [40]. In summary, the roles of B7-H4, B7-H5, B7-H6, and B7-H7 in allergic asthma are long overdue to be determined.

2.5 ILDR2 in the immune response

Ildr2 (Ig-like domain-containing receptor 2), the gene encoding the murine ortholog (formerly designated “Lisch-like”) was originally identified as a modifier of susceptibility to type 2 diabetes in obese mice [41]. Its expression level was associated with reduced β- cell number and reproduction and with persistent mild hypoinsulinemic hyperglycemia [41]. A new immunomodulatory function of this B7-like homolog protein has been recently reported by Hecht and associates [42]. They showed that the administration of a recombinant ILDR2-mFc protein to mice displayed a therapeutic effect in a model of rheumatoid arthritis. It induced an increase in the IgG1/IgG2a ratio which suggested a shift from the proinflammatory pro-rheumatic Th1 responses to anti-inflammatory Th2 responses. The ILDR2 upregulation was reported previously for DC cultures when they were stimulated to become DC2-like cell that promotes Th2 response [43]. Therefore, ILDR2 has a promoting effect on allergic diseases, however, it has never been investigated directly.

3. Neuroimmune semaphorins in asthma

Several neuronal guidance proteins, known as semaphorin molecules, function in the immune system. This dual tissue performance has led to them being defined as “neuroimmune semaphorins” [44]. They have been shown to regulate T cell activation by serving as immune checkpoints (Figure 3) [12]. Neuroimmune semaphorins are either constitutively or inducibly expressed on immune cells. The T cell co-stimulatory action of neuroimmune semaphorins requires the presence of two signals: signal one provided by TCR/MHC engagement and signal two arises from B7/CD28 interaction. Thus, neuroimmune semaphorins serve as a “signal three” for immune cell activation by supporting their polarization, expansion, differentiation, and regulating the intensity of immune response. This book chapter summarizes the current knowledge on the structure and receptors for several neuroimmune semaphorins involved in the immune response and their role in allergic asthma.

Figure 3.

Neuroimmune semaphorins in T cell – DC crosstalk. a. Sema3A. Sema3A inhibits T cell activation. Low constitutive levels of Sema3A on DC are upregulated with cell activation. DC surface-expressed and soluble Sema3A inhibit T cell proliferation presumably acting through NRP-1. Sema3A inhibits DC activation and chemotaxis. Inducible T cell-expressed and soluble Sema3A use NRP-1 and NRP-2 as ligand-binding receptors and NRP-associated Plexin A1 and A2 as signaling receptors to regulate DC activation and chemotaxis. b. Sema3E. Sema3E regulates DC subsets. Higher numbers of CD11b + DC and lower numbers of CD103+ DC were detected in the lungs of Sema3E−/− mice at the steady-state condition and after allergen sensitization. The DC receptor involved in such Sema3E action is Plexin D1. c. Sema4A. Sema4A-mouse Tim2 (mTim2) or human ILT4 (hILT4) pathways costimulate T cells. Sema4A on DC directly binds mTim-2 or hILT4 on T cells. This leads to optimal T cell activation, proliferation and cytokine production. d. Sema4D. Distinct receptor-dependent effects of T cell-expressed Sema4D on DC functions. Sema4D costimulates T cells. Sema4D serves as an indirect costimulatory molecule for T cell activation. Sema4D on T cells stimulates DC to accelerate their activation and maturation. Stimulated DC, in their turn, enhance T cell activation. The main receptor for such Sema4D action is believed to be CD72. Sema4D costimulates DC. T cell-expressing and soluble Sema4D ligation of DC-expressing Plexin B1 and B2 receptors stimulates DC proinflammatory cytokine production and migration. e. Sema6D. Sema6D acts as indirect T cell costimulatory molecule. T cell expressed Sema6D activates DC through Plexin A1 receptor. Polyclonally- or Ag-stimulated T cells upregulate Sema6D expression. Sema6D stimulates T cell viability, proliferation and cytokine production on late stages of immune response. f. Sema7A. Sema7A in T cell-DC interaction. Indirect T cell stimulation by T cell expressed Sema7A ligation of Plexin C1 on DC.

