Key Points
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Asthma is a complex syndrome that includes many disease phenotypes and endotypes.
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Allergic asthma and Type 2 asthma represent the most common phenotype with lung eosinophilia. Both ILC2 and Th2 cells, as well as type 2 cytokine-producing natural killer (NK) T cells, may contribute to this form.
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In addition to Th1 and Th2 cells, other T cell subsets contribute to the development of allergic asthma at different stages including Th1, Th17, Th9, Th22 and different populations of T REG cells.
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The innate immune system participates in the initiation and maintenance of both allergic and nonallergic asthma. Activated NKT cells in the lung produce proinflammatory cytokines contributing to bronchial hyperreactivity.
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Epithelial cell activation essentially contributes to the inflammatory burden.
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Remodeling in asthma includes basement membrane thickening, myofibroblast differentiation, smooth muscle hyperplasia, epithelial activation and angiogenesis, and is controlled by the immune system.
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Immune tolerance with the induction of T and B regulatory cells is effective in treatment and prevention in mouse models and represents the major mechanism of action of allergen-specific immunotherapy (AIT) for the treatment of allergic rhinitis and asthma in humans.
Asthma is a very common chronic disorder of the airways characterized by variable and recurring symptoms, airflow obstruction, bronchial hyperresponsiveness (BHR) and underlying inflammation. It is a complex syndrome that develops after environmental exposures such as innocuous allergens, infectious agents and air pollutants in genetically susceptible individuals with differences in severity, co-morbidities, natural history and treatment response. The asthma syndrome encompasses several disease subtypes defined by distinct pathophysiologic mechanisms, called endotypes. Some examples of these asthma endotypes include aspirin-sensitive asthma (ASA), allergic bronchopulmonary mycosis (ABPM), allergic asthma, asthma predictive indices (API), late-onset asthma in adulthood and cross-country skier’s asthma. Among them, allergic asthma is one of the best characterized. Recent advances have significantly contributed to our knowledge of the mechanisms underlying this endotype. It is characterized by an inflammatory immune response with high levels of T helper cell type 2 (Th2) lymphocytes, type 2 innate lymphoid cells, eosinophils and basophils together with activation of the tissue cells, particularly epithelium and smooth muscle cells, that leads to mucus production, mucosal edema, reversible airway obstruction, BHR and airway remodeling. Allergic asthma is associated with specific IgE sensitization to indoor and outdoor allergens, and sometimes with elevated total serum IgE levels, which represent major risk factors for the development of asthma and persistent wheezing in children.
Mechanisms of the Allergic Inflammatory Response
The immune response in allergic asthma consists of two main phases: (1) sensitization and memory and (2) the effector phase, which can be further subdivided into the immediate-phase response (IPR) and the late-phase response (LPR). During the sensitization phase of asthma the differentiation and clonal expansion of allergen-specific CD4 + Th2 cells producing IL-4 and IL-13 are essential to induce class switch to the ε immunoglobulin heavy chain in B cells and the production of allergen-specific IgE antibodies (Abs). Allergen-specific IgE binds to the high-affinity FcεRI on the surface of mast cells and basophils, thus leading to the patient’s sensitization ( Figure 28-1 ). A memory pool of allergen-specific T and B cells is also generated. The IPR, which is also called the type I hypersensitivity response, occurs after new encounters with the causative allergen, which induces cross-linking of the IgE-FcεRI complexes on sensitized effector cells, leading to the release of anaphylactogenic mediators responsible for the classical symptoms of IPR, which induce increased vascular permeability, extravasation of fluid into the tissues and smooth muscle contraction ( Figure 28-2 ). If contact with the allergen persists, the LPR occurs 6 to 12 hours later. Activated allergen-specific Th2 cells produce IL-4, IL-5, IL-9 and IL-13, which play a key role in the maintenance of allergen-specific IgE levels, eosinophilia, recruitment of inflammatory cells to inflamed tissues, production of mucus and decreased threshold of contraction of smooth muscles, leading to increased inflammation associated with BHR, a cardinal feature of asthma ( Figure 28-2 ). Recently, other cytokines including thymic stromal lymphopoietin (TSLP), IL-25, IL-31 and IL-33 produced in epithelial cells have been shown to participate in the Th2 response and inflammation.
Th2 Cells and Th2 Cytokines
CD4 + Th2 cells are present in lung biopsy specimens and bronchoalveolar lavage (BAL) fluid from patients with allergic asthma and play a prominent role in the initiation and development of the disease. Although several cell types produce Th2 cytokines, including mast cells, basophils, natural killer T (NKT) cells and the recently identified type 2 innate lymphoid cells (ILC2), Th2 lymphocytes are still considered fundamental in allergic asthma. Initially, mouse models demonstrated that depletion of CD4 + T cells prevents the development of asthma.
