Patent Application
20190041401
This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named “2879-186_SequenceListing_ST25”, has a size in bytes of 5 KB, and was recorded on 25 Mar. 2016. The information contained in the text file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).
The present invention is directed toward novel methods to identify, predict and/or treat subjects having severe and/or persistent asthma.
Allergic asthma is characterized by a T helper 2 (Th2) immune response and the presence of allergen-specific Immunoglobulin E (IgE) antibodies (Robinson D S, et al. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992; 326:298-304; Burrows B, et al. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989; 320:271-7). IgE mediated mast cell activation plays an important role in asthmatic patients. This conclusion is supported by the efficacy of omalizumab in many patients with refractory asthma (Strunk R C, and Bloomberg G R. Omalizumab for asthma. N Engl J Med 2006; 354: 2689-95). The differentiation of T cells into various T helper cell populations is a functional but not terminal differentiation state. This was clearly demonstrated in early studies by Coffman and colleagues, who showed that fully differentiated Th2 cells could produce Th1 cytokines in the presence of appropriate stimulants (Coffman R L, et al. Reversal of polarized T helper 1 and T helper 2 cell populations in murine leishmaniasis. Ciba Found Symp 1995; 195:20-3; Mocci S, and Coffman R L. Induction of a Th2 population from a polarized Leishmania-specific Th1 population by in vitro culture with IL-4. J Immunol 1995; 154:3779-87.3). In recent years, this plasticity of T helper cell function has drawn attention (Yang X O, et al. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs. Immunity 2008; 29:44-56; Koenen H J, et al. Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 2008; 112:2340-52; Lee Y K, et al. Late developmental plasticity in the T helper 17 lineage. Immunity 2009; 30:92-107; Wei G, et al. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD41 T cells. Immunity 2009; 30:155-67; Zhou L, et al. Plasticity of CD41 T cell lineage differentiation. Immunity 2009; 30:646-55). Studies have shown interconversion of T helper cells, especially interconversion of regulatory T and Th17 cells. Furthermore, studies have demonstrated the presence of dual-positive Th2/Th17 cells in the blood and tissue of asthmatic patients (Cosmi L, et al. Identification of a novel subset of human circulating memory CD4(1) T cells that produce both IL-17A and IL-4. J Allergy Clin Immunol 2010; 125:222-30, e1-4; Wang Y H, et al. A novel subset of CD4(1) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma. J Exp Med 2010; 207:2479-91) and healthy subjects (Malmhall C, et al. Immunophenotyping of circulating T helper cells argues for multiple functions and plasticity of T cells in vivo in humans-possible role in asthma. PLoS One 2012; 7:e40012).
Although allergens play an important role, there are other environmental factors, such as infection, and a broad range of chemical and physical factors that contribute to exacerbation of asthma. Many of the latter factors are likely to elicit a Th17-type immune response. A particular matter of interest is the qualitative difference between Th2 and Th17 cells in their response to glucocorticoids. IL-17 production by Th17 cells has been shown to be less susceptible to inhibition by glucocorticoids compared with IL-4 and IL-5 production by Th2 cells (McKinley L, et al. TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice. J Immunol 2008; 181:4089-97). Thus the emergence of Th2/Th17 cells in the airways could make asthma less responsive to glucocorticoid treatment. A number of articles have reported increased presence of IL-17 in lung biopsy specimens and sputum from asthmatic patients (Pene J, et al. Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes. J Immunol 2008; 180:7423-30; Barczyk A, et al. Interleukin-17 in sputum correlates with airway hyperresponsiveness to methacholine. Respir Med 2003; 97:726-33; Bullens D M, et al. IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx? Respir Res 2006; 7:135; Molet S, et al. IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. J Allergy Clin Immunol 2001; 108:430-8; Al-Ramli W, et al. T(H)17-associated cytokines (IL-17A and IL-17F) in severe asthma. J Allergy Clin Immunol 2009; 123:1185-7; Agache I, et al. Increased serum IL-17 is an independent risk factor for severe asthma. Respir Med 2010; 104:1131-7; Wong C K, et al. Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin Exp Immunol 2001; 125:177-83; Vazquez-Tello A, et al. Induction of glucocorticoid receptor-beta expression in epithelial cells of asthmatic airways by T-helper type 17 cytokines. Clin Exp Allergy 2010; 40:1312-22). Increased expression of IL-17 was associated with severe asthma. However, there are no reports on the presence of dual-positive Th2/Th17 cells in bronchoalveolar lavage (BAL) fluid from asthmatic patients. Thus there are a number of reasons to study and determine the significance of Th2/Th17 cells in asthmatic patients.
In addition, asthma is a chronic illness (Jackson D J, et al. Asthma exacerbations: origin, effect, and prevention. J Allergy Clin Immunol 2011; 128:1165-74; Lemanske R F, and Busse W W. Asthma: clinical expression and molecular mechanisms. J Allergy Clin Immunol 2010; 125(Suppl):S95-102) with airway hyperreactivity and remodeling persisting in asymptomatic patients with normal pulmonary function (Townley R G, et al. Bronchial sensitivity to methacholine in current and former asthmatic and allergic rhinitis patients and control subjects. J Allergy Clin Immunol 1975; 56:429-42; Shapiro G G, et al. Methacholine bronchial challenge in children. J Allergy Clin Immunol 1982; 69:365-9). Because of the perennial nature of some allergens, it has been difficult to ascertain whether continuous allergen exposure is necessary for the persistence of asthma. Longitudinal observations in patients with occupational asthma indicate that asthma persists in most patients years after removal from occupational exposure (Malo J L, et al. Natural history of occupational asthma: relevance of type of agent and other factors in the rate of development of symptoms in affected subjects. J Allergy Clin Immunol 1992; 90:937-44; Moller D R, et al. Persistent airways disease caused by toluene diisocyanate. Am Rev Respir Dis 1986; 134:175-6). These results suggest that repetitive allergen exposure establishes a biochemical mechanism that sustains asthma in the absence of the inciting allergen. Tremendous progress has been made in uncovering the mechanisms surrounding the inception and development of asthma through animal model studies (Kips J C, et al. Murine models of asthma. Eur Respir J 2003; 22:374-82; Kumar R K, and Foster P S. Modeling allergic asthma in mice: pitfalls and opportunities. Am J Respir Cell Mol Biol 2002; 27:267-72). However, very little is known about the mechanisms regulating the persistence of chronic asthma.
