The Importance of Dendritic Cells in Asthma
The Importance of Dendritic Cells in Asthma
Since DCs are the major drivers of immune polarization, they may pose as potential therapeutic targets in immune diseases including allergic asthma, HIV and cancer.
The process of targeting molecules is one of the most important features of DC-driven immune response. Based on the expression of their surface molecules, DCs recognize antigens. Modulation of this interaction to a favorable conduit could have a therapeutic potential. This may be done in a two-way targeting of the interactions between antigens and PRRs. Recombinant protein or synthetic structure generation of known allergens and their modification to generate a tolerant immune response could be a promising target. Second, blocking atopy-committed PRRs and inducing tolerance-producing PRRs on DCs could further strengthen this approach. Among the soluble PRRs, collectins (collagen-containing c-type lectins), surfactant protein D (SP-D) binds to CD14 impeding Der p binding and induction of NF-κB through TLR4. SP-D can also regulate the DC-SIGN expression to amend the airway inflammation. Mite allergen-specific immunotherapy could change the expression of co-stimulatory molecules (CD86, HLA-DR and TLR4) on DCs to modify the pathophysiological features of allergic asthma. However, this therapy comes with numerous side effects. To compensate for the side effects, one group of investigators proposed to use carbohydrate-modified allergens in combination with targeting DCs with hypoallergenic substances. This requires low dose of allergen-specific immunotherapy with reduced risk of side effects. Indeed, efficient uptake of mannan-conjugates by DCs results in a shift from IgE to IgG in B-lymphocytes. NLRP10, a NOD-like receptor, lacks the general ligand binding LRR domain found in other members, and it has been found to be a negative regulator of other NOD-like receptors. It may modulate DCs through an inflammasome to regulate caspase-1 activity leading to reduced Th2 response.
Another approach could be the targeting of key molecules involved in DC maturation. On antigenic exposure and completion of proper signaling repertoire, DCs mature to a specific phenotype. Identification of signaling events responsible for the development of DCs into an atopic or a tolerogenic phenotype could be promising in targeting them for a desirable response. Transcription factor E2-2 is required in the maintenance of interferon-producing mature pDCs, and its deletion can result in an immunogenic mDC phenotype. TLR7/9 signaling has been shown to induce production of IFN-α in pDCs. Similarly, expression of CCR9 is a key event in the maturation of DCs to pDCs. Through receptor agonists, genetic manipulation or stem cell therapy, the population of pDCs can be restored for homeostasis and tolerance. Experimental evidences suggest hypermethylation of multiple genes, including zbtb41 and Foxo6, in CD11c splenic DCs in allergen-naïve offspring of asthmatic mother, suggesting possible therapeutic target with epigenetic modifications. However, site-specific methylation or demethylation cannot be achieved at this point, limiting its therapeutic application in allergic asthma.
We found that the administration of Fms-like tyrosine kinase 3 ligand (Flt3-L) reverses the AHR and airway inflammation in murine models of allergic asthma using OVA, HDM or cockroach antigens. The administration of Flt3-L increased regulatory DCs (CD11cCD8αCD11b) and Tregs (CD4CD25ICOSFoxp3IL-10) in addition to a decrease in Th17 (CD4IL-17IL-23RROR-γtCD25) cells in the lungs. This treatment could be a promising therapy for allergic asthma. Neuropeptide Y (NPY) levels have been found to be elevated in the lungs of allergic asthmatic patients, mostly in chronic condition. Activation of NPY receptor-1 (NPY-Y1) on DCs is needed for efficient antigen uptake, and T-cell priming. In addition, NPY regulates the motility of DCs. Recently, we found the expression of NPY-Y receptors on structural and immune cells of the airways suggesting a potential paracrine–autocrine signaling in these cell types leading to exacerbation of asthma. Thus, targeting a specific NPY receptor may also be a therapeutic target to mitigate asthma.