3.1 Sema3A and Sema3E

Sema3A, previously known as chick collapsin 1, SemD, or Sema III, was discovered in the 1990s. In the nervous system, it functions either as a repulsive agent for axonal outgrowth or an attractive agent for apical dendrite growth [45, 46, 47, 48]. Sema3A is a glycoprotein with an Ig-like C2-type domain, a PSI (cysteine-rich module in extracellular portion) domain, and a Sema domain. Antipenko and associates [49] reported the crystal structure of Sema3A and identified a neuropilin (NRP) binding site and a potential plexin interaction site. Further studies demonstrated the physiologic receptors for Sema3A which consist of NRP/Plexin complexes where NRP1 serves as a ligand-binding receptor whereas Plexin A1 functions as a signaling receptor [50, 51]. The secreted 95 kDa forms of Sema3A can further undergo a proteolytic cleavage forming the 65 kDa forms [49], which have decreased activity toward neurons [52, 53]. The cryoEM of extracellular complex of Sema3A, PlexinA4, and NRP1 at 3.7 Å resolution demonstrated a large symmetric 2:2:2 molecular assembly in which each subunit makes multiple interactions with others [54].

The immunomodulatory role of Sema3A in allergic asthma has been extensively studied by the laboratory of Dr. Vadasz at Technion, Israel [55, 56, 57]. When examining the serum levels of Sema3A in asthmatic patients with different stages of disease severity they have determined that Sema3A was significantly downregulated in both severe and moderate asthmatic patients when compared to that of healthy individuals [55]. Low levels of Sema3A correlated with asthma severity. Purified CD4 + T cells from asthmatic patients were incubated with recombinant human (rh) Sema3A protein for 24 h what led to a higher number of Treg cells as compared to similarly conditioned cell cultures from healthy controls [55]. Moreover, rhSema3A affected Treg cells directly by inducing a higher Foxp3 expression. Considering the results of these clinical studies and established downregulatory role of Treg cells in asthma, it is logical to conclude that Sema3A plays an inhibitory role in allergic disease in part by inducing and stabilizing Treg cells. Indeed, the low expression of Sema3A was noticed in the nasal epithelium in the animal model of allergic rhinitis as compared to control mice [58]. Re-introduction of recombinant Sema3A to the mouse nose alleviated sneezing and nasal rubbing symptoms in allergic mice. When rhSema3A was administered intraperitoneally to the mice treated with allergen, a downregulation of lung inflammatory response and angiogenesis was observed [56, 57]. However, the full understanding of the mechanisms of lung inflammation and angiogenesis suppression by Sema3A is still ill-defined. In summary, these experiments indicated that sema3A is a potential novel therapeutic agent for the treatment of bronchial asthma.

Sema3E (originally termed M-SemaH) was first identified in the metastatic cell lines using a differential display technique which allowed to identify 2 splice variants encoding the same 775 a.a. protein [59]. The protein consists of a putative signaling sequence in NH- terminus followed by a large semaphorin domain, a c2 immunoglobulin-like domain at the amino acids 595–659, approximately 20 residues serving as a transmembrane domain, and positively charged residues in the COOH-terminus [59]. Sema3E contains 13 conserved cysteine residues and 3 potential A’-glycosylation sites. The amino acid sequence of Sema3E was found to be 82% identical to the reported partial sequence of chick collapsin 5 and 44–48% to all other members of the subclass III of the family [59]. Also, the AU-rich motif (AUUUA) conferring protein instability has been defined.