In the majority of mouse models, asthma resolves spontaneously in 1 to 2 weeks (Duez C, et al. Fas deficiency delays the resolution of airway hyperresponsiveness after allergen sensitization and challenge. J Allergy Clin Immunol 2001; 108:547-56; Haworth O, et al. NK cells are effectors for resolvin E1 in the timely resolution of allergic airway inflammation. J Immunol 2011; 186:6129-35; Leech M D, et al. Resolution of Der p1-induced allergic airway inflammation is dependent on CD41CD251Foxp31 regulatory cells. J Immunol 2007; 179:7050-8). Repetitive allergen exposure in alternative models induces tolerance (Duez C, et al. Fas deficiency delays the resolution of airway hyperresponsiveness after allergen sensitization and challenge. J Allergy Clin Immunol 2001; 108:547-56; Kumar R K, et al. Reversibility of airway inflammation and remodeling following cessation of antigenic challenge in a model of chronic asthma. Clin Exp Allergy 2004; 34:1796-802; Schramm C M, et al. Chronic inhaled ovalbumin exposure induces antigen-dependent but not antigen-specific inhalational tolerance in a murine model of allergic airway disease. Am J Pathol 2004; 164:295-304). In others, cessation of repetitive allergen exposure results in resolution of inflammation (Chen Z G, et al. Neutralization of TSLP inhibits airway remodeling in a murine model of allergic asthma induced by chronic exposure to house dust mite. PLoS One 2013; 8:e51268; Henderson W R Jr, et al. A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am J Respir Crit Care Med 2002; 165:108-16; Johnson J R, Wiley, et al. Continuous exposure to house dust mite elicits chronic airway inflammation and structural remodeling. Am J Respir Crit Care Med 2004; 69:378-85). The longest study period to date (Johnson J R, Wiley, et al.) has demonstrated attenuated airway remodeling and airway hyperreactivity persisting for 9 weeks after cessation of repetitive dust mite antigen exposure.
Recent studies have identified a novel IL-5/IL-13-producing type 2 innate lymphoid cell (ILC2) population in mice (Moro K, et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(1)Sca-1(1) lymphoid cells. Nature 2010; 463:540-4; Neill D R, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010; 464:1367-70; Saenz S A, et al. IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses. Nature 2010; 464:1362-6) and human subjects (Mj€osberg J M, et al. Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol 2011; 12:1055-62; Spits H, and Di Santo J P. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nat Immunol 2011; 12:21-7). Although numerous studies have established the importance of ILC2s in the initiation of airway eosinophilic inflammation, (Barlow J L, et al. IL-33 is more potent than IL-25 in provoking IL-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction. J Allergy Clin Immunol 2013; 132:933-41; Bartemes K R, et al. IL-33-responsive lineage-CD251 CD44(hi) lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs. J Immunol 2012; 88:1503-13; Doherty T A, et al. STAT6 regulates natural helper cell proliferation during lung inflammation initiated by Alternaria. Am J Physiol Lung Cell Mol Physiol 2012; 303:L577-88; Halim T Y, et al. Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation. Immunity 2012; 36:451-63; Kim H Y, et al. Innate lymphoid cells responding to IL-33 mediate airway hyperreactivity independently of adaptive immunity. J Allergy Clin Immunol 2012; 129:216-27, e211-6; Petersen B C, et al. Interleukin-25 induces type 2 cytokine production in a steroid-resistant interleukin-17RB1 myeloid population that exacerbates asthmatic pathology. Nat Med 2012; 18:751-8) the role of ILC2s in maintenance of existing airway hyperreactivity has not been established, and their involvement in the airways of asthmatic patients has not been examined.
One embodiment of the present invention is method of treating severe asthma in a subject comprising obtaining a sample from the subject; detecting the presence of dual T helper 2/T helper 17 (Th2/Th17) cells in the sample from the subject; determining the frequency of the dual Th2/Th17 cells in the sample from the subject; determining the frequency of total cluster differentiation 4 (CD4) T helper cells in the sample from the subject; comparing the frequency of the dual Th2/Th17 cells to the frequency of the total CD4 T helper cells in the sample, wherein a frequency of greater than 5% of the dual Th2/Th17 cells as compared to the frequency of the total CD4 T helper cells further comprises detecting the presence of Th2 cells in the sample, determining the frequency of the detected Th2 cells in the sample and further comparing the frequency of the dual Th2/Th17 cells to the frequency of the Th2 cells, wherein a higher frequency of Th2/Th17 cells as compared to the frequency of the Th2 cells identifies the subject as having severe asthma; and administering to the subject identified as having severe asthma a compound selected from the group consisting of a bronchodilator, corticosteroid, leukotriene antagonist, anti-cytokine antibody, anti-cytokine receptor antibody, anti-IgE antibody, an antibiotic, a phosphodiesaterease inhibitor, an anti-MEK compound and combinations thereof for treating the subject.
In one aspect of the invention, the detecting the dual Th2/Th17 cells in the sample comprises detecting expression of CD4 (CD: cluster of differentiation antigen), CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells, also known as CD294, and G protein coupled receptor 44) and CCR6 (CC chemokine receptor 6), wherein co-expression of CD4, CRTH2 and CCR6 indicates the presence of the dual Th2/Th17 cells.
In still another aspect of the invention, detecting the presence of the dual Th2/Th17 cells comprises detecting expression of CD4, IL4 and IL17, wherein co-expression of CD4, interleukin-4 (IL4) and interleukin-17 (IL17) indicates the presence of dual Th2/Th17 cells.
In yet another aspect of the invention, detecting the presence of the dual Th2/Th17 cells comprises detecting expression of interleukin-1b (IL1b), wherein expression of IL1b indicates the presence of dual Th2/Th17 cells.
In another aspect of the invention, detecting the dual Th2/Th17 cells comprises determining the expression level of the complement factor C3 and/or C3a in the sample from the subject, wherein an elevated expression level of C3 or C3a as compared to the expression level of C3 and/or C3a from a healthy control, indicates the presence of dual Th2/Th17 cells.
In still another aspect of the invention, detecting the Th2 cells in the sample comprises detecting expression of CRTH2 on CD4 T cells in the sample, wherein expression of CRTH2 indicates the presence of the Th2 cells.
In yet another aspect of the invention, the step of determining the frequency of the dual Th2/Th17 cells, the CD4 T cells and the Th2 cells in the sample is flow cytometry.