Chemokine receptors on DCs direct them to the required site based on their ligand concentration, released from different immune cells including epithelium. Understanding of the expression and activity of different chemokine receptors on DCs at different maturation stages as well as in different DC phenotype would enable us to develop therapy based on chemoattraction of DCs. The chemokine receptor CCR8 expressed on DC is of major importance in allergic asthma. Both mature and immature DCs express CCR8, but only mature DCs respond to CCL1. CCR8 antagonists, such as diazospiroundecane, can inhibit migration of DCs, T lymphocytes and eosinophils to reduce inflammation in allergic asthma. Bellinghausen et al. showed that enhanced production of CCL18 by tolerogenic DCs suppresses the Th2-driven airway inflammation. Thus, therapies targeting DCs to express CCL18 could be beneficial in allergic asthma. Therapy directed to express more CXCR3/4, which is specific for pDCs could bring tolerance in allergic people. Furthermore, expression of CXCL9-12, ligands for the chemokine receptors, could attract the therapeutic pDCs to the site. The major drawback in this approach is that both HIV and cancer use the same chemokine receptors for invasion and migration. So, therapy devised should be carefully selected and studied extensively before application.
Antigen presentation through MHC molecules present on DCs requires TCR and other co-stimulatory molecules. If the immune synapse between these cells is incomplete or insufficient, polarization of T-lymphocytes to a specific phenotype for a proper immune response in abated. The important aspect in targeting this molecular interaction is that this interaction is integral to our host defense against pathogens. However, therapeutic potential of this interaction could be significant if the targeting a site modulates the response instead of stopping it. Targeting PD-L2 on DCs, which directly reduces the IL-12p40 production on allergen exposure, could modulate IL-13, and thereby help in ameliorating the DC-driven allergic response. By adoptive transfer of naturally occurring Tregs (CD4CD25) and inducible Tregs (CD4CD25) isolated from lungs and spleen of BALB/c mice into cockroach antigen-sensitized and challenged mice, we found that PD-1 regulates AHR and airway inflammation, and thus could be a target for therapeutic intervention. Gamma-interferon-inducible lysosomal thiol reductase (GILT) reduces disulfide bonds in proteins taken up by DCs for optimal processing and presentation on MHC class II molecules. Targeting GILT could reduce the Der p 1 antigen processing and presentation ameliorating the allergic response associated with HDM.
Microbiome-induced immunomodulation could be another potential therapeutic approach where microorganisms inducing tolerance can be used in atopic patients. As we discussed in earlier section that some of these microorganisms, including H. pylori, modulate the host immune response to evade and disseminate. Virulence factors of H. pylori, GGT and VacA help in the reprogramming of DCs to a tolerogenic phenotype (Figure 7). Downregulation of co-stimulatory molecules on DCs and polarization of Tregs further strengthens the immune response away from Th2. This survival trait can be harnessed for host benefit by selecting harmless strains that may induce tolerance and introduce them in an atopic individual (Figure 7). As the bacterium is harmless, it would not cause damage to the host, but by inducing tolerance it may relegate the allergic phenotype. S. sciuri W620 isolated from farm dust has been shown to have protective effect in murine HDM and OVA models for allergic airway inflammation, possibly through the activation of TLR2 and NOD2 on DCs. Viral infections, such as RSV, may prime the immune system to respond inappropriately to allergens. One of the ways to reduce eosinophilia in these cases is by blocking cysteinyl leukotrienes in DCs to reduce the production of RANTES (CCL5).
(Enlarge Image)
Figure 7.
Bacteria-induced tolerance in dendritic cells. Gut microbiome contain bacteria which may try to evade the immune system by reprogramming dendritic cells (DCs) and the immune responses in host. Helicobacter pylori produces HP-NAP, GGT and VacA to polarize Th1 or Treg cells through PRRs. Production of Tregs results in a tolerogenic response in the host. These Tregs travel to the lung draining lymph nodes and protects the host from allergen-mediated allergic asthma by blocking Th2 cells.
