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Please note this is a comparison between Version 1 by Monique Vogel and Version 6 by Conner Chen.
Allergic diseases represent a global health and economic burden of increasing significance. The lack of disease-modifying therapies besides specific allergen immunotherapy (AIT) which is not available for all types of allergies, necessitates the study of novel therapeutic approaches. Exosomes are small endosome-derived vesicles delivering cargo between cells and thus allowing inter-cellular communication. Since immune cells make use of exosomes to boost, deviate, or suppress immune responses, exosomes are intriguing candidates for immunotherapy.
- exosomes
- allergy
- immunotherapy
1. Allergy and Allergy Immunotherapy
Allergic diseases are a global issue as more and more people are affected by allergies [1]. Type I hypersensitivity is characterized by abnormal IgE-mediated inflammation in response to harmless antigens called allergens caused by a lack of immune tolerance coupled with the expansion of TH2 cells that drive IgE responses from B cells [2,3]. Allergen-specific IgE sensitizes mast cells and basophils by binding to the high-affinity IgE receptor FcεRI [4]. Upon secondary contact with the allergen, those cells degranulate and release inflammatory mediators [5]. Symptomatic treatment options for allergies involve down-regulation of the mediators released by mast cells or basophils (e.g., anti-histamine) or aim to down-regulate IgE levels, such as the monoclonal anti-IgE antibody Omalizumab [6]. The only disease modifying treatment available for some but not all allergies is allergen-specific immunotherapy (short AIT or SIT). AIT is a repeated immunization approach that aims to re-educate the immune system and generate tolerance towards the allergen [7,8]. Mechanistically, AIT induces regulatory T cells and B cells that are able to produce anti-inflammatory cytokines, such as IL-10 and TGF-β. This leads to suppression in TH2 responses but increased IgG4 production [9,10,11]. A novel approach in allergy immunotherapy is to boost immune responses by eliciting a non-allergic, but rather anti-viral/bacterial TH1-like response in an attempt to shift the immune response [12]. In general, it is accepted that IgG antibodies can suppress IgE-mediated effector functions by competing for the allergen epitope thus neutralizing IgE, or by ligation of inhibitory FcγRIIb receptors on mast cells/basophils [13,14,15]. FcγRIIb receptors contain immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domains that shut down FcεRI-dependent effector cell activation [16,17,18]. Additionally, FcγRs and IgG can promote the internalization of IgE thus preventing IgE-dependent activation of mast cells and basophils [19]. However, further research in this area is still required as IgG can also contribute to inflammation, depending on the IgG subclass and type of FcγR receptor involved [20]. In summary, AIT is a viable therapy that still requires improvement as it is not available for all allergens and often bears the risk of side effects caused by allergen application, which can degranulate allergic effector cells via FcεRI-displayed IgE. Therefore, there is a need for novel treatment strategies that reduce side effects while maintaining efficacy.
Allergic diseases are a global issue as more and more people are affected by allergies [1]. Type I hypersensitivity is characterized by abnormal IgE-mediated inflammation in response to harmless antigens called allergens caused by a lack of immune tolerance coupled with the expansion of TH2 cells that drive IgE responses from B cells [2][3]. Allergen-specific IgE sensitizes mast cells and basophils by binding to the high-affinity IgE receptor FcεRI [4]. Upon secondary contact with the allergen, those cells degranulate and release inflammatory mediators [5]. Symptomatic treatment options for allergies involve down-regulation of the mediators released by mast cells or basophils (e.g., anti-histamine) or aim to down-regulate IgE levels, such as the monoclonal anti-IgE antibody Omalizumab [6]. The only disease modifying treatment available for some but not all allergies is allergen-specific immunotherapy (short AIT or SIT). AIT is a repeated immunization approach that aims to re-educate the immune system and generate tolerance towards the allergen [7][8]. Mechanistically, AIT induces regulatory T cells and B cells that are able to produce anti-inflammatory cytokines, such as IL-10 and TGF-β. This leads to suppression in TH2 responses but increased IgG4 production [9][10][11]. A novel approach in allergy immunotherapy is to boost immune responses by eliciting a non-allergic, but rather anti-viral/bacterial TH1-like response in an attempt to shift the immune response [12]. In general, it is accepted that IgG antibodies can suppress IgE-mediated effector functions by competing for the allergen epitope thus neutralizing IgE, or by ligation of inhibitory FcγRIIb receptors on mast cells/basophils [13][14][15]. FcγRIIb receptors contain immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domains that shut down FcεRI-dependent effector cell activation [16][17][18]. Additionally, FcγRs and IgG can promote the internalization of IgE thus preventing IgE-dependent activation of mast cells and basophils [19]. However, further research in this area is still required as IgG can also contribute to inflammation, depending on the IgG subclass and type of FcγR receptor involved [20]. In summary, AIT is a viable therapy that still requires improvement as it is not available for all allergens and often bears the risk of side effects caused by allergen application, which can degranulate allergic effector cells via FcεRI-displayed IgE. Therefore, there is a need for novel treatment strategies that reduce side effects while maintaining efficacy.