In another aspect of the invention, a ratio of dual Th2/Th17 cells to Th2 cells of greater than 1 indicates a higher level of dual Th2/Th17 cells as compared to the level of Th2 cells.
Yet another embodiment of the present invention is a method of treating a subject having persistent asthma comprising obtaining a sample from the subject; determining the frequency of Type-2 cytokine-producing innate lymphoid (ILC2) cells in the sample, comparing the frequency of ILC2 cells from the subject to a control level, wherein an increased frequency of ILC2 from the subject as compared to the control identifies the subject as having persistent asthma; and administering to the subject a compound selected from the group consisting of a bronchodilator, corticosteroid, leukotriene antagonist, anti-cytokine antibody, anti-cytokine receptor antibody, anti-IgE antibody, an antibiotic, a phosphodiesaterease inhibitor, an anti-MEK compound and combinations thereof for treating the subject. In one aspect, the method further comprises determining the expression level of interleukin-33 (IL33) in the sample from the subject, wherein an increased level of IL33 as compared to the IL33 expression level from the control indicates the greater severity of the persistent asthma.
In one aspect of the invention, the greater the increase in the frequency of ILC2 cells as compared to the control indicates greater severity of the persistent asthma.
In still another aspect of the invention, the frequency of the ILC2 cells is determined by flow cytometry.
In yet another aspect, the expression level of IL33 is determined by ELISA.
Another embodiment of the present invention is a method of treating a subject having steroid resistant asthma comprising obtaining a sample from the subject; determining the expression level of mitogen-activated protein kinase (MEK) in the sample, comparing the expression level of MEK from the subject to an expression level of MEK from a control, wherein an increased expression level of MEK from the subject as compared to the control level identifies the subject as having steroid resistant asthma; and administering to the subject an anti-MEK compound or a non-steroid compound for treating the subject.
In one aspect, the greater the increase in the expression level of MEK from the subject as compared to the control level indicates greater severity of steroid resistance.
In yet another aspect, the expression level of MEK is determined by flow cytometry or ELISA.
In any of the embodiments of the invention described herein, the fluid is selected from bronchoalveolar lavage fluid (BAL), peripheral blood, nasal washing and induced sputum.
In one aspect of the invention a kit for determining the expression level of one or more genes selected from the group consisting of CD4, CRTH2, CCR6, IL-4, IL-17, IL1b, C3, C3a, ILC2, IL33, and MEK, wherein the kit comprises a component selected from the group consisting of an antibody, an antisense RNA molecule, and a molecular probe, and a molecular tag, wherein in the component detects the expression of the one or more genes.
This invention generally relates to improved methods and kits for identifying/predicting and/or treating severe asthma and/or persistent asthma in subjects by analyzing for the presence of certain cells and/or by determining the expression of one or more genes as disclosed herein. The inventors have made the surprising finding that asthma is associated with a higher frequency of dual-positive Th2/Th17 cells in BAL fluid and that the Th2/Th17predominant subgroup of asthmatic patients manifests steroid (such as glucocorticoids) resistance in vitro. This subgroup was also determined to have the greatest airway obstruction and hyperreactivity compared with Th2predominant and Th2/Th17low subgroups. Th2 cells (also referred to as TH2) and Th2/Th17 cells (also referred to as dual Th2/Th17 cells or TH2/TH17 cells) are subgroups of CD4 T helper cells. Asthma is hererogenous, thus some subjects have only Th2 cells, others have both Th2 and Th2/Th17, and yet others have no Th2 or Th2/Th17 cells. Those who have both Th2 and Th2/Th17 cells may fall into categories or subgroups (or endotypes): Th2-predominant (Th2predominant) and Th2/Th17-predominant (Th2/Th17predominant). The inventors have made the surprising finding that of these subjects, the Th2/Th17predominant subgroup have been found to have more severe asthma, thus the Th2/Th17predominant asthma is more severe than Th2predominant asthma and a further subgroup of Th2/Th17low. A predominant subgroup is defined by having a higher amount or frequency of a specific subgroup of cells (Th2, Th2/Th17 cells) compared to the amounts of one or more other subgroups. For example, a Th2/Th17predominant subgroup is a subgroup in which the subject(s) has been determined to have a higher amount or frequency of dual Th2/Th17 cells as compared to the amount of Th2 cells. A Th2predominant subgroup is a subgroup in which the subject(s) has been determined to have higher amount or frequency of Th2 cells as compared to the amount of dual Th2/Th17 cells. A predominant subgroup has greater than 5% of the total T cells belonging to that specific subgroup. A low subgroup is defined by having 5% or less of total T cells belonging to that subgroup. For example, a Th2/Th17low subgroup is a subgroup in which the subject(s) has been determined to have a frequency of Th2/Th17 cells that is 5% or less than the total CD4 T helper cells.
In addition, the inventors have found that elimination of T cells though antibody-mediated depletion or lethal irradiation and transplantation of recombination-activating gene (Rag1)−/− bone marrow in mice with chronic asthma resulted in resolution of airway inflammation but not airway hyperreactivity or remodeling. Elimination of T cells and type 2 innate lymphoid cells (ILC2s) through lethal irradiation and transplantation of Rag2−/−: γc−/− bone marrow or blockade of interleukine-33 (IL-33) resulted in resolution of airway inflammation and hyperreactivity. Persistence of asthma was found to require multiple interconnected feedback and feed-forward circuits between ILC2s and epithelial cells. Additionally, epithelial IL-33 was found to induce ILC2s, a rich source of IL-13. The latter directly induced epithelial IL-33, establishing a positive feedback circuit. IL-33 autoinduced, generating another feedback circuit. IL-13 upregulated IL-33 receptors and facilitated IL-33 autoinduction, thus establishing a feed-forward circuit. Elimination of any component of these circuits resulted in resolution of chronic asthma. In agreement with the foregoing, IL-33 and ILC2 levels were increased in the airways of asthmatic patients. In addition, IL-33 levels correlated with disease severity. The inventors describe a critical network of feedback and feed-forward interactions between epithelial cells and ILC2s involved in maintaining chronic asthma. Although T cells contributed to the severity of chronic asthma, they were redundant in maintaining airway hyperreactivity and remodeling.
The present invention provides for a method of identifying and/or treating a subject having severe asthma by detecting the presence of Th2/Th17 cells as well as detecting the presence of Th2 cells in a sample from the subject and determining and measuring and comparing the level of the Th2/Th17 cells to the level of Th2 cells in the sample, wherein a higher level or frequency of Th2/Th17 cells as compared to the level of the Th2 cells in the biological sample from the subject, identifies the subject as having severe asthma. In one aspect, the subject identified as having severe asthma is then treated. As used herein severe asthma is defined as sustained xacerbation of asthma that has not been found to respond to standard treatments of asthma with bronchodilators (inhalers) and steroids.