IL-4 plays a major role in the development of protective immune responses to helminths and other extracellular parasites, but it also has a central role in the regulation of allergic asthma, being the major stimulus for the differentiation of antigen-stimulated naïve T cells into Th2 cells. IL-4 is also essential for human and mouse B cell switch to IgE and IgG4 or IgG1, respectively, and increases the expression of class II MHC molecules, CD23 and IL-4R in B cells. IL-4 increases the production of cysteinyl leukotrienes from IgE-primed mast cells, and together with TNF-α the expression of vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells. IL-4- and IL-4Rα-deficient mice have severely compromised Th2 differentiation and their serum levels of IgG1 and IgE are strongly reduced.
IL-5 is another central Th2 cytokine, which is simultaneously produced with IL-4 under the control of the transcription factor GATA-3 with a central role in allergic asthma. IL-5, together with IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), leads to growth, activation, differentiation, survival and mobilization of eosinophils to the lungs as a key feature of asthma. Eotaxin-2, an eosinophil chemokine, seems to be crucial for IL-5-induced IL-13 production and BHR and has a role in remodeling. IL-5 levels and eosinophils are increased in BAL and in biopsies of asthmatics and they correlate with severity of the disease. Eosinophils produce cysteinyl leukotrienes and may enhance the production of IL-13, which can directly induce BHR. IL-5-deficient mice are resistant to induction of experimental asthma. The treatment of asthma with anti-IL-5 antibodies (mepolizumab, reslizumab) reduced blood eosinophilia and sputum eosinophils, but few or no effects on asthma symptoms were observed in the initial clinical trials. Recent studies reported significant reductions in exacerbation rates in refractory eosinophilic asthma and steroid-dependent asthma with sputum eosinophilia.
IL-13 is another Th2 cytokine that has been shown to play a critical role in asthma. IL-13 shares with IL-4 one receptor chain, IL-4Rα. IL-13 does not promote Th2 differentiation, because T cells do not express the IL-13 receptor. IL-13 is also able to activate eosinophils and mast cells, recruits eosinophils and prolongs their survival. It up-regulates the levels of CD23 and MHC II on B-cells and induces the expression of different adhesion molecules on monocytes. IL-13 also plays an important role in tissue remodeling and fibrosis, and TGF-β has been linked to these effects. The role of IL-13 in asthma is supported by epidemiologic data showing that IL-13 polymorphisms lead to a higher frequency of asthma exacerbations in childhood and elevated total IgE and blood eosinophilia. IL-13 knockout mice fail to mount a profound goblet cell hyperplasia without affecting IL-4- and IL-5-producing cells, mast cell cytokine production and IgE levels. Specific overexpression of IL-13 in the lung leads to typical features of asthma. Although all these findings indicate that IL-13 is a critical cytokine required for the development of asthma, an IL-4 mutant protein blocking the binding of both IL-4 and IL-13 to IL-4Rα was able to reduce the severity of the LPR, but not BHR in clinical trials. This suggests that other factors, in addition to IL-13/IL-4, function to induce BHR in patients with asthma.
The Development of the Th2 Response
There are two types of IL-4 binding receptors: the type I and type II IL-4R. Both types have the IL-4Rα chain in common. Type I IL-4R binds IL-4 exclusively and consists of IL-4Rα (CD124) and the common gamma chain γc (CD132), which is also a receptor for IL-2, IL-7, IL-9, IL-15 and IL-21. Type II IL-4R binds IL-4 and also IL-13 and consists of the IL-4Rα chain and the IL-13Rα1 chain. Whereas the signals of the type II IL-4R are mediated by signal transduction and activator of transcription (STAT3), the signals of the type I IL-4R are transduced by STAT6. Binding of IL-4 to the IL-4 receptor complex promotes intracellular signaling cascades involving several Jaks members that culminate with the phosphorylation and activation of STAT6 that enhance Th2 cell differentiation. The binding of IL-13 to its receptors, which consist of IL-13Rαl and IL-13Rα2 and the IL-4Rα chain, also results in phosphorylation of STAT-6. STAT-6 then translocates to the nucleus and binds to STAT-6 transcriptional elements or interacts with additional Th2-associated transcription factors such as GATA-3, which is selectively expressed in Th2 cells and is critical for Th2 cytokine expression. Increased expression of both STAT-6 and GATA-3 has been observed in bronchial mucosa of asthmatic patients.