The extensive work by Movassagh and associates from the laboratory of Dr. Gounni at the University of Manitoba, Canada [60] defined the effect of Sema3E deficiency in experimental mouse model of asthma. Such deficiency resulted in substantial airway eosinophilia in untreated Sema3E^−/− mice whereas the numbers of alveolar macrophages, T, B, NK, and NKT cells were comparable to those in WT mice. Therefore, the absence of Sema3E predisposed mice to allergic inflammation. Indeed, repeated inhalational exposure to HDM increased many components of asthmatic response in Sema3E^−/− mice. This increase involved peribronchial inflammation, AHR to methacholine challenges, goblet cell hyperplasia, collagen deposition, and Th2/Th17 cytokine levels. All these features of asthmatic response were significantly downregulated when recombinant Sema3E was administered to the allergen sensitized mice intranasally [61]. A higher frequency of CD11b + pulmonary DC, a Th2- promoting subtype of DC, was observed in Sema3E−/− mice in both, the steady-state and allergen sensitized conditions as compared to WT control animals. When adoptively transferred to naïve mice, these Sema3E−/− CD11b + DC were able to induce the highest allergic lung inflammatory response especially when the DC recipients were Sema3E−/− mice. While examining the generated bone marrow chimeric mice, the authors defined the contribution of Sema3E on bone marrow-derived inflammatory cells in allergen-induced lung pathology. This work aligns with their previous study demonstrating Sema3E-mediated inhibition of human ASM cell proliferation and migration and defining the signaling pathways involved in such effect [62]. Moreover, their recent study clearly demonstrated a suppressed Sema3E expression in human severe asthma using bronchial biopsy and lung tissue histology specimens [63]. These data suggest that Sema3E plays an important regulatory role in allergic asthma. Targeting this molecule could be a novel approach to treat allergic asthma.

3.2 Sema4A and Sema4D

The Sema4A molecule is a 761 aa long glycoprotein of 150 kDa molecular weight with an NH2-terminal 32 aa signal peptide, a Sema domain, and an Ig domain of the C2 type (both 651 aa), a hydrophobic 21 aa transmembrane region, and a 57 aa cytoplasmic tail (Swissprot Accession # Q9H3S1). Its functions are the most complicated, diverse, and least studied. Sema4A has six known receptors (reviewed in 12). Sema4A exists in both membrane-bound and soluble forms [64, 65]. On the cell surface, it is expressed as a monomer and a dimer [65].

The role of Sema4A in asthma has been evaluated in the laboratory of Dr. Chapoval at the University of Maryland, USA [64, 66, 67]. It has been shown previously that lung-specific vascular endothelial growth factor (VEGF) expression induced asthma-like pathologies in the murine lungs [68, 69]. The experimental models of OVA-induced and VEGF-mediated allergic airway inflammation were used to assess the changes in expression of immune semaphorins and their receptors in mouse lung tissues [64]. We reported Sema4A expression was detected on bronchial epithelial cells, smooth muscle cells, and accessory-like cells. Both external allergen and lung local VEGF upregulated the expression of Sema4A and its receptors in the lung tissue. Allergen treatment led to a detection of a whole Sema4A protein plus its dimer in the bronchoalveolar (BAL) fluids under inflammation which was not found in the control mouse group. In vivo allergic response which consisted of eosinophilic BAL and lung tissue infiltration, mucous cell hyperplasia, AHR to methacholine challenges, sera Ag-specific IgG1/IgG2b/IgE contents, and IL-13 levels in BAL, sera, and cell cultures, was significantly upregulated in Sema4A−/− mice as compared to similarly treated WT mice [66]. In our next study, we employed in vivo re-introduction of rhSema4A to Sema4A-deficient and sufficient mice before the allergen challenge which was sufficient to downregulate the number of BAL eosinophils and the levels of BAL cytokines such as IL-6, IL-17A and TNFα [67]. Moreover, using rhSema4A in a chronic model of allergen exposure, we showed that it retains a potent anti-inflammatory effect even when lung tissue damage and remodeling are established [67]. The observed in vivo critical regulatory effect of Sema4A in acute and chronic allergic responses suggests that Sema4A-related pathways may be used for an immunotherapeutic asthma intervention.

A recent study by Lynch and associates [70] examined the role of Sema4A in respiratory syncytial virus (RSV)-induced bronchiolitis which is a predisposition for asthma. The authors used BDCA2-diphtheria toxin receptor (DTR) transgenic mice to induce the specific and reversible depletion of plasmacytoid DC (pDC) with intraperitoneal DT injections. They showed that pDC depletion in neonatal, but not adult, mice increased bronchiolitis severity and was sufficient to evoke an asthma-like phenotype upon viral challenge thus conforming that severe bronchiolitis in early life predisposes to subsequent asthma upon viral exposure. They also demonstrated that pDC from virus-infected mice expand Foxp3 + NRP1+ Treg cells and such expansion is effectively inhibited by the use of anti-Sema4A neutralizing Ab. Moreover, NRP1+ Treg cells transfer from infected to naïve mice prevents the recipients from viral bronchiolitis and subsequent asthma. This study further strengthens the importance of the Sema4A-mediated Treg cells expansion pathway and its important role in asthma protection and/or suppression.