The dual Th2/Th17 cells can be identified and/or detected by detecting the expression of a combination of genes. Such a combination of genes includes cluster of differentiation antigen 4 (CD4), chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2; also known as CD294, and G protein coupled receptor 44) and CC chemokine receptor 6 (CCR6). In one aspect, co-expression of all three genes (CD4, CRTH2 and CCR6) indicates or serves as a surrogate for the presence of the dual Th2/Th17 cells. Another combination of genes includes CD4, IL4 and IL17, wherein detection of the co-expression of all three of these genes indicates the presence of Th2/Th17 cells. Once co-expression is identified or detected, the frequency of Th2/Th17 cells is determined. If the frequency of the Th2/Th17 cells is determined to be less than 5% of the total CD4 T cells in the sample, then this subgroup or endotype is a Th2/Th17low subgroup. If however, the frequency of the Th2/Th17 cells is determined to be at least 5% or greater of the total CD4 T cells in the sample, then the frequency of Th2 cells is determined and a comparison is then performed between the frequency of the Th2/Th17 cells and the Th2 cells. If the frequency of the Th2/Th17 cells is higher compared to the frequency of the Th2 cells, this subgroup or endotype is a Th2/Th17predominant. If however, the frequency of the Th2 cells is higher compared to the frequency of the Th2/Th17, then the subgroup is referred to as a Th2predominant subgroup or endotype. When comparing the determined levels or frequency of the dual Th2/Th17 cells to the determined levels or frequency of the Th2 cells, a ratio of greater than 1 indicates a higher level or frequency of dual Th2/Th17 cells as compared to Th2 cells.
In addition, the inventors have found that the dual Th2/Th17 cells can be detected by determining the expression level of complement factor C3 and/or C3a. The inventors have determined that an elevated expression level of C3 and/or C3a in a subject as compared to the expression level of C3 and/or C3a from a healthy control indicates the presence of Th2/Th17 cells.
In order to detect and/or determine the level or frequency of Th2 cells in a sample, expression of CRTH2 on CD4 T cells in the sample is detected. If expression of CRTH2 is detected, then Th2 cells are present in the sample.
The level or frequency of dual Th2/Th17 cells and/or the level or frequency of Th2 cells and/or the total CD4 T cells in a sample can be determined and/or measured by methods such as flow cytometry, ELISA, real-time PCR or immunofluorescence/immunocytochemical staining or a combination thereof. In a preferred aspect, flow cytometry is used.
Another embodiment of the present invention is a method to identify and/or predict and/or treat a subject having persistent asthma by determining the frequency of ILC2 cells in a sample from the subject wherein an increased frequency of ILC2 cells from the subject as compared to a non-allergic healthy control identifies and/or predicts that the subject has persistent asthma. In one aspect the identified subject is then treated. The inventors have found that the greater the frequency of ILC2 cells from the subject as compared to the control indicates greater severity of the persistent asthma. In one aspect of this embodiment, the method further comprises determining the expression level of IL33 in the sample from the subject, wherein an increased level of IL33 (for example >490 pg/ml of bronchoalveolar lavage fluid) as compared to the level that is present in bronchoalveolar lavage from non-allergic healthy control subjects, indicates greater severity of the persistent asthma. In one aspect, the frequency of the ILC2 cells in the sample from the subject is determined by flow cytometry. In another aspect, the expression level of IL33 is the sample from the subject is determined by ELISA.
A further embodiment of the present invention is a method to identify and/or predict and/or treat a subject having steroid resistant asthma by determining the expression level of MEK ERK kinase 1 (MEK), wherein an increased expression level of MEK as compared to a non-allergic healthy control identifies and/or predicts that the subject has steroid resistant asthma. In one aspect the identified subject is then administered an anti-MEK compound or a non-steroid compound. The inventors have found that the greater the increase in the expression level of MEK as compared to the control MEK expression level indicates greater severity of steroid resistance. In one aspect, the expression level of MEK is determined by methods such as flow cytometry and/or ELISA.
In one aspect of the methods of the invention, the sample is from bronchoalveolar lavage fluid (BAL), peripheral blood (including serum), nasal washing or induced sputum.
A subject having persistent asthma means that the subject has asthma symptoms every 30 day. The subject may need to use a rescue inhaler daily and typically a subject's normal activities are affected by symptoms such as wheezing, shortness of breath and/or chest tightness. Persistent asthma can be mild, moderate or severe.
As used herein, the term “expression”, when used in connection with detecting the expression of a gene, can refer to detecting transcription of the gene (i.e., detecting mRNA levels) and/or to detecting translation of the gene (detecting the protein produced). To detect expression of a gene refers to the act of actively determining whether a gene is expressed or not. This can include determining whether the gene expression is upregulated as compared to a control, downregulated as compared to a control, or unchanged as compared to a control or increased or decreased as compared to a reference level. Therefore, the step of detecting expression does not require that expression of the gene actually is upregulated or downregulated or increased or decreased, but rather, can also include detecting that the expression of the gene has not changed (i.e., detecting no expression of the gene or no change in expression of the gene). In addition, the expression level of one or more genes disclosed herein that are strongly correlated with IL-13 can be differentially expressed.
In addition to the methods already disclosed, expression of transcripts and/or proteins can be measured by any of a variety of known methods in the art. For RNA expression, methods include but are not limited to: extraction of cellular mRNA and Northern blotting using labeled probes that hybridize to transcripts encoding all or part of the gene; amplification of mRNA using gene-specific primers, polymerase chain reaction (PCR), and reverse transcriptase-polymerase chain reaction (RT-PCR) and/or RNA Ampliseq, followed by quantitative detection of the product by any of a variety of means; multiplexed quantitative PCR enrichment of cDNA amplicons, followed by conversion of amplicons to sequence libraries and Next-generation based sequencing of libraries to generate digital count expression data; extraction of total RNA from the cells, which is then labeled and used to probe cDNAs or oligonucleotides encoding the gene on any of a variety of surfaces; in situ hybridization; and detection of a reporter gene.