2. The Biology of Exosomes
The biology of exosomes was reviewed by Kalluri & LeBleu [21]. In brief, Extracellular vesicles (EVs) are small membrane blebs with a diameter of approximately 40 nm–1 µm released from all cell types. They are found in different fluids, such as plasma, urine, semen, bronchial fluid, and synovial fluid. Exosomes (40–160 nm) are of endosomal origin, which distinguishes them from ectosomes that bud from the surface of plasma membranes. Exosomes are surrounded by a bilayer lipid membrane composed of cholesterol, phosphatidylserines, and sphingolipids, which confer protection against proteases and RNases [22]. Exosome surface proteins include the classical exosome markers, the Tetraspanin proteins CD9, CD63, CD81, CD82, as well as adhesion molecules and the immune regulator molecules major histocompatibility complex (MHC) Class I and II [23]. Tetraspanins are involved in cell penetration, invasion, and fusion events allowing exosomes to provide their targets with molecules by transferring membrane material by fusing to target cells without the need of direct cell-cell contact. Besides their surface proteins, exosomes can carry a variety of cargo proteins. Exosomes are enriched with endosomal proteins from parent cells implicated in exosome biogenesis including ESCRT-related proteins (Alix and TSG101) and cytoplasmic proteins, such as Annexins and Rab GTPases, responsible for membrane transport and fusion of exosomes to the cell membrane. Additionally, exosomes contain heat shock proteins (e.g., HSP90, HSP70) which help peptide loading on MHC, and metabolic enzymes, such as ATPase or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [24]. Besides their protein cargo, exosomes are loaded with cargo RNA. Different types of RNAs are enriched in exosomes including coding messenger RNA and non-coding RNAs, such as ribosomal RNA (rRNA) and miRNA. Those miRNAs can act as regulator molecules in recipient cells to alter gene expression at the post-transcriptional level by targeting mRNA transcripts [25,26]. In summary, exosomes contain a variety of molecules that are involved in key cellular functions that play a role in physiological and pathological processes [27].
The biology of exosomes was reviewed by Kalluri & LeBleu [21]. In brief, Extracellular vesicles (EVs) are small membrane blebs with a diameter of approximately 40 nm–1 µm released from all cell types. They are found in different fluids, such as plasma, urine, semen, bronchial fluid, and synovial fluid. Exosomes (40–160 nm) are of endosomal origin, which distinguishes them from ectosomes that bud from the surface of plasma membranes. Exosomes are surrounded by a bilayer lipid membrane composed of cholesterol, phosphatidylserines, and sphingolipids, which confer protection against proteases and RNases [22]. Exosome surface proteins include the classical exosome markers, the Tetraspanin proteins CD9, CD63, CD81, CD82, as well as adhesion molecules and the immune regulator molecules major histocompatibility complex (MHC) Class I and II [23]. Tetraspanins are involved in cell penetration, invasion, and fusion events allowing exosomes to provide their targets with molecules by transferring membrane material by fusing to target cells without the need of direct cell-cell contact. Besides their surface proteins, exosomes can carry a variety of cargo proteins. Exosomes are enriched with endosomal proteins from parent cells implicated in exosome biogenesis including ESCRT-related proteins (Alix and TSG101) and cytoplasmic proteins, such as Annexins and Rab GTPases, responsible for membrane transport and fusion of exosomes to the cell membrane. Additionally, exosomes contain heat shock proteins (e.g., HSP90, HSP70) which help peptide loading on MHC, and metabolic enzymes, such as ATPase or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [24]. Besides their protein cargo, exosomes are loaded with cargo RNA. Different types of RNAs are enriched in exosomes including coding messenger RNA and non-coding RNAs, such as ribosomal RNA (rRNA) and miRNA. Those miRNAs can act as regulator molecules in recipient cells to alter gene expression at the post-transcriptional level by targeting mRNA transcripts [25][26]. In summary, exosomes contain a variety of molecules that are involved in key cellular functions that play a role in physiological and pathological processes [27].