Sema4D, also known as Cluster of Differentiation 100 (CD100), was the first semaphorin with defined expression and function in the immune system ([71, 72], reviewed in [12, 44, 73]). Several studies pointed to its critical regulatory role in the immune system ([74, 75], reviewed in [12, 44, 73, 76]). Sema4D consists of an NH2- terminal signal peptide, a sema domain, an Ig domain of the C2 type, a hydrophobic transmembrane region, and a cytoplasmic tail [71, 72]. The molecule’s crystal modeling demonstrates the presence of a conserved seven-blade β-propeller structure [77] which is the structure of a conserved sema domain and is shared by all semaphorin family members. There is an 88% amino acid identity between human and murine Sema4D homologs [72]. Sema4D exists in both, membrane-bound and soluble forms, which are both biologically active [78, 79].

The recent report from Dr. Chapoval’s laboratory at the University of Maryland has demonstrated an important regulatory role of Sema4D in asthma pathogenesis [80]. We exposed Sema4D-deficient and WT mice to OVA injections and challenges in the well-defined mouse model of OVA-induced experimental asthma. Sema4D-deficient mice demonstrated a significant decrease in eosinophilic airway infiltration after allergen challenge relative to WT mice. This reduced allergic inflammatory response was associated with decreased BAL Th2 and Th17 cytokine levels. The reduced T cell proliferation in OVA₃₂₃₋₃₃₉-restimulated Sema4D−/− cell cultures suggested lower T cell activation. Sema4D deficiency led to the increased number of Treg cells in mice after the allergen challenge. Surprisingly, Sema4D deficiency had no effect on airway hyperreactivity (AHR) to methacholine challenges in either acute or chronic experimental disease settings. Moreover, the lung DC number and activation were not affected by Sema4D deficiency. Our research data provided new insight into Sema4D biology and defined Sema4D as an important regulator of Th2-driven lung inflammation and as a potential target for disease immunotherapy.

3.3 Sema6D and Sema7A

Molecular cloning, mapping, and functional analysis of Sema6D together with Sema6C have been carried out more recently if compared to other semaphorins with costimulatory properties ([81], reviewed in [12]). Amino acid sequence alignment analysis of human semaphorin (HSA)SEMA6C, rat Sema6C, and mouse Sema6C showed the existence of the class VI semaphorin characteristic of the extracellular domain and PSI domain, which differ from all known members of semaphorin family. Predicted structure (HSA)SEMA6D isoforms were compared with related semaphorin proteins. Five isoforms of SEMA6D have been isolated and the significance of the alternatively spliced variants was evaluated by RT-PCR and Northern blots. The expression of different isoforms was found to be regulated in a tissue- and development-dependent manner. Sema6D consists of a signal peptide, a PSI domain, a transmembrane segment, an Ig domain, and a sema domain. Sequence analysis has shown that the translated polypeptides are composed of a 1–21 aa signal peptide followed by a 59–477 aa sema domain, a 508–563 aa PSI domain, a transmembrane segment, and a long cytoplasmic region.