Methods to measure protein expression levels generally include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of the protein including but not limited to enzymatic activity or interaction with other protein partners. Binding assays are also well known in the art. For example, a BIAcore machine can be used to determine the binding constant of a complex between two proteins. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (O'Shannessy et al., 1993, Anal. Biochem. 212:457; Schuster et al., 1993, Nature 365:343). Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA); or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR).
In addition, the presence of cells and/or the frequency of cells are detected. The frequency of the cells such as CD4 T cells and/or Th2 and/or Th2/T17 and/or ILC2 is determined by a method including by not limited to flow cytometery.
When comparing the expression level of any one or more genes as disclosed herein, it is to be understood that the expression level of the any one or more genes is compared with the same gene or genes from the reference or control. For example, if the expression level of CRTH2 and CCR6 are to be determined or analyzed, then the expression level of CRTH2, and CCR6 from the subject would be compared to the expression level of CRTH2, and CCR6 from a healthy control. The expression level of any one or more genes as disclosed herein is considered altered if the expression level of the one or more genes as compared to the expression level of the same one or more genes from the reference or healthy control is increased or decreased (upregulated or downregulated).
In one aspect, the expression levels of at least one, at least two, or at least three, of the genes are altered (i.e. increased, decreased or be a combination of expression levels) as compared to the corresponding genes in a control, wherein one or more of the gene expression levels can be increased (or the genes are upregulated) as compared to the control expression level. In one aspect, the gene expression level of the one or more genes is at least about a 2 fold, at least about a 3 fold, at least about a 4 fold, at least about a 5 fold, at least about a 10-fold, at least about a 20 fold, at least about a 25 fold, at least about a 30 fold, at least about a 40 fold or at least about a 50 fold difference from the expression level of the healthy control.
As used herein, reference to a control (or reference), means a subject (or group of subjects) who is a relevant control to the subject being evaluated by the methods of the present invention. The control can be matched in one or more characteristics to the subject. More particularly, the control can be matched in one or more of the following characteristics, gender, age, disease state, including a healthy control that has been determined to be disease-free. The control expression level used in the comparison of the methods of the present invention can be determined from one or more relevant control subjects. Additionally, a control can be a healthy individual or group of individuals that have been determined to not have an airway disease or condition such as asthma.
The invention also provides for treating the subject. In some aspects, the subjects can be treated by administration of one or more compounds including but not limited to bronchodilators (such as beta agonists and anti-cholinergics), corticosteroids, leukotriene antagonists, anti-cytokine antibodies, anti-cytokine receptor antibodies, anti-IgE antibody, antibiotics, a phosphodiesaterease inhibitor, an anti-MEK compound (such as Trametinib, Selumetinib and Cobimetinib) and combinations thereof.
The invention also provides for a kit for determining expression level of one or more genes described herein (CD4, CRTH2, CCR6, IL-4, IL-17, IL1b, C3, C3a, IL33, and MEK). The kit can comprise one or more components selected from an antibody, an antisense RNA molecule, and a molecular probe or tag, wherein in the component detects the expression and/or frequency of the one or more genes. The kit further comprises pharmaceutically acceptable carriers.
The presence of allergic sensitivity (skin test result positivity or IgE-specific antibody in serum) can be detected in a vast majority, but not all, asthmatic patients (Romanet-Manent S, et al. Allergic vs nonallergic asthma: what makes the difference? Allergy 2002; 57:607-13). In addition to IgE, mild-to moderate eosinophilia is a characteristic feature of asthma. Interestingly, some nonallergic asthmatic patients also have blood eosinophilia. Both IgE antibody and eosinophilia are driven by a Th2-type immune response. Thus the presence of Th2 cells in lung tissue and BAL fluid is anticipated and has previously been reported (Robinson D, et al. Activation of CD41 T cells, increased Th2-type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma. J Allergy Clin Immunol 1993; 92:313-24; Krug N, et al. Cytokine profile of bronchoalveolar lavage-derived CD4(1), CD8(1), and gammadelta T cells in people with asthma after segmental allergen challenge. Am J Respir Cell Mol Biol 2001; 25:125-31; Thunberg S, et al. Allergen provocation increases TH2-cytokines and FOXP3 expression in the asthmatic lung. Allergy 2010; 65:311-8; Walker C, et al. Activated T cells and cytokines in bronchoalveolar lavages from patients with various lung diseases associated with eosinophilia. Am J Respir Crit Care Med 1994; 150:1038-48; Leung D Y, et al. Dysregulation of interleukin 4, interleukin 5, and interferon gamma gene expression in steroidresistant asthma. J Exp Med 1995; 181:33-40; Huang S K, et al. IL-13 expression at the sites of allergen challenge in patients with asthma. J Immunol 1995; 155:2688-94; Brightling C E, et al. TH2 cytokine expression in bronchoalveolar lavage fluid T lymphocytes and bronchial submucosa is a feature of asthma and eosinophilic bronchitis. J Allergy Clin Immunol 2002; 110:899-905). The median frequency of BAL IL-4+CD4 T cells in asthmatic patients in published reports has ranged from 5% 40 to 9% (Krug N, et al. Cytokine profile of bronchoalveolar lavage-derived CD4(1), CD8(1), and gammadelta T cells in people with asthma after segmental allergen challenge. Am J Respir Cell Mol Biol 2001; 25:125-31). The inventors have observed a median frequency of 11% IL-4+CD4 T cells in BAL fluid from asthmatic patients. The relatively higher frequency could be due to the difference in severity of asthma and the number of patients studied. The previous studies were performed on a small number (n=11-12) of patients whose asthma was relatively mild (median forced expiratory volume (FEV1), 90% to 100%). The inventors studied 52 patients with severe asthma whose median FEV1 was 73.5%. Th2-type cytokines, especially IL-5 and IL-13, have been recovered from BAL fluid obtained from asthmatic patients, (Krug N, et al. Cytokine profile of bronchoalveolar lavage-derived CD4(1), CD8(1), and gammadelta T cells in people with asthma after segmental allergen challenge. Am J Respir Cell Mol Biol 2001; 25:125-31; Thunberg S, et al. Allergen provocation increases Th2-cytokines and FOXP3 expression in the asthmatic lung. Allergy 2010; 65:311-8) although some studies did not observe any increase in levels of these cytokines (Bossley C J, et al. Pediatric severe asthma is characterized by eosinophilia and remodeling without T(H)2 cytokines. J Allergy Clin Immunol 2012; 129:974-82.e13; Batra V, et al. Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on alpha-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts. Clin Exp Allergy 2004; 34:437-44). IL-4 and especially IL-13 act on epithelial cells and induce transcription of specific genes (Zhu Z, et al. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999; 103:779-88; Striz I, et al. IL-4 induces ICAM-1 expression in human bronchial epithelial cells and potentiates TNFalpha. Am J Physiol 1999; 277:L58-64). Microarray analysis of airway epithelial cells from asthmatic patients demonstrated an increase in IL-4/IL-13-responsive genes (Woodruff P G, et al. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am J Respir Crit Care Med 2009; 180:388-95). However, a significant number of asthmatic patients did not show an increase in IL-4/IL-13-responsive genes. On the basis of these findings, Woodruff et al. have identified two subgroups of asthma: TH2high and TH2low.