This involves multiple signaling pathways involving various cytokines, chemokines and pro-inflammatory mediators. Modulation of these interactions in favor of tolerance rather than allergy can be achieved through targeting the molecules involved in such interaction at multiple stages. Both AP-1 and NF-κB, the redox-sensitive transcription factors, are involved in multiple inflammatory pathways. Targeting these transcription factors could attenuate allergic response in asthmatics. Lysophosphatidic acid (LPA) overexpression in DCs of murine model of AHR hinders NF-κB to negatively regulate DC activation and allergic airway inflammation. The adoptive transfer of perforin-sufficient CD8 T cells could mitigate the allergic response through secretion of IFN-γ and TNF-α. Additionally, they may target the allergen-presenting mDCs and deplete them to reduce the antigen presentation to Th2 cells and thereby decreasing the eosinophil infiltration. Level of type III IFN-λ2 (IL-28A), an antiviral cytokine, has been found to be low in allergic asthmatics. Exploiting this finding, studies have shown that overexpression of IL-28 in mDCs may lead to a Th1 polarization rather than Th2 in murine model of asthma. Complement molecule, C5a protects from AHR by regulating DCs during allergen sensitization, whereas it acts as a potent pro-allergic molecule during the effector phase. Between the two receptors of C5a, C5aR and C5a receptor-like 2 (C5L2), C5L2 deficiency in mice has protective effects on the development of AHR and airway inflammation. C5L2 could be another potential target for therapy against allergic asthma.
Vitamin D has emerged as a potent immunomodulator in recent years. It modulates the immune cells through its active form calcitriol, also known as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The inactive form of vitamin D, 25(OH)D3, is used as an indicator of circulating vitamin D level in an individual. However, effectiveness depends on the potential of a cell to contain the metabolizing enzyme, 1α-hydroxylase (CYP27B1), to convert inactive 25(OH)D3 into active 1,25(OH)2D3. Upregulation of CYP27B1 has been seen in DCs upon maturation with LPS or with T cell in an immune synapse. Involvement of vitamin D binding protein (carrier protein), vitamin D receptor (VDR) and CYP24A1 (catabolizing enzyme) are also important in maintaining the effects of vitamin D in a particular cell. Recently, it was demonstrated that through vitamin D binding protein, level of free 25(OH)D3 available to DCs governs the balance between allergic and tolerogenic response to allergens. Increase in intracellular vitamin D levels drive DCs to reduce expression of co-stimulatory molecules including CD80, CD83, CD86 and HLA-DR. In addition, it also decreases the levels of TNF-α and IL-12. This effect may be correlated with reduced nuclear transport of activated RelA, an NF-κB subunit through importin-α3 (nuclear transporter) due to a decreased expression of importin-α3 under low levels of vitamin D.
With many interactions known to stimulate DCs to induce tolerance and activate Tregs, the nature of precise signaling events remains obscure. Along with vitamin D, vitamin A also has some role in immunomodulation. DCs express alcohol dehydrogenase (ADH) to oxidize vitamin A to retinaldehyde, which then is converted to retinoic acid in the presence of retinal dehydrogenase (RALDH). Retinoic acid is a known inducer of Foxp3 Tregs. Unlike ADH, only a specific set of DCs (mostly CD103) express RALDH. Retinoic acid increases the expression of RALDH in DCs in an autocrine manner through RAR/RXR signaling. In addition, certain microbial stimuli have been shown to induce RALDH expression through TLR-ERK pathway to induce tolerance via retinoic acid. Intestinal DCs deficient in β-catenin has low levels of RALDH and reduced IL-10 production to assist Th2 polarization.
Wnt ligands bind to frizzled receptor to cause accumulation of β-catenin in the cytoplasm and translocation to the nucleus, which in turn regulates a number of genes to induce tolerance. Thus, activation of β-catenin signaling programs DCs to a tolerogenic type and helps in Treg production. Experiments with monocyte-derived DCs have revealed that TAM receptor (MerTK, Tyro3 and Axl) activation can lead to an upregulation in the expression of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 via IFNR/STAT1 signaling. This in turn causes suppression of TLR-mediated inflammatory signals, and supports Foxp3 Tregs to induce tolerance.
Ig-like receptors ILT3 and ILT4 can be activated by vitamin D, and immunosuppressant cytokines IDO and IL-10. ILT3 activation leads to phosphorylation of phosphatases, SHP-1 and SHIP-1, to cause inhibition of NF-κB and p38/MAPK signaling pathways. This results in the development of tolerogenic DCs that assist Tregs to induce tolerance.
Potential Checkpoints for Therapeutic Intervention
Since DCs are the major drivers of immune polarization, they may pose as potential therapeutic targets in immune diseases including allergic asthma, HIV and cancer.