The Th1/Th2 Paradigm
After the discovery of Th1 and Th2 cells in 1986, it was suggested that a Th2 response underlies the development of allergic diseases, and that Th1 responses are predominant in infections and autoimmunity ( Figure 28-3 ). Following these initial findings, the general dogma was that a switch toward a Th1 response would be required for successful treatment of allergies by AIT and a switch toward a Th2 response would be beneficial for treatment of autoimmunity. Th1 cells secrete IFN-γ, particularly induced by IL-12, which is secreted from dendritic cells (DCs). Th2 cells secrete IL-4, IL-5 and IL-13 induced by IL-4. Th1 cells were thought to balance Th2 responses and protect against allergic diseases, as has been shown in models of infection with intracellular bacteria. In humans, infants with higher levels of IFN-γ in their cord blood were demonstrated to be less likely to develop atopy. Furthermore, expression of T-bet, the master switch transcription factor for Th1 development and IFN-γ production, is decreased in the airways of patients with asthma. Moreover, patients with allergic asthma display low levels of the Th1-driven cytokine IL-12, as well as lower expression of IL-12R.
Beyond the Th2 Paradigm in Allergies and Asthma
The Th2 paradigm of asthma explains many features of asthma, but there are many other observations that cannot be explained exclusively by this paradigm. For example, non-Th2 factors such as IFN-γ, neutrophils and IL-17 are present in the airways of many patients with asthma, particularly patients with severe asthma and corticosteroid-resistant asthma, suggesting that IFN-γ and IL-17 are proinflammatory cytokines. Depending on the stage of inflammation they contribute to ongoing allergic asthma and Th1 and Th17 are not necessarily polarized as cells that oppose Th2 cells ( Figure 28-2 ). It was demonstrated that Th1 cells contribute to exacerbation of the LPR by inducing apoptosis of the airway epithelium in atopic patients, and that neutralization of IL-17 and Th17-related functions in an experimental asthma model reduces neutrophilia, while increasing eosinophil infiltration in the lung. In addition, two novel Th cell subsets have been identified according to their cytokine signature, Th9 and Th22 cells ( Figure 28-2 ). However, their exact contribution to the initiation and continuation of allergic asthma needs to be further explored. The prevalence of Th1- and Th17-mediated autoimmune diseases such as type 1 diabetes, inflammatory bowel disease and multiple sclerosis has significantly increased, as has that of atopic diseases, in westernized cultures over the past decades. This might be partially explained by environmental changes that have occurred in westernized cultures that could have altered the function of a specific T cell population with suppressive capacity – regulatory T cells (T REG ; see below) – thus enhancing the development of not only Th2- but also Th1- and Th17-mediated diseases.
Other observations that cannot be explained by the Th2 paradigm of asthma are: (1) low levels of Th2 cells in the airways of patients with severe asthma and with steroid-resistant asthma; (2) that viral infection, exercise and air pollution are common nonallergic factors that normally induce symptoms of asthma; (3) that only around 30% to 40% of patients with allergic rhinitis develop asthma; (4) therapies targeting Th2 factors in clinical trials have not always been as effective as predicted. Therefore, other innate and adaptive inflammatory pathways must be considered beyond the Th1/Th2 paradigm to explain the development of asthma.
New T Cell Subsets
Regulatory T Cells and Immune Tolerance
T REG cells comprise a group of different T cell subsets with suppressive capacity that are essential for the induction of immune tolerance ( Figure 28-4 ). T REG cells can be broadly divided into two main groups: (1) the thymus-derived naturally occurring CD4 + CD25 + forkhead box protein 3 (FOXP3) + T REG cells, also called natural T REG (nT REG ) cells, and (2) the inducible T REG (iT REG ) cells. nT REG cells constitutively express high levels of the alpha chain of the IL-2 receptor (CD25) and the suppressor costimulatory molecules CTLA4 and PD1. The expression levels of GITR, CD103 and CD122 on nT REG cells correlate with their suppressive activity. In mice, FOXP3, the master transcription factor for T REG cell generation, is specifically expressed by nT REG cells, but in humans it might be also expressed in activated T cells. Mutations in FOXP3 lead to the immune dysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) or the X-linked autoimmune and allergic dysregulation syndrome (XLAAD), diseases characterized by severe autoimmune and allergic phenotypes. Similar phenotypes are reported for scurfy mice due to FOXP3 mutations and impaired capacity to generate functional T REG cells. iT REG cells generated in the periphery after antigenic stimulation are characterized by high levels of IL-10 production and play a key role in the maintenance of healthy immune response to allergens. iT REG cells suppress effector T cell responses by mechanisms that depend on IL-10 and also TGF-β, and produce perforin and granzymes to kill antigen-presenting cells. In humans, Type 1 iT REG (Tr1) cells inhibited the proliferation and cytokine responses of naïve as well as established Th1 and Th2 cells, including allergen-specific Th2 cell lines. Healthy and allergic individuals display three different allergen-specific T cell subtypes as Th1, Th2 and Tr1 in different ratios. The imbalance between Th2 and Tr1 cells, depending on the dominant subset, may induce allergy development or recovery. Immune tolerance to venom allergens is an appropriate model for high-dose tolerance to allergens in humans. During the exposure to venom allergen, venom-specific IL-10-secreting Tr1 cells are clonally differentiated from allergen-specific Th1 and Th2 cells. Interestingly, histamine receptor 2, which is also up-regulated on specific Th2 cells, suppresses allergen-stimulated T cells and enhances IL-10 production related to the tolerance mechanism. Nonallergic beekeepers have an approximately 1,000 times higher allergen-specific IgG4 versus allergen-specific IgE ratio compared to bee venom allergic individuals. Another tolerance model with cat allergen also showed elevated levels of allergen-specific IgG4 levels after exposure to high-dose cat allergen. This also represents immune tolerance to the Th2 type immune response to specific allergen. Together these outcomes may infer that pets in the house may induce tolerance and decrease the risk of asthma.