The role of Sema6D in asthma has never been investigated. Based on the published data claiming a costimulatory role of Sema6D in T cell activation, we assume it regulates a disease severity. Regulation of T cell activation by Sema6D was examined in vitro and in vivo [82]. Upon T cell activation, after an initial decrease in Sema6D mRNA expression, they observed its stable upregulation and, later, a protein expression on the surface of T cells. This upregulation was relevant to both anti-CD3/CD28-stimulated and Ag-stimulated T cells. Using Sema6D-Ig, the authors identified Plexin A1 on DC as a Sema6D receptor. Interestingly, when anti-Sema6D blocking Ab was added to the cell cultures, it affected T cell proliferation in late stages (4–6 days of culture) whereas in the early stages (2–4 days), T cell viability and proliferation, as well as cytokine (IL-2) production, were not different from those without Ab in the culture. Specific targeting of Sema6D decreased T cell activation in vivo in the OTII cell adoptive transfer model. In this model, OTII T cells were obtained from the aTCR-transgenic strain that contains rearranged TCR-Vα and -Vβ genes in the germline DNA encoding a TCR specific for chicken ovalbumin (OVA) peptide 323–339 bound to I-A molecules in a context of H-2b haplotype (CD45.2). These CD45.2 cells were adoptively transferred to congenic B6-Ly5.2/Cr (CD45.1) recipients. The splenocyte proliferation was assessed as an expansion of donor OTII T cells in the recipient mice and expressed as the percentage of TCR+ CD4+ CD45.2+ cells in the isolated splenocyte population. The donor cell numbers were significantly lower when the recipient (CD45.1) mice received Sema6D-Ig at the time of cell plus OVA protein injections. Interestingly, Sema6D-Ig treatment did not affect an early T cell activation (day 4) but significantly reduced CD45.2+ T cell expansion on day 7. It is still unclear, however, if Plexin A1 is the only functional receptor for Sema6D on DC.

It is well established that macrophage polarization is a result of and a contributor to asthma pathogenesis [83]. Macrophages consist of more than 70% of lung cells and increased M2 macrophage polarization mirrored by increased Th2 response leads to further heightening of asthma pathology [83]. Macrophages and DCs expressed high levels of Sema6D [84]. Sema4D deficiency led to a downregulation of M2 polarization by bone marrow-derived macrophages accompanied by significant reductions in expression of Arg1, chitinase 3 like-1 (Chi3l1), Retnla, and Il10, as determined by qRT-PCR [84]. In vivo, Sema6D^−/− mice demonstrated an exaggerated inflammatory response to LPS-induced sepsis accompanied by elevated levels of proinflammatory cytokines, including IL-12p40, TNF, and IL-6 as compared to WT mice. This study indirectly demonstrated the important role of Sema6D in asthma in part by regulation of macrophage polarization and activation.

The cDNA clone containing the entire coding sequence of the Sema7A gene and its molecular characteristics were first reported by Yamada and associates [85]. The human Sema7A cDNA clones were identified through the screening of a plasmid library generated from a leukemic T cell line. The 1998-base pairs of the cloned DNA’s open reading frame encoded a 666 aa protein. This protein contained a 46 aa signal peptide and a 19 aa GPIanchor glycophosphatidylinositol linkage motif. The membrane-anchoring form of Sema7A was 602 aa long. The estimated molecular mass of the nonglycosylated form was 68 kDa. The authors located an “RGD (Arg-Gly-Asp) cell attachment sequence and the five potential N-linked glycosylation sites on the membrane-anchoring form”. The expression of a native Sema7A form in transfected cells was confirmed by immunoprecipitation and flow cytometry analyses of cell transfectants. The Sema7A gene was identified on chromosome 15 (15q23–24) by radiation hybrid mapping. The 88.0% similarity at the nucleotide level was detected between murine and human Sema7A or 89.3% similarity at the amino acid level of corresponding proteins [86]. Both human and mouse SEMA7A contain a seven-bladed β-propeller semaphorin N- terminus domain, a plexin, semaphorin, and integrin domain (PSI), an immunoglobulin-like domain, and the characteristic for this particular semaphorin molecule GPI anchor in their C-terminus [87].