Most differentiated T helper cells manifest plasticity and acquire additional functional features (Mocci S, and Coffman R L. Induction of a Th2 population from a polarized Leishmania-specific Th1 population by in vitro culture with IL-4. J Immunol 1995; 154:3779-87; Yang X O, et al. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs. Immunity 2008; 29:44-56; Koenen H J, et al. Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells. Blood 2008; 112:2340-52; Lee Y K, Turner, et al. Late developmental plasticity in the T helper 17 lineage. Immunity 2009; 30:92-107; Wei G, et al. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD41 T cells. Immunity 2009; 30:155-67; Zhou L, et al. Plasticity of CD41 T cell lineage differentiation. Immunity 2009; 30:646-55. Th2 cells can acquire the ability to produce Th17 cytokines without losing their ability to produce Th2 cytokines (Cosmi L, et al. Identification of a novel subset of human circulating memory CD4(1) T cells that produce both IL-17A and IL-4. J Allergy Clin Immunol 2010; 125:222-30, e1-4). The dual-positive cells emerge from Th2 cells in the presence of Th17− inducing cytokines: IL-1b, IL-6, and IL-21 (Wang Y H, et al. A novel subset of CD4(1) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma. J Exp Med 2010; 207:2479-91). These dual-positive blood cells also express CCR6. The frequency of the Th2/Th17 population is increased in peripheral blood from asthmatic patients (Wang Y H, et al.). Using a multicolor flow cytometric approach, the inventors detected Th2, Th17, and dual-positive Th2/Th17 cells in BAL fluid from asthmatic patients. The differentiation of Th2 and Th17 is regulated by GATA3 and RORgt, respectively. Immunofluorescence studies of BAL cells showed co-expression of IL-4 and IL-17, as well as nuclear colocalization of GATA3 and RORgt, supporting the Th2/Th17 phenotype. IL-4 and IL-6 play a crucial role in induction of Th2 and Th17, respectively. They do so by inducing phosphorylation of STAT6 and STAT3. The inventors have been able to demonstrate the presence of dual-positive pSTAT3/pSTAT6 cells, further confirming the dual Th2/Th17 phenotype.
One area of interest is the mechanism of differentiation of dual-positive Th2/Th17 cells. Both Th2 and Th17 cells could give rise to Th2/Th17 cells. However, the frequency of single-positive Th17 cells was either less than that of Th2 cells or undetectable in the study subjects. This contrasted with much higher frequency of Th2 cells in BAL fluid from most asthmatic patients. Furthermore, IL-17 production in dual-positive cells was usually associated with IL-4high CD4 T-cell counts. These findings favor Th2 cells as the precursors for Th2/Th17 cells. The generation and sustenance of Th2 cells require the AP1 transcription factor JunB (Hartenstein B, et al. Th2 cell-specific cytokine expression and allergen-induced airway inflammation depend on JunB. EMBO J 2002; 21:6321-9). Some recent publications have demonstrated that JunB forms a trimolecular complex with basic leucine zipper ATF-like (Batf) and interferon regulatory factor (IRF) (Coffman R L, et al. Reversal of polarized T helper 1 and T helper 2 cell populations in murine leishmaniasis. Ciba Found Symp 1995; 195:20-33; Glasmacher E, et al. A genomic regulatory element that directs assembly and function of immunespecific AP-1-IRF complexes. Science 2012; 338:975-80; Li P, et al. BATF-JUN is critical for IRF4-mediated transcription in T cells. Nature 2012; 490:543-6). This complex binds to the so-called AP1-IRF composite element (AICE) in the IL-17 gene promoter and plays an important role in IL-17 gene induction (Ciofani M, et al. A validated regulatory network for Th17 cell specification. Cell 2012; 151:289-303). The inventors have previously shown that JunB is a MEK-inducible protein (Liang Q, et al. IL-2 and IL-4 stimulate MEK1 expression and contribute to T cell resistance against suppression by TGF-beta and IL-10 in asthma. J Immunol 2010; 185:5704-13). Thus IL-4high Th2 cells with a high JunB level could, under Batf-inducing conditions (eg, stimulation with IL-1), begin to form complexes with Batf and IRF4 and lead to IL-17 production. MEK also induces the PEA-3 (polyoma enhancer activator 3) family transcription factor Etv4 (Fontanet P, et al. Pea3 transcription factor family members Etv4 and Etv5 mediate retrograde signaling and axonal growth of DRG sensory neurons in response to NGF. J Neurosci 2013; 33: 15940-51; Li X, et al. MEK is a key regulator of gliogenesis in the developing brain. Neuron 2012; 75:1035-50). The latter is required for Th17 induction (Pham D, et al. The transcription factor Etv5 controls TH17 cell development and allergic airway inflammation. J Allergy Clin Immunol 2014; 134:204-14). Thus MEK-driven JunB and Etv4 may promote IL-17 production in IL-4high Th2 cells.