3. The Biomedical Application of Exosomes
EVs, and specifically exosomes have been a fast-rising topic in the development of novel biomedical therapeutics due to their relevance as cellular communicators. The recent advances in engineering and application of exosomes were reviewed in depth by Perocheau et al. [28]. Of specific interest to the field of immunology is the fact that immune responses are regulated by exosomes as antigens, MHC complexes, and co-stimulatory molecules are transferred between immune cells. Furthermore, the physiological nature of exosomes as delivery agents has been viewed as a potential advantage compared to other therapeutics that can elicit anti-drug responses [29]. Other advantages include their non-toxicity, their wide distribution in biological fluids, the ability to induce functional responses in specific target cells, their variability in cargo as well as their ability to easily cross biological barriers. The fact that exosome function is dysregulated in a variety of diseases makes their direct therapeutic potential even more intriguing [30]. It is established that exosomes play a major role during various steps of cancer growth and metastasis [31]. Another example are viruses, such as Human T-cell Lymphotropic Virus Type-1 (HTLV-1) in which viral proteins packaged into EVs promote the development of inflammation and enhance viral spread in organs and peripheral blood [32]. Of interest for this review is the fact that exosomes also seem to play a role in the dysregulated communication in allergy and allergic asthma [33]. Recently, several exosome-based immunotherapeutics have entered clinical trials, demonstrating the promise of this technology. The various candidates are produced in mammalian cell culture or cow’s milk. Whereas some candidates are used unmodified others have been specifically engineered, for example by expressing the pro-inflammatory cytokine IL-12 or Stimulator of Interferon Genes (STING) which aim to promote the identification and killing of cancer cells by the immune system [34,35]. Furthermore, exosomes can be used to cross the blood-brain barrier in order to deliver siRNA for reducing the expression of genes, such as BACE which is a potential gene involved in Alzheimer’s disease [36]. Exosomes are also used as a delivery system shielding other established therapeutics from anti-drug responses by the immune system [37]. Even though exosome technology has progressed quickly in recent years, the main hurdle left is the large-scale production and purification of exosomes. Current exosome purification methods include density gradient and ultracentrifugation, chromatography or precipitation using chemicals polymers or capture by antibodies which have been compared by Konoshenko et al. [38]. In conclusion, while this new technology is still evolving, several exosome-based therapeutics are moving into clinics thus highlighting their potential as a new generation of therapeutics.