Interestingly, it has recently been shown that functional allergen-specific T REG cells are generated in human tonsils by mechanisms partially depending on plasmacytoid dendritic cells (pDCs) and that triggering of TLR4 or TLR8 and proinflammatory cytokines, such as IL-1β and IL-6, breaks allergen-specific T cell tolerance in human tonsils and peripheral blood. Considering that the relatively large lingual tonsil is not removed by tonsillectomy and remains intact for life, these data suggest that the tonsils are the organs where immune tolerance induction during successful sublingual immunotherapy (SLIT) may take place, thus representing a potential novel target for future therapeutic interventions.
Th17 Cells
A new T cell lineage secreting large quantities of IL-17A, also known as IL-17, was identified and called Th17 cells. Th17 cells are essential for the elimination of extracellular pathogens and they might also play a role in the development of psoriasis, Crohn’s disease and rheumatoid arthritis. They produce proinflammatory cytokines such as IL-17A, IL-17F, IL-22 or IL-26 after activation. The main cytokines involved in Th17 development and expansion include TGF-β, IL-6, IL-1β, IL-21 and IL-23. The retinoic acid receptor-related orphan receptor γt/C2 (RORγt/RORC2), in mice and humans respectively, is the master transcription factor involved in Th17 cell development. Several studies in mouse models and human data suggest that Th17 cells play a pathogenic role in the development of allergic diseases. Th17 cells contribute to neutrophilic inflammation in acute airway inflammation models. IL-17 is demonstrated to be the main cytokine driving the granulocyte influx observed in the lungs of allergic asthma models. In humans, it was shown that Th17 cells contribute to allergic airway disease by inducing airway smooth muscle cell migration. Genetic polymorphism studies demonstrated an association of IL-17 and asthma.
Th9 and Th22 Cells
Recent findings showed that TGF-β alone converts Th2 cells into selective producers of IL-9 (Th9), and that in combination with IL-4 it is able to promote the generation of Th9 cells. IL-9 significantly contributes to the development of allergic asthma by directly acting on T cells, B cells, mast cells, eosinophils, neutrophils and epithelial cells and promoting eosinophilic inflammation, BHR, elevated IgE levels and increased mucus secretion ( Figure 28-2 ). The expression of IL-9 and IL-9 receptor is increased in bronchial tissue of atopic asthmatic subjects. Supporting this role, mice selectively overexpressing IL-9 in the lung developed many features that resembled human asthma.
Th22 cells represent a novel Th cell subset characterized by particularly high production of IL-22, which might be also produced by other T cells such as Th0 and Th17 cells. The exact role of IL-22 in asthma is not fully understood yet and further research is required. It was reported that Th22 cells together with Th17 cells contribute to enhance migration of airway smooth muscle cells, thus increasing the accumulation of such cells in asthma ( Figure 28-2 ). As previously discussed, Th17 cells induce BHR in steroid-resistant asthma, but whether this effect might also be partially due to IL-22 remains elusive. IL-22 could also inhibit allergic airway inflammation in the effector phase by altering the function of DCs and inhibiting IL-25 production from lung epithelial cells.
B Regulatory Cells and Allergen Tolerance
Very recent findings demonstrated that IL-10-secreting B regulatory (Br1) cells might also play an essential role in the generation of a healthy immune response to allergens. Human IL-10-secreting Br1 cells are able to suppress antigen-specific CD4 + T cell proliferation. In addition, the major bee venom allergen phospholipase A (PLA)-specific B cells from nonallergic beekeepers showed increased expression of IL-10 and IgG4 and the frequency of IL-10-secreting PLA-specific B cells increased in allergic patients receiving allergen-specific immunotherapy. These data provide novel information on IL-10-secreting Br1 cells in allergic inflammation in humans.