A previous study has demonstrated that Th2/Th17 cells induce a more severe form of experimental asthma in an adoptive transfer model in mice when compared with Th2 and Th17 cells (Wang et al.). The inventors have observed more severe airway obstruction and airway hyperreactivity in the Th2/Th17predominant subgroup. An explanation for the increased severity of asthma could be the presence of Th2/Th17 cells. However, the mean fluorescence intensity (MFI) of IL-4 in Th2/Th17 cells from the Th2/Th17predominant subgroup is higher than that in Th2 cells from the Th2predominant subgroup. These data are support a higher level of IL-4 production by the Th2/Th17 cells in the Th2/Th17predomiant subgroup. Thus the severity of asthma in this subgroup could be due to increased IL-4 production. On the other hand, IL-17 production by Th2/Th17 cells is likely to change the quality of airway inflammation and function. IL-17 is known to directly affect airway epithelial cells, fibroblasts, and smooth muscle cells (Barczyk A, et al.; Bullens D M, et al.; Molet S, et al.; Al-Ramli W, et al.; and Agache I, et al). In agreement, the inventors have observed a significant negative correlation between BAL IL-17 levels and FEV1. All asthmatic patients were taking high-dose inhaled steroids at the time of the study. Despite this treatment, they had sustained airway obstruction, suggesting relative steroid resistance. Th17 cells have previously been shown to be resistant to glucocorticoids.14 The inventors' results show that the dual-positive Th2/Th17 cells are also resistant to dexamethasone. This resistance may contribute to the refractory nature of their asthma. A number of molecular mechanisms have been implicated in glucocorticoid resistance. Reduced glucocorticoid receptor (GR) phosphorylation by p38 mitogen-activated protein kinase (α and γ) leads to reduced nuclear translocation and confers resistance (Irusen E, et al., p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma. J Allergy Clin Immunol 2002; 109:649-57). However, Th17 cells manifest normal nuclear translocation of GR receptor (McKinley L, et al. TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice. J Immunol 2008; 181:4089-97). The anti-inflammatory action of the GR is facilitated by histone deacetylase 2 (HDAC2), a chromatin-remodeling enzyme. Patients with severe asthma have reduced HDAC2 levels, which could contribute to glucocorticoid resistance (Ito K, et al. Expression and activity of histone deacetylases in human asthmatic airways. Am J Respir Crit Care Med 2002; 166:392-6). The 6 isoform of phosphatidylinositol-3 kinase phosphorylates HDAC2 and contributes to its degradation, which results in steroid resistance (To Y, et al. Targeting phosphoinositide-3-kinase-delta with theophylline reverses corticosteroid insensitivity in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2010; 182:897-904). The transcription factor c-Fos (a component of AP1) directly antagonizes GR and confers resistance (Adcock I M, et al. Abnormal glucocorticoid receptor-activator protein 1 interaction in steroid-resistant asthma. J Exp Med 1995; 182:1951-8). c-Fos is downstream of the MEK-extracellular signal-regulated kinase 1/2 signaling pathway. The inventors have found that severe asthma is associated with increased expression of MEK1 in CD4 T cells (Liang Q, et al. IL-2 and IL-4 stimulate MEK1 expression and contribute to T cell resistance against suppression by TGF-beta and IL-10 in asthma. J Immunol 2010; 185:5704-13). Increased expression of MEK1 confers resistance against a broad spectrum of endogenous homeostatic regulators (i.e., TGF-β, IL-10, and glucocorticoids) through induction of c-Fos and JunB. Inhibition of MEK1 reverses this resistance. The inventors demonstrate that Th2/Th17 cells express higher levels of MEK1, and these MEK1high CD4 T cells are resistant to dexamethasone. The inventor's BAL CD4 T-cell analysis allows them to identify 3 distinct subgroups of asthma: Th2predominant, Th2/Th17predominant, and Th2/Th17low. Woodruff et al. (2009) identified only Th2high and Th2low subgroups of asthma based on epithelial microarray data. The inventors of the present invention have identified a subgroup of Th2predominant patients in whom BAL Th2 cells co-express IL-17. This finding has mechanistic and clinical implications. It is well known that asthma exacerbation is frequently triggered by respiratory tract infection (Jackson D J, et al. Asthma exacerbations: origin, effect, and prevention. J Allergy Clin Immunol 2011; 128:1165-74). A subgroup of asthmatic patients has chronic lung infections with Mycoplasma and Chlamydia species (Johnston S L, and Martin R J. Chlamydophila pneumoniae and Mycoplasma pneumoniae: a role in asthma pathogenesis? Am J Respir Crit Care Med 2005; 172:1078-89). Many environmental factors, such as air pollution, smoke, and chemicals, nonspecifically aggravate asthma (To T, et al. The air quality health index and asthma morbidity: a population-based study. Environ Health Perspect 2013; 121:46-52; Perez L, et al. Chronic burden of near-roadway traffic pollution in 10 European cities (APHEKOM network). Eur Respir J 2013; 42:594-605). Infections and these environmental factors are known to elicit inflammatory cytokines, such as IL-1b and IL-6 (Tabarani C M, et al. Novel inflammatory markers, clinical risk factors and virus type associated with severe respiratory syncytial virus infection. Pediatr Infect Dis J 2013; 32:e437-42; Thompson A M, et al. Baseline repeated measures from controlled human exposure studies: associations between ambient air pollution exposure and the systemic inflammatory biomarkers IL-6 and fibrinogen. Environ Health Perspect 2010; 118:120-4). Both IL-1b and IL-6 induce differentiation of Th2/Th17 cells from Th2 cells. Thus a typical patient with allergic asthma with a Th2predominant endotype could trend toward a Th2/Th17predominant endotype over time if he or she has recurrent respiratory tract infections, were exposed to the aforementioned environmental toxicants, or both. The invasive nature of bronchoscopy and the potential for adverse events together are a major deterrence for patient participation. Induced sputum, which is less invasive, is an alternative approach to obtaining airway cells. However, the number of cells obtained through induced sputum is much less than that through BAL. Isolation of cells from the sputum requires harsh mucolytic treatment, which can affect cellular function and viability. Flow cytometric analysis of sputum cells has shown greater variability.
Finally, the subjects in the study took routine medications at the time of BAL. Medications can affect T-cell numbers and their cytokine expression. Unfortunately, there is no alternative. The institutional review board does not allow discontinuation of medications, especially in patients with severe asthma, whose disease can deteriorate without medication.
As demonstrated in the examples presented below, the inventors have found an increased frequency of dual-positive Th2/Th17 cells in BAL fluid from asthmatic patients. The increased expression of Th2/Th17 cells and one of its cytokines, IL-17, are associated with heightened airway hyperreactivity and airway obstruction, two objective features of asthma severity. Severe asthma manifests relative steroid resistance. The inventors provide a mechanistic explanation for this resistance. Th2/Th17 cells express high levels of MEK1, which is associated with steroid resistance. The identification of a TH2/TH17predominant endotype in addition to the previously recognized TH2predominant and Th2low endotypes of asthma has pathogenetic and therapeutic implications.