Targeting the Molecules Involved in Antigen Recognition
The process of targeting molecules is one of the most important features of DC-driven immune response. Based on the expression of their surface molecules, DCs recognize antigens. Modulation of this interaction to a favorable conduit could have a therapeutic potential. This may be done in a two-way targeting of the interactions between antigens and PRRs. Recombinant protein or synthetic structure generation of known allergens and their modification to generate a tolerant immune response could be a promising target. Second, blocking atopy-committed PRRs and inducing tolerance-producing PRRs on DCs could further strengthen this approach. Among the soluble PRRs, collectins (collagen-containing c-type lectins), surfactant protein D (SP-D) binds to CD14 impeding Der p binding and induction of NF-κB through TLR4. SP-D can also regulate the DC-SIGN expression to amend the airway inflammation. Mite allergen-specific immunotherapy could change the expression of co-stimulatory molecules (CD86, HLA-DR and TLR4) on DCs to modify the pathophysiological features of allergic asthma. However, this therapy comes with numerous side effects. To compensate for the side effects, one group of investigators proposed to use carbohydrate-modified allergens in combination with targeting DCs with hypoallergenic substances. This requires low dose of allergen-specific immunotherapy with reduced risk of side effects. Indeed, efficient uptake of mannan-conjugates by DCs results in a shift from IgE to IgG in B-lymphocytes. NLRP10, a NOD-like receptor, lacks the general ligand binding LRR domain found in other members, and it has been found to be a negative regulator of other NOD-like receptors. It may modulate DCs through an inflammasome to regulate caspase-1 activity leading to reduced Th2 response.
DC Maturation
Another approach could be the targeting of key molecules involved in DC maturation. On antigenic exposure and completion of proper signaling repertoire, DCs mature to a specific phenotype. Identification of signaling events responsible for the development of DCs into an atopic or a tolerogenic phenotype could be promising in targeting them for a desirable response. Transcription factor E2-2 is required in the maintenance of interferon-producing mature pDCs, and its deletion can result in an immunogenic mDC phenotype. TLR7/9 signaling has been shown to induce production of IFN-α in pDCs. Similarly, expression of CCR9 is a key event in the maturation of DCs to pDCs. Through receptor agonists, genetic manipulation or stem cell therapy, the population of pDCs can be restored for homeostasis and tolerance. Experimental evidences suggest hypermethylation of multiple genes, including zbtb41 and Foxo6, in CD11c splenic DCs in allergen-naïve offspring of asthmatic mother, suggesting possible therapeutic target with epigenetic modifications. However, site-specific methylation or demethylation cannot be achieved at this point, limiting its therapeutic application in allergic asthma.
We found that the administration of Fms-like tyrosine kinase 3 ligand (Flt3-L) reverses the AHR and airway inflammation in murine models of allergic asthma using OVA, HDM or cockroach antigens. The administration of Flt3-L increased regulatory DCs (CD11cCD8αCD11b) and Tregs (CD4CD25ICOSFoxp3IL-10) in addition to a decrease in Th17 (CD4IL-17IL-23RROR-γtCD25) cells in the lungs. This treatment could be a promising therapy for allergic asthma. Neuropeptide Y (NPY) levels have been found to be elevated in the lungs of allergic asthmatic patients, mostly in chronic condition. Activation of NPY receptor-1 (NPY-Y1) on DCs is needed for efficient antigen uptake, and T-cell priming. In addition, NPY regulates the motility of DCs. Recently, we found the expression of NPY-Y receptors on structural and immune cells of the airways suggesting a potential paracrine–autocrine signaling in these cell types leading to exacerbation of asthma. Thus, targeting a specific NPY receptor may also be a therapeutic target to mitigate asthma.