EVs, and specifically exosomes have been a fast-rising topic in the development of novel biomedical therapeutics due to their relevance as cellular communicators. The recent advances in engineering and application of exosomes were reviewed in depth by Perocheau et al. [28]. Of specific interest to the field of immunology is the fact that immune responses are regulated by exosomes as antigens, MHC complexes, and co-stimulatory molecules are transferred between immune cells. Furthermore, the physiological nature of exosomes as delivery agents has been viewed as a potential advantage compared to other therapeutics that can elicit anti-drug responses [29]. Other advantages include their non-toxicity, their wide distribution in biological fluids, the ability to induce functional responses in specific target cells, their variability in cargo as well as their ability to easily cross biological barriers. The fact that exosome function is dysregulated in a variety of diseases makes their direct therapeutic potential even more intriguing [30]. It is established that exosomes play a major role during various steps of cancer growth and metastasis [31]. Another example are viruses, such as Human T-cell Lymphotropic Virus Type-1 (HTLV-1) in which viral proteins packaged into EVs promote the development of inflammation and enhance viral spread in organs and peripheral blood [32]. Recently, several exosome-based immunotherapeutics have entered clinical trials, demonstrating the promise of this technology. The various candidates are produced in mammalian cell culture or cow’s milk. Whereas some candidates are used unmodified others have been specifically engineered, for example by expressing the pro-inflammatory cytokine IL-12 or Stimulator of Interferon Genes (STING) which aim to promote the identification and killing of cancer cells by the immune system [33][34]. Furthermore, exosomes can be used to cross the blood-brain barrier in order to deliver siRNA for reducing the expression of genes, such as BACE which is a potential gene involved in Alzheimer’s disease [35]. Exosomes are also used as a delivery system shielding other established therapeutics from anti-drug responses by the immune system [36]. Even though exosome technology has progressed quickly in recent years, the main hurdle left is the large-scale production and purification of exosomes. Current exosome purification methods include density gradient and ultracentrifugation, chromatography or precipitation using chemicals polymers or capture by antibodies which have been compared by Konoshenko et al. [37]. In conclusion, while this new technology is still evolving, several exosome-based therapeutics are moving into clinics thus highlighting their potential as a new generation of therapeutics.
4. Exosomes in the Regulation of Immune Responses
The transfer of exosomes between immune cells can have a strong functional effect on immune responses and either promote, deviate or suppress immune responses [39,40,41]. Many studies have shown that dendritic cells modulate CD4+, as well as CD8+ immune responses via exosomes that carry MHCI or MHCII molecules as well as co-stimulatory molecules CD8/CD86 [42,43,44]. Interestingly, extracellular vesicles containing intact p-MHC complexes pre-loaded with antigen-derived peptide can also lead to direct antigen presentation to T cells without the need for antigen processing, this mechanism of antigen presentation is referred to as “cross-dressing” [45,46,47]. In cross-dressing, DC-derived exosomes can be recaptured by DC and can remain on the cell surface to be directly presented by DC or internalized to be reloaded on endogenous MHC-class I molecules. Besides the exosomal transfer of p-MHCII and native antigens, RNA cargo provides a fundamental mechanism for intercellular communication [25]. Donor cells package mRNA or small non-coding micro RNAs (miRNAs) into exosomes. In the exosome-receiving cell, mRNAs can be translated into proteins and miRNAs can post-transcriptionally regulate target mRNAs. The transfer of miRNA exists in immune cells as means for antigen presenting cells (APCs) communication and activation [48,49,50]. Interestingly, viruses can highjack this system as EBV-infected B cells transfer viral miRNAs to DCs that silence immune-stimulatory molecules [51]. Regulatory T cells can suppress CD4+ T cell proliferation and cytokine production by transferring miRNA via exosomes which block gene expression [52,53]. Mesenchymal stem cells (MSC) are of high interest in the treatment of inflammatory diseases as they produce anti-inflammatory exosomes that can suppress DC maturation, T cell activation and promote regulatory T cells and B cells [54,55,56,57]. Cancer cells use exosomes carrying tumor antigens or inhibitory molecules that can suppress the activation of DCs, T cells and NK cells in the tumor microenvironment [58,59,60]. Due to the high variety of immunomodulatory properties, exosomes have the potential to specifically address different types of diseases depending on immunopathology [61,62,63].