The extensive examination of a costimulatory function of Sema7A in T cell proliferation established this neuroimmune semaphorin as an inhibitor of T cell activation [88]. Sema7A^−/− T cells demonstrated an enhanced proliferation upon Ag re-stimulation in vitro. However, no significant differences were observed in WT and Sema7A−/− DC maturation induced by various TLR ligands. Moreover, Sema7A deficiency in DC did not affect their ability to activate WT T cells. Furthermore, Sema7A^−/− Treg cells were functional and actively comparably to WT Treg cells suppressed experimental colitis in vivo. This further points to a specific defect in the naive CD4 T cells associated with Sema7A deficiency. The proposed model of Sema7A function in T cell signaling is that Sema7A interacts with the components of the TCR complex and with a putative receptor on APCs which stabilizes TCR/CD3 complex and promotes inhibitory signals that limit T cell proliferation. The role of Sema7A in asthma has been reported in two recent publications [89, 90]. Sema7A was found to be expressed on the surface of circulating eosinophils and upregulated on bronchoalveolar lavage eosinophils obtained after segmental bronchoprovocation of asthmatic patients with allergen. Moreover, among all BAL cells, eosinophils were the predominant source of Sema7A. Among the members of the IL-5-family cytokines such as IL-3, IL-5, and GM-CSF, Sema7A protein on the surface of blood eosinophils was increased most by IL-3 exposure. The adherence of IL-3-activated eosinophils to the plate-bound receptor plexin C1 was doubled from the initial 30% in inactivated cells to 60% which proves the functional effect of Sema7A expressed on the eosinophil surface. Interestingly and relevant to asthma pathophysiology, a recombinant Sema7A induced alpha-smooth muscle actin production in human bronchial fibroblasts. These studies established semaphorin 7A as an important modulator of eosinophil profibrotic functions in the airway remodeling of patients with chronic asthma. The interference with the described pathway holds the potential to modulate asthma inflammation in the future (Tables 1 and 2).

B7 family member	Role in asthma	Function	Reference
B7–1/B7–2	Stimulatory	Stimulates T cell activation and inflammatory cytokines production	[17, 18]
B7-H1 (PD-L1)/B7-DC (PD-L2)	Inhibitory	Downregulates inflammatory cytokines production and airway hyperreactivity	[22, 25, 26]
B7-H2	Stimulatory	Induces Th2 cytokines and IgE productions	[28, 29]
B7-H3	Stimulatory	Increases Th2 and Th17 cytokine production	[35, 36]
B7-H4	Unknown
B7-H5	Unknown
B7-H6	Unknown
B7-H7	Unknown
ILDR2	Stimulatory	Promotes Th2 response	[42, 43]

Table 1.

Role of the B7 family members in asthma.

Semaphorin	Role in asthma	Function	Reference
Sema3A	Inhibitory	Stimulates Treg cells.; Low serum levels correlate with asthma severity; Downregulates lung inflammatory response	[55, 56, 57, 58]
Sema3E	Inhibitory	Sema3E deficiency upregulates asthmatic response, led to a high frequency of Th2 promoting DC. Sema3E inhibits ASM cell proliferation. Low lung tissue expression is associated with higher asthma severity.	[60, 61, 62, 63]
Sema4A	Inhibitory	Sema4A deficiency led to increases in many asthma parameters. Recombinant Sema4A applications in vivo led to a suppression of asthmatic response. Stimulates Treg cells in vitro and in vivo, induces new Treg cells in the in vitro CD4 + T cell cultures	[66, 67]
Sema4D	Stimulatory	Sema4D deficiency led to a lower lung inflammatory response to allergen challenges, lower T cell activation, and increased number of Treg cells	[80]
Sema6D	Unknown
Sema7A	Inhibitory	Expressed on eosinophils. Regulates ASM contractility. Eosinophils are predominant source of Sema7A in the lungs. Lung Sema7A expression is upregulated by allergen bronchoprovocation	[89, 90]

Table 2.

Role of neuroimmune semaphorins in asthma.

4. Conclusions

Analysis of costimulatory molecules critically involved in asthma, a chronic respiratory Th2-driven disease, will help us to underline the immune mechanisms of disease development and progression. A complete understanding of these mechanisms will guide the development of novel therapeutic strategies to combat asthma and related allergies. Studies aimed to characterize the functions of several B7 family members and semaphorin family members in allergic asthma are either incomplete or ongoing. Further studies of the interplays between different individual costimulatory pathways should provide clearer insights into the disease pathology and guide the development of precise therapeutics.

Acknowledgments

S.P.C is supported by SemaPlex LLC and by NIH/NIAID RO1 AI076736 and RO1 AI143845 grants where she is a co-investigator. A.I.C. is supported by the Ministry of Science and Higher Education of the Russian Federation grant No. FZMW-2020-0007.