In a recent study analyzing the role of IL-10 in particular, solely IL-10-overexpressing human B cells acquired a prominent immunoregulatory profile comprising up-regulation of suppressor cytokine signaling-3 (SOCS3), glycoprotein A repetitions predominant (GARP), CD25 and PD-L1. Concurrently, their secretion profile was characterized by a significant reduction in proinflammatory cytokines (TNF-α, IL-8 and MIP-1α) and augmented production of antiinflammatory IL-1RA and vascular endothelial growth factor (VEGF). IL-10-overexpressing B cells secreted less IgE, and potently suppressed proinflammatory cytokines in peripheral blood mononuclear cells, maturation of monocyte-derived dendritic cells (promoting a tolerogenic phenotype) and antigen-specific proliferation in vitro.
Innate Inflammatory Mechanisms in Asthma
Compelling experimental evidence has demonstrated that asthma does not exclusively depend on Th2 adaptive immune responses, and it is increasingly seen as a disease that has a strong innate immune component. Today, it is accepted that the classical Th2 cytokine signature of allergic asthma might not simply reflect an adaptive Th2 cellular response. Different innate immune system cells in the lung such as epithelial cells, DCs, other airway cells, neutrophils, eosinophils, NK cells and NKT cells as well as ILC2 also significantly contribute to the initiation and maintenance of allergic asthma. In contrast to the adaptive immune system, the response mounted by the innate inflammatory system does not involve memory and is less sensitive to corticosteroids, which could explain the apparent resistance to steroid treatments in many forms of asthma and severe asthma. The innate immune system cells express a wide range of pattern recognition receptors (PRRs) that allow them to recognize molecular patterns released from pathogens (pathogen-associated molecular patterns, PAMPs) or from damaged tissues (damage-associated molecular patterns, DAMPs). Innate immune system cells respond to environmental insults such as cigarette smoke that may directly damage respiratory tissues, activating damage PRRs, or to respiratory viruses activating Toll-like receptor (TLR)3, TLR7 or TLR8, which leads to inflammation, and synergize to significantly exacerbate lung inflammation. In the same way, other PRRs including C-type lectin receptors, scavenger receptors or nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) could also contribute to enhance inflammation after exposure to protease-containing allergens, pathogens or pollution. Lung epithelial cells do not only represent a structural barrier, they also contribute to mount proper immune responses by secreting different types of mediators such as chemokines (RANTES/CCL5, eotaxin/CCL11 and MCP-1/CCL2), growth factors (platelet-derived growth factor, fibroblast growth factor and endothelins), nitric oxide that increases airway inflammation, as well as cytokines such as IL-25, IL-33 and TSLP (see below) that may activate innate cells, including mast cells, basophils and NKT cells or ILC2.
IL-25 , also known as IL-17E, is a member of the IL-17 cytokine family produced by Th2 polarized T cells and in vitro cultured mast cells and epithelial cells. Recent data suggest a crucial role for IL-25 in asthma by favoring the production of Th2 cytokines, enhancing IgE synthesis, inducing mucus production and epithelial hypertrophy or augmenting the numbers of eosinophils in blood. IL-25 mediates its effects through the induction of Th2 cytokine production (IL-4, IL-5 and IL-13) in non-B/non-T (NBNT) c-kit + FcεRI − cells in mesenteric lymph nodes, in a subset of natural killer T (NKT) cells and in ILC2. Eosinophils and basophils from atopic individuals have also been described as sources of IL-25. The latter may maintain reactivity of Th2 central memory cells that express the IL-25R upon stimulation by the innate immune system. In mouse models, IL-25 expressed in the lungs of sensitized mice upon antigen inhalation is sufficient to induce allergic diseases of the airways and administration of anti-IL-25 monoclonal antibodies (mAbs) reduced IL-5 and IL-13 production, eosinophilic infiltration, goblet hyperplasia and BHR. This indicates that IL-25 is a potent inducer of Th2 type immunity in the lung by its effects on several different cell types.
IL-33 is a cytokine that plays a significant role in the regulation of mucosal immune responses of the airways. It is mainly produced by bronchial epithelial cells, but also by fibroblasts and smooth muscle cells. The receptor for IL-33 is ST2, which is mainly expressed on Th2 cells, mast cells, some NKT cells and mucosal ILC2. IL-33 levels are increased in the serum of allergic patients suffering from anaphylaxis and different studies demonstrated that IL-33 potently activates human eosinophils and mediates direct degranulation of mast cells in the absence of allergen. In mice, IL-33 is able to generate airway inflammation through a process depending on IL-5-producing T cells but not IL-4. Administration of neutralizing antibodies or transfer of soluble ST2 impairs Th2 type inflammation in asthma in mice. Administration of exogenous IL-33 leads to lymphocyte-independent airway hyperreactivity and goblet cell hyperplasia in mice.