The transfer of exosomes between immune cells can have a strong functional effect on immune responses and either promote, deviate or suppress immune responses [38][39][40]. Many studies have shown that dendritic cells modulate CD4+, as well as CD8+ immune responses via exosomes that carry MHCI or MHCII molecules as well as co-stimulatory molecules CD8/CD86 [41][42][43]. Interestingly, extracellular vesicles containing intact p-MHC complexes pre-loaded with antigen-derived peptide can also lead to direct antigen presentation to T cells without the need for antigen processing, this mechanism of antigen presentation is referred to as “cross-dressing” [44][45][46]. In cross-dressing, DC-derived exosomes can be recaptured by DC and can remain on the cell surface to be directly presented by DC or internalized to be reloaded on endogenous MHC-class I molecules. Besides the exosomal transfer of p-MHCII and native antigens, RNA cargo provides a fundamental mechanism for intercellular communication [25]. Donor cells package mRNA or small non-coding micro RNAs (miRNAs) into exosomes. In the exosome-receiving cell, mRNAs can be translated into proteins and miRNAs can post-transcriptionally regulate target mRNAs. The transfer of miRNA exists in immune cells as means for antigen presenting cells (APCs) communication and activation [47][48][49]. Interestingly, viruses can highjack this system as EBV-infected B cells transfer viral miRNAs to DCs that silence immune-stimulatory molecules [50]. Regulatory T cells can suppress CD4+ T cell proliferation and cytokine production by transferring miRNA via exosomes which block gene expression [51][52]. Mesenchymal stem cells (MSC) are of high interest in the treatment of inflammatory diseases as they produce anti-inflammatory exosomes that can suppress DC maturation, T cell activation and promote regulatory T cells and B cells [53][54][55][56]. Cancer cells use exosomes carrying tumor antigens or inhibitory molecules that can suppress the activation of DCs, T cells and NK cells in the tumor microenvironment [57][58][59]. Due to the high variety of immunomodulatory properties, exosomes have the potential to specifically address different types of diseases depending on immunopathology [60][61][62].
5. Exosomes in Allergic Sensitization and Inflammation
Exosomes play a major role in allergy including sensitization, allergen presentation and TH2 polarization, and the recruitment and activation of macrophages and eosinophils. Allergic sensitization is driven by barrier disruption of skin or lung where inflammatory signals from epithelial cells, including thymic stromal lymphopoietin (TSLP)/IL-25/IL-33, are thought to activate type 2 innate lymphoid cells (ILC2) and thus drive type 2 immunity [63][64]. Interestingly, it was shown that TSLP-activated DCs release OX40L expressing exosomes that drive CD4+ TH2 proliferation and differentiation [65]. Exosomes are involved in asthmatic inflammation which has been reviewed in depth by Cañas et al. [66]. MicroRNAs seem to play a significant role in the asthmatic process, which was supported by findings that miRNA is differentially expressed in the sputum of asthma patients [67][68]. Another interesting study showed differences in 24 exosomal miRNAs in bronchoalveolar fluids (BAL) of allergic versus asthmatic patients [69]. The miRNA-17-92 cluster (miRNA-17-5p, miRNA-17-3p, miRNA-18a, miRNA-19a, miRNA-19b, miRNA-20a, and miRNA-92-1) was shown to be an important general regulator of T cell biology [70] and among the different miRNAs in the cluster, miR-19 is specifically upregulated in CD4+ T cells from asthmatic patients compared to healthy individuals [71]. Like the miR-17-92, the miR-23 cluster plays a role in T cell function and in particular in controlling TH2 differentiation by targeting IL-4 and GATA3 [72]. Upon allergen exposure, exosomes released from epithelial cells induce the proliferation and the chemotaxis of macrophages during asthmatic inflammation [73]. Recently, a study showed that in epithelial exosomes, contactin-1 (CNTN1) is involved in the activation and recruitment of monocyte-derived dendritic cells and T-cell responses in allergic asthma [74]. Likewise, eosinophil-derived exosomes promote eosinophil migration, augment adhesion by a specific increase of adhesion molecules, such as ICAM-1 and induce reactive oxygen species (ROS) and nitric oxide (NO) production in an autocrine fashion [75]. Additionally, this leads to alveolar epithelial cell (AEC) death, delay wound repair and increase airway smooth muscle cell proliferation which causes airway obstruction and tissue remodeling [76]. Exosome production is also increased by airway allergen exposure as it was shown that PBMCs from house dust mites (HDM) allergic patients produce higher numbers of exosomes in response to HDM re-stimulation and HDM-induced exosomes were also shown to contain altered cargo/properties than exosomes produced in unstimulated PBMCs [77][78]. An interesting report showed that DCs are able to package native cat allergen Fel d 1 into exosomes [79]. B cell-derived exosomes were reported to carry processed birch allergen Bet V 5 peptide/MHCII complexes that can stimulate proliferation, IL-5, and IL-13 production from BET v 1 specific T cells lines [80]. Hence, even though detailed mechanistic studies are still required to better understand the exact role of exosomes in allergy, they have been shown to be involved in a number of key allergic processes due to their immunomodulatory function and are thus attractive candidates for the development of novel therapeutics in allergic diseases (