Chemokine Receptors
Chemokine receptors on DCs direct them to the required site based on their ligand concentration, released from different immune cells including epithelium. Understanding of the expression and activity of different chemokine receptors on DCs at different maturation stages as well as in different DC phenotype would enable us to develop therapy based on chemoattraction of DCs. The chemokine receptor CCR8 expressed on DC is of major importance in allergic asthma. Both mature and immature DCs express CCR8, but only mature DCs respond to CCL1. CCR8 antagonists, such as diazospiroundecane, can inhibit migration of DCs, T lymphocytes and eosinophils to reduce inflammation in allergic asthma. Bellinghausen et al. showed that enhanced production of CCL18 by tolerogenic DCs suppresses the Th2-driven airway inflammation. Thus, therapies targeting DCs to express CCL18 could be beneficial in allergic asthma. Therapy directed to express more CXCR3/4, which is specific for pDCs could bring tolerance in allergic people. Furthermore, expression of CXCL9-12, ligands for the chemokine receptors, could attract the therapeutic pDCs to the site. The major drawback in this approach is that both HIV and cancer use the same chemokine receptors for invasion and migration. So, therapy devised should be carefully selected and studied extensively before application.
Antigen Presentation
Antigen presentation through MHC molecules present on DCs requires TCR and other co-stimulatory molecules. If the immune synapse between these cells is incomplete or insufficient, polarization of T-lymphocytes to a specific phenotype for a proper immune response in abated. The important aspect in targeting this molecular interaction is that this interaction is integral to our host defense against pathogens. However, therapeutic potential of this interaction could be significant if the targeting a site modulates the response instead of stopping it. Targeting PD-L2 on DCs, which directly reduces the IL-12p40 production on allergen exposure, could modulate IL-13, and thereby help in ameliorating the DC-driven allergic response. By adoptive transfer of naturally occurring Tregs (CD4CD25) and inducible Tregs (CD4CD25) isolated from lungs and spleen of BALB/c mice into cockroach antigen-sensitized and challenged mice, we found that PD-1 regulates AHR and airway inflammation, and thus could be a target for therapeutic intervention. Gamma-interferon-inducible lysosomal thiol reductase (GILT) reduces disulfide bonds in proteins taken up by DCs for optimal processing and presentation on MHC class II molecules. Targeting GILT could reduce the Der p 1 antigen processing and presentation ameliorating the allergic response associated with HDM.
Immune Modulation Through Microbiome
Microbiome-induced immunomodulation could be another potential therapeutic approach where microorganisms inducing tolerance can be used in atopic patients. As we discussed in earlier section that some of these microorganisms, including H. pylori, modulate the host immune response to evade and disseminate. Virulence factors of H. pylori, GGT and VacA help in the reprogramming of DCs to a tolerogenic phenotype (Figure 7). Downregulation of co-stimulatory molecules on DCs and polarization of Tregs further strengthens the immune response away from Th2. This survival trait can be harnessed for host benefit by selecting harmless strains that may induce tolerance and introduce them in an atopic individual (Figure 7). As the bacterium is harmless, it would not cause damage to the host, but by inducing tolerance it may relegate the allergic phenotype. S. sciuri W620 isolated from farm dust has been shown to have protective effect in murine HDM and OVA models for allergic airway inflammation, possibly through the activation of TLR2 and NOD2 on DCs. Viral infections, such as RSV, may prime the immune system to respond inappropriately to allergens. One of the ways to reduce eosinophilia in these cases is by blocking cysteinyl leukotrienes in DCs to reduce the production of RANTES (CCL5).
(Enlarge Image)
Figure 7.
Bacteria-induced tolerance in dendritic cells. Gut microbiome contain bacteria which may try to evade the immune system by reprogramming dendritic cells (DCs) and the immune responses in host. Helicobacter pylori produces HP-NAP, GGT and VacA to polarize Th1 or Treg cells through PRRs. Production of Tregs results in a tolerogenic response in the host. These Tregs travel to the lung draining lymph nodes and protects the host from allergen-mediated allergic asthma by blocking Th2 cells.