Thymic stromal lymphopoietin ( TSLP ) is another cytokine mainly produced by human lung and skin epithelial cells that acts on DCs, increasing the expression of different costimulatory molecules, including OX40-L, which in turn promotes the generation of IL-4-, IL-5- and IL-13-producing T cells and inhibiting IL-10 and IFN-γ. In addition, TSLP-activated DCs induce the production of Th2-attracting chemokines and activation-regulated chemokine (TARC) and monocyte-derived chemokine (MDC). The expression of TSLP is significantly increased in the asthmatic airways and in the skin of atopic dermatitis patients, respectively, and correlates with disease severity. These data demonstrate that TSLP is also a Th2-promoting cytokine that significantly contributes to the pathogenesis of human asthma.
Antigen-Presenting Cells
Antigen-presenting cells (APCs) are able to capture environmental allergens in the airways, skin or mucosa, migrate to the nearest lymph nodes and present the processed antigenic peptides to T cells. The most potent stimulators of naïve T cells are DCs lining the mucous membranes of the airways. In contrast, alveolar macrophages, which are abundant in the lung, phagocytize antigens, but they are not able to up-regulate the expression of CD80 or CD86 costimulatory molecules and actively tolerize CD4 + T cells. In humans, circulating DCs can be broadly divided into two groups: (1) myeloid dendritic cells (mDCs), and (2) plasmacytoid dendritic cells (pDCs). mDCs can be further divided into type 1 mDCs expressing BDCA1 and type 2 mDCs expressing BDCA3. Both mDCs and pDCs express a different repertoire of TLRs and display a diverse cytokine signature after microbial stimulation. mDCs induce naïve CD4 + T cells to produce large quantities of IFN-γ but few Th2 cytokines, whereas pDCs were initially described as inducer CD4 + T cells to produce Th2 cytokines but not IFN-γ. For a long time, it was believed that only immature or partially mature DCs generate functional T REG cells and that mature DCs induce specific effector Th cells after encountering different stimuli in specific environments. Recent findings indicate that fully mature pDCs are also able to induce functional T REG cells in humans, thus indicating that pDCs constitute a unique DC subset exhibiting intrinsic tolerogenic capacity. In mice, depletion and adoptive transfer of pulmonary pDCs in experiments demonstrated this DC subset to be essential for the prevention of allergic sensitization and asthma development.
Mast Cells and Neutrophils
Mast cells play a key role in both the IPR and LPR effector phases of allergic asthma. Sensitized mast cells are activated in an IgE-dependent manner after new encounters with the offending allergens contributing to the IPR. Mast cells contribute to the development of BHR in asthmatic individuals, who show significantly higher numbers of activated mast cells compared to healthy individuals. During the LPR, eosinophils, basophils, neutrophils and activated T cells massively infiltrate the exposed areas and trigger potent inflammatory responses which, depending also on the IPR, contribute to the generation of BHR and the chronic symptoms of asthma. IL-17-mediated neutrophil infiltration is a very important feature of severe asthma that has been correlated with disease severity.
Basophils
In addition to the classical role of basophils during the IPR, several studies have demonstrated that they also express class II MHC molecules and are able to prime naïve CD4 + T cells into Th2 cells. They produce large quantities of IL-4, IL-13 and TSLP and it was shown that they could also play a role in the sensitization phase of allergic asthma by enhancing adaptive immunity and Th2 responses. A recent study demonstrated that inflammatory DCs were necessary and sufficient for induction of Th2 immunity and features of asthma, whereas basophils were not required, thus suggesting a model whereby DCs initiate and basophils amplify Th2 immunity to house dust mite allergen.
Natural Killer T Cells
Due to their unique expression of the invariant T cell receptor (TCR) and their capacity to rapidly produce cytokines after activation, the natural killer T (NKT) cells are considered as a cell subset belonging to the innate immune system with the capacity to amplify adaptive immune responses. NKT cells might be involved in the development of BHR, and different subsets of NKT cells were described in different models of asthma. For the development of BHR in a model of allergic asthma, NKT cells producing IL-4 and IL-13 were required ( Figure 28-2 ). In an asthma model induced with ozone to mimic air pollution, NKT cells producing IL-17 were required to induce neutrophil infiltration in the airways, however, in a model of virus-induced BHR, CD4 − NKT cells were required. In both cases, Th2 cells and adaptive immunity were necessary for NKT cells to promote BHR, which might help to explain some forms of nonallergic asthma. The frequency of NKT cells in the lungs of asthma patients appears to be highly variable and related to asthma severity and symptom control.