Exosomes play a major role in allergy including sensitization, allergen presentation and TH2 polarization, and the recruitment and activation of macrophages and eosinophils. Allergic sensitization is driven by barrier disruption of skin or lung where inflammatory signals from epithelial cells, including thymic stromal lymphopoietin (TSLP)/IL-25/IL-33, are thought to activate type 2 innate lymphoid cells (ILC2) and thus drive type 2 immunity [64,65]. Interestingly, it was shown that TSLP-activated DCs release OX40L expressing exosomes that drive CD4+ TH2 proliferation and differentiation [66]. Exosomes are involved in asthmatic inflammation which has been reviewed in depth by Cañas et al. [67]. MicroRNAs seem to play a significant role in the asthmatic process, which was supported by findings that miRNA is differentially expressed in the sputum of asthma patients [68,69]. Another interesting study showed differences in 24 exosomal miRNAs in bronchoalveolar fluids (BAL) of allergic versus asthmatic patients [70]. The miRNA-17-92 cluster (miRNA-17-5p, miRNA-17-3p, miRNA-18a, miRNA-19a, miRNA-19b, miRNA-20a, and miRNA-92-1) was shown to be an important general regulator of T cell biology [71] and among the different miRNAs in the cluster, miR-19 is specifically upregulated in CD4+ T cells from asthmatic patients compared to healthy individuals [72]. Like the miR-17-92, the miR-23 cluster plays a role in T cell function and in particular in controlling TH2 differentiation by targeting IL-4 and GATA3 [73]. Upon allergen exposure, exosomes released from epithelial cells induce the proliferation and the chemotaxis of macrophages during asthmatic inflammation [74]. Recently, a study showed that in epithelial exosomes, contactin-1 (CNTN1) is involved in the activation and recruitment of monocyte-derived dendritic cells and T-cell responses in allergic asthma [75]. Likewise, eosinophil-derived exosomes promote eosinophil migration, augment adhesion by a specific increase of adhesion molecules, such as ICAM-1 and induce reactive oxygen species (ROS) and nitric oxide (NO) production in an autocrine fashion [76]. Additionally, this leads to alveolar epithelial cell (AEC) death, delay wound repair and increase airway smooth muscle cell proliferation which causes airway obstruction and tissue remodeling [77]. Exosome production is also increased by airway allergen exposure as it was shown that PBMCs from house dust mites (HDM) allergic patients produce higher numbers of exosomes in response to HDM re-stimulation and HDM-induced exosomes were also shown to contain altered cargo/properties than exosomes produced in unstimulated PBMCs [78,79]. An interesting report showed that DCs are able to package native cat allergen Fel d 1 into exosomes [80]. B cell-derived exosomes were reported to carry processed birch allergen Bet V 5 peptide/MHCII complexes that can stimulate proliferation, IL-5, and IL-13 production from BET v 1 specific T cells lines [81]. Hence, even though detailed mechanistic studies are still required to better understand the exact role of exosomes in allergy, they have been shown to be involved in a number of key allergic processes due to their immunomodulatory function and are thus attractive candidates for the development of novel therapeutics in allergic diseases (
Figure 1).
Figure 1. Exosomes in allergic sensitization and inflammation. Allergic sensitization is thought to be driven by allergen entry through epithelium in a damaged or inflamed environment. Epithelial cells release TSLP/IL-33/IL-25 activating DCs and ILC2 cells which initiate type 2 immunity (TH2). Additionally, epithelial cells release exosomes containing contactin-1 (CNTN1) that play a role in recruiting macrophages and mo-DCs [74]. DC-derived and B cell-derived exosomes containing allergen, p-MHCII complexes and co-stimulatory molecules can amplify the TH2 milieu [65][80]. ILC2 cells activate and recruit eosinophils via IL-5 resulting in exosome release that promote ROS production in eosinophils and further eosinophil recruitment [75]. Mast cell-derived exosomes can amplify TH2 responses by stimulating lymphocytes via co-stimulatory molecules, such as OX40L, LFA-1 and CD40L [81][82].
).