Interaction of DCs with Immune Cells
This involves multiple signaling pathways involving various cytokines, chemokines and pro-inflammatory mediators. Modulation of these interactions in favor of tolerance rather than allergy can be achieved through targeting the molecules involved in such interaction at multiple stages. Both AP-1 and NF-κB, the redox-sensitive transcription factors, are involved in multiple inflammatory pathways. Targeting these transcription factors could attenuate allergic response in asthmatics. Lysophosphatidic acid (LPA) overexpression in DCs of murine model of AHR hinders NF-κB to negatively regulate DC activation and allergic airway inflammation. The adoptive transfer of perforin-sufficient CD8 T cells could mitigate the allergic response through secretion of IFN-γ and TNF-α. Additionally, they may target the allergen-presenting mDCs and deplete them to reduce the antigen presentation to Th2 cells and thereby decreasing the eosinophil infiltration. Level of type III IFN-λ2 (IL-28A), an antiviral cytokine, has been found to be low in allergic asthmatics. Exploiting this finding, studies have shown that overexpression of IL-28 in mDCs may lead to a Th1 polarization rather than Th2 in murine model of asthma. Complement molecule, C5a protects from AHR by regulating DCs during allergen sensitization, whereas it acts as a potent pro-allergic molecule during the effector phase. Between the two receptors of C5a, C5aR and C5a receptor-like 2 (C5L2), C5L2 deficiency in mice has protective effects on the development of AHR and airway inflammation. C5L2 could be another potential target for therapy against allergic asthma.
Vitamin D
Vitamin D has emerged as a potent immunomodulator in recent years. It modulates the immune cells through its active form calcitriol, also known as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The inactive form of vitamin D, 25(OH)D3, is used as an indicator of circulating vitamin D level in an individual. However, effectiveness depends on the potential of a cell to contain the metabolizing enzyme, 1α-hydroxylase (CYP27B1), to convert inactive 25(OH)D3 into active 1,25(OH)2D3. Upregulation of CYP27B1 has been seen in DCs upon maturation with LPS or with T cell in an immune synapse. Involvement of vitamin D binding protein (carrier protein), vitamin D receptor (VDR) and CYP24A1 (catabolizing enzyme) are also important in maintaining the effects of vitamin D in a particular cell. Recently, it was demonstrated that through vitamin D binding protein, level of free 25(OH)D3 available to DCs governs the balance between allergic and tolerogenic response to allergens. Increase in intracellular vitamin D levels drive DCs to reduce expression of co-stimulatory molecules including CD80, CD83, CD86 and HLA-DR. In addition, it also decreases the levels of TNF-α and IL-12. This effect may be correlated with reduced nuclear transport of activated RelA, an NF-κB subunit through importin-α3 (nuclear transporter) due to a decreased expression of importin-α3 under low levels of vitamin D.
Signaling Pathways to Induce Tolerance in DCs & Activate Tregs
With many interactions known to stimulate DCs to induce tolerance and activate Tregs, the nature of precise signaling events remains obscure. Along with vitamin D, vitamin A also has some role in immunomodulation. DCs express alcohol dehydrogenase (ADH) to oxidize vitamin A to retinaldehyde, which then is converted to retinoic acid in the presence of retinal dehydrogenase (RALDH). Retinoic acid is a known inducer of Foxp3 Tregs. Unlike ADH, only a specific set of DCs (mostly CD103) express RALDH. Retinoic acid increases the expression of RALDH in DCs in an autocrine manner through RAR/RXR signaling. In addition, certain microbial stimuli have been shown to induce RALDH expression through TLR-ERK pathway to induce tolerance via retinoic acid. Intestinal DCs deficient in β-catenin has low levels of RALDH and reduced IL-10 production to assist Th2 polarization.
Wnt ligands bind to frizzled receptor to cause accumulation of β-catenin in the cytoplasm and translocation to the nucleus, which in turn regulates a number of genes to induce tolerance. Thus, activation of β-catenin signaling programs DCs to a tolerogenic type and helps in Treg production. Experiments with monocyte-derived DCs have revealed that TAM receptor (MerTK, Tyro3 and Axl) activation can lead to an upregulation in the expression of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 via IFNR/STAT1 signaling. This in turn causes suppression of TLR-mediated inflammatory signals, and supports Foxp3 Tregs to induce tolerance.
Ig-like receptors ILT3 and ILT4 can be activated by vitamin D, and immunosuppressant cytokines IDO and IL-10. ILT3 activation leads to phosphorylation of phosphatases, SHP-1 and SHIP-1, to cause inhibition of NF-κB and p38/MAPK signaling pathways. This results in the development of tolerogenic DCs that assist Tregs to induce tolerance.
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