Natural Killer Cells
NK cells encompass different subsets of lymphocytes that do not express CD3, CD4 or CD8. They are essential for killing tumor and virus-infected cells as well as in controlling certain microbial infections. Subsets of NK cells (CD56 bright CD16 dull ) are able to produce high levels of cytokines including IFN-γ, tumor necrosis factor (TNF)-α, TGF-β, IL-5, IL-10 and IL-22 after stimulation. NK cells contribute to exacerbate airway inflammation and increase Th2 cytokines and eosinophilia as demonstrated after depletion experiments in a mouse model. In contrast, TLR9-L-activated NK cells produced high levels of IFN-γ, suggesting a protective role in the development of asthma. Activated NK cells also produced IL-22, which in turn favored the production of antimicrobial peptides and enhanced epithelial cell integrity. In addition, a tiny NK cell subset that secretes mainly Th2 cytokines but not IFN-γ has been shown to contribute to IgE production in humans.
γδ Cells
T cells expressing γδ T cell receptors (γδ T cells) are normally found in high numbers in mucosal tissues, where the contact with allergenic proteins occurs. Although their main function seems to be associated with the generation of immune responses against bacterial antigens, they were also shown to play an important role in allergic sensitization. After allergen challenge, a population of γδ T cells producing Th2-type cytokines was described. In humans, the role of γδ T cells in asthma is controversial; some studies report increased γδ T cell numbers in BAL fluid from patients with asthma, whereas others show decreased numbers of peripheral blood γδ cells.
CD8 + T Cells
CD8 + T cells can be classified as type 1 (producing IFN-γ) or type 2 (producing IL-4 and IL-5). Exogenous allergens are cross-presented to allergen-specific CD8 + cells through class I pathways, but the precise role of CD8 + cells in asthma is not clear and may depend on the relative numbers of both types in the lungs and blood of asthmatic individuals. It was initially suggested that type 2 CD8 + cells may contribute to asthma pathogenesis. Type 1 CD8 + cells were shown also to enhance asthma symptoms, but they could also display a protective role by eliminating allergen-specific Th2 cells.
Type 2 Innate Lymphoid Cells (ILC2)
Type 2 innate lymphoid cells (ILC2) were initially described in the gut but they are also abundant in lungs and mucosa. They have been shown to produce large quantities of Th2 cytokines after activation with IL-25 and IL-33, playing an important role in virus-induced BHR and allergic asthma. ILC2s require the transcription factors RORα and GATA3 for their development and mainly produce IL-5, IL-9 and IL-13 after activation. Different studies demonstrated that ILC2s play an important role in BHR induction after influenza virus infection through a process depending on IL-33 produced by activated alveolar macrophages. In addition, other studies have demonstrated in mice a role for ILC2 in the pathophysiology of asthma and allergic inflammation. These data could help to explain virus-induced and allergen-induced BHR and asthma through a common pathway to generate Th2 responses.
Airway Remodeling in Asthma
Asthmatic airways are characterized by structural airway changes known as airway remodeling, including smooth muscle hypertrophy, goblet cell hyperplasia, subepithelial fibrosis and angiogenesis. Epithelial and mesenchymal cells are in close contact, forming a truly epithelial-mesenchymal unit that coordinates the initiation of proper responses to injury in the lung. This unit coordinates growth and the response to damage after injury from the environment, potential alterations of which are related to the development of asthma. For this important role, they use cytokines and growth factors such as TGF-β, epithelial growth factor (EGF) and VEGF. One of the main features associated with persistent asthma is structural remodeling due to the conversion of mesenchymal cells into myofibroblasts, producing large amounts of interstitial collagens and leading to fibrosis and thickening of the subepithelial basement membrane (lamina reticularis). TGF-β produced by resident tissue induces the synthesis of collagen I and inhibits collagenase production in an autocrine manner, thus contributing particularly to airway remodeling and fibrosis in the pathogenesis of asthma. TGF-β also plays an important role in the control of airway inflammation and restoration of healthy immune responses to allergens. Other alterations observed in the airway include thickening of the bronchial wall, mucus hypersecretion, hyperplasia and hypertrophy of the smooth muscle layer, and neovascularization. Genetic polymorphisms in asthma susceptibility have been described for several genes including ADAM33 and filaggrin ( FLG ) genes.
The Role of Cell Trafficking and Migration in Pulmonary Inflammation
The homing of the inflammatory cells to the lung is a key aspect in the development of asthma and lung inflammation. Cell trafficking to the lung in asthma is a very complex and redundant process that involves different cytokines, chemokines, adhesion molecules and matrix metalloproteinases (MMPs).
During allergic inflammation, several cells in the lung rapidly produce the chemokines MCP-1/CCL2, MCP-2/CCL8 and eotaxin/CCL11, and basophils expressing the chemokine receptors CCR2, 3 and 4 are the first cells to be recruited. Patients with allergic asthma have significantly higher expression levels of CCR3 and eotaxin/CCL11. The chemokines MCP-3/CCL7, MCP-4/CCL13 and VCAM-1 and MadCAM-1 also direct the recruitment of eosinophils expressing CCR3, CXCR4 and α4β1/VLA-4 and α4β7/LPAM-1 integrins into the lung. Once inflammatory cells are recruited to the lung, migration into the tissues requires firm adhesion. Extravasation/diapedesis is a complex process that is also regulated by different integrins, chemokines and cytokines through the regulation of the expression of proteinases such as MMPs and matrix-degrading enzymes that allow the leukocytes to penetrate through the basement membrane and into the tissue stroma. In a mouse model of asthma, the expression of MMP2 and MMP9 is significantly increased in BAL fluid after allergen challenge. Supporting these data, inhibition of MMP2 with TIMP-2 impaired the egression of eosinophils from the lungs into the airway lumen and the MMP2-deficient mouse model died of asphyxia after allergen challenge due to severe airway inflammation.
Epithelial Cell Activation and Barrier Function in Asthma
The epithelial barrier function of bronchial epithelial cells in the asthmatic lung, sinus epithelial cells in the sinus tissue of chronic rhinosinusitis patients as well as keratinocytes in the skin of atopic dermatitis patients have been demonstrated to be defective. These recent studies suggest that tissue integrity is disturbed in patients so that allergens, bacterial toxins and other particles are able to penetrate the epidermis and the lung epithelium, where they may activate the immune system and lead to severe chronic inflammation in both diseases. Epithelial tight junctions (TJs) are responsible for the regulation of paracellular flux and epithelial impermeability. TJs consist of different transmembrane and scaffold adapter proteins and form the most apical intercellular junction essential for barrier function between epithelial cells. In addition, they prevent foreign particles, such as allergens, from entering into the subepithelial layers. In contrast, opening of TJs can lead to drainage of inflammatory cells toward the lumen, supporting the resolution of pathologic processes. Consequently, they can be considered as gatekeepers that could contribute both to aggravation of inflammation-related tissue damage or resolution of inflammation via drainage. It has been shown that TJs are disrupted in airways of patients with asthma as assessed by biopsies, as well as in air-liquid interface epithelial cell cultures from the asthmatic bronchi.
The Role of Respiratory Viruses in Asthma Development and Exacerbations
A large number of epidemiologic studies have shown that asthma exacerbations with acute airway obstruction and wheezing are associated with infections triggered by specific respiratory viruses such as rhinoviruses and, to a lesser extent, respiratory syncytial virus. The persistence of respiratory viruses, virus load and virus co-infections have been related to more severe respiratory illnesses. In addition, high rates of respiratory bacterial infections have been associated with asthma exacerbations, indicating that in general respiratory infections may exacerbate rather than prevent the development of asthma. Other studies reported the opposite effect, suggesting that infections might contribute, with different mechanisms during sensitization and effector phases preventing the development of asthma or enhancing symptoms of already existing asthma.
Rhinovirus
Rhinovirus, the common cold virus, is the most frequent type of viral infection associated with asthma exacerbations. The detailed underlying immunologic mechanisms are not completely known, but infection of epithelial and bronchial endothelial cells with rhinovirus generates a plethora of proinflammatory mediators that contribute to the worsening of asthma episodes. At the T cell level, human rhinovirus infections might contribute to the generation of Th2 cells and inhibit Th1 or IL-10-producing T REG cells. Additionally, virus-specific CD8 + cells producing type 2 cytokines may develop during viral infections. Rhinovirus infections in atopic asthmatic patients lead to more severe and prolonged lower respiratory tract symptoms compared to nonatopic subjects. Clinical trials showed that this might be due to impaired innate and adaptive immune responses in the airways of asthmatic patients.
Respiratory Syncytial Virus
Respiratory syncytial virus (RSV) is the most common viral infection during the first 3 years of life, leading to acute viral bronchiolitis associated with the subsequent development of recurrent wheezing. In addition, allergic asthma increases the risk of RSV infection of the lower respiratory tract and hospitalization, and early wheezing is also a strong risk factor for subsequent RSV hospitalization. RSV infection impairs the capacity of epithelial cells to induce T REG cells that are able to suppress undesired adaptive immune responses in the respiratory mucosa, and consequently effector T cells are activated leading to airway inflammation. RSV is also able to promote airway remodeling, which in turn increases the susceptibility of the lung epithelium to initiate allergic responses to inhaled allergens. Treatment of children hospitalized with RSV bronchiolitis with the antiviral ribavirin decreased the risk of subsequent allergic sensitization and development of asthma. In the same way, treatment with palivizumab, a monoclonal antibody preventing RSV infection, of premature infants also reduced subsequent recurrent wheezing.
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