Persistence of asthma is generally considered to be mediated by allergen-specific memory T cells (Corry D B, et al. Requirements for allergen-induced airway hyperreactivity in T and B cell-deficient mice. Mol Med 1998; 4:344-55). The depletion of allergen-specific memory T cells in the model of the inventors did not eliminate airway hyperreactivity and remodeling. However, their absence reduced airway hyperreactivity and especially inflammation. On the other hand, the depletion of ILC2s led to resolution of all features of asthma. Although many previous studies demonstrated the ability of ILC2s to induce airway eosinophilic inflammation independent of T cells, this is the first demonstration of ILC2s inducing sustained airway hyperreactivity. The inventors substantiated the role of ILC2s through gain-in-function (adoptive transfer) and loss-of-function (lethal irradiation followed by Rag2−/−: γc−/− marrow transplantation and anti-IL-33 treatment) approaches. Epithelial cells and ILC2s established three distinct positive feedback and feed-forward mechanisms to sustain asthma, as summarized in
An important feature of these positive feedback and feedforward circuits is that they are interconnected. There is strong mathematic and experimental evidence that interconnected positive feedback circuits induce ultrasensitivity and bistability (Chang D E, et al. Building biological memory by linking positive feedback loops. Proc Natl Acad Sci USA 2010; 107:175-80; Shin S Y, et al. Functional roles of multiple feedback loops in extracellular signal-regulated kinase and Wnt signaling pathways that regulate epithelial-mesenchymal transition. Cancer Res 2010; 70:6715-24). Ultrasensitivity is manifested when a linear input generates a sigmoidal output. Ultrasensitivity induces system bistability. A system is considered bistable when it is “on” (active) in the absence of any input (Xiong W, and Ferrell J E Jr. A positive-feedback-based bistable ‘memory module’ that governs a cell fate decision. Nature 2003; 426:460-5; Markevich N I, et al. Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades. J Cell Biol 2004; 164:353-9; Srividhya J, et al., Open cascades as simple solutions to providing ultrasensitivity and adaptation in cellular signaling. Phys Biol 2011; 8:046005). The effect of interconnected feed-forward circuits is illustrated by the differential effect of IL-13 versus IL-4 or IL-17 on IL-33 autoinduction. Although all three cytokines directly induced IL-33, only IL-13 produced synergy through its feed-forward effect on IL-33R expression. Previously, the inventors demonstrated that repetitive stimulation of epithelial cells with IL-13 led to extracellular signal-regulated kinase (ERK) 1/2 bistability through the establishment of a signaling feedback circuit (LiuW, et al. Establishment of extracellular signal-regulated kinase 1/2 bistability and sustained activation through Sprouty 2 and its relevance for epithelial function. Mol Cell Biol 2010; 30:1783-99). ERK1/2 bistability primed epithelial cells for heightened cytokine production. ERK1/2 signaling is likely relevant because its phosphorylation is increased in airway epithelial cells from asthmatic patients (Liu W, et al. Cell-specific activation profile of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, and p38 mitogen-activated protein kinases in asthmatic airways. J Allergy Clin Immunol 2008; 121:893-902.e2). Thus IL-13 could establish multiple feedback and feed-forward circuits for sustained IL-33 production.
The inventors have demonstrated the biological relevance of their findings in human asthma. In agreement with previous reports, (Prefontaine D, et al. Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells. J Immunol 2009; 183:5094-103; Prefontaine D, et al. Increased IL-33 expression by epithelial cells in bronchial asthma. J Allergy Clin Immunol 2010; 125:752-4), the inventors showed increased IL-33 levels in BAL fluid from asthmatic patients. The IL-33 level negatively correlated with the airway flow volume. For the first time, the inventors demonstrate a significant increase in ILC2 numbers in BAL fluid from asthmatic patients.
Previously, increased ILC2 numbers were reported in nasal polyps from patients with chronic rhinosinusitis (Mj€osberg J M, et al. Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol 2011; 12:1055-62; Shaw J L, et al. IL-33-responsive innate lymphoid cells are an important source of IL-13 in chronic rhinosinusitis with nasal polyps. Am J Respir Crit Care Med 2013; 188:432-9) and in peripheral blood from asthmatic patients (Bartemes K R, et al. Enhanced innate type 2 immune response in peripheral blood from patients with asthma. J Allergy Clin Immunol 2014; 134:671-8.e4). The frequency in polyps ranged from 0.1% to 3.6% of CD45+ cells and that in blood ranged from 0.01% to 0.03% of mononuclear cells. The frequency of ILC2s in BAL fluid from asthmatic patients was similar to that in polyps but was higher than that in peripheral blood.
The novel findings of the inventors as provided and discussed herein and presented in the examples below have implications for chronic illnesses in general. These results indicate that recurrent episodes of any acute illness can establish feedback and feed-forward circuits. Once established, these feedback circuits sustain the disease process during intervals between acute episodes, which facilitates the transition of an acute illness to a chronic one.
The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations which occur to the skilled artisan are intended to fall within the scope of the present invention. All references cited in the present application are incorporated by reference herein to the extent that there is no inconsistency with the present disclosure.
In this example the expression of Th2, Th17, and dual-positive Th2/Th17 cells in BAL fluid from patients with treatment-refractory asthma and their response to glucocorticoids was analyzed. In addition, the clinical relevance of Th2/Th17 cells in asthmatic patients is shown.
Patients were allowed to continue their routine medication. Bronchoscopy and BAL were performed, as described previously (Good J T Jr, et al. Refractory asthma: importance of bronchoscopy to identify phenotypes and direct therapy. Chest 2012; 141:599-606). BAL fluid was processed immediately. Cells were isolated by means of centrifugation. Supernatant fluid was placed into aliquots in small samples and frozen. Cells were either cultured or fixed immediately in 4% paraformaldehyde and processed for flow cytometry. For cultures, cells were washed and divided into 2 treatment groups: medium or dexamethasone (107 mol/L). The cells were cultured in RPMI 1640 with 10% FBS overnight. The next day, the cells were washed and fixed in paraformaldehyde. Monensin (2 μmol/L) was added 6 hours before fixing.
Cells were stained with the following antibodies. Allophycocyaninlabeled mouse anti-human CD4 (clone RPA-T4), phycoerythrin (PE)-Cy7-labeled mouse anti-human IL-4 (clone MP4 25D2), allophycocyanin/Cy7-labeled mouse anti-human IL-17 (clone BL168), mouse anti-CD3ε (clone OKT3), mouse anti-CD68 (clone Y1/82A), mouse anti-CD163 (clone GH1/61), anti-chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2; clone BM16), mouse anti-CCR6 (clone G034E3), and rat anti-IL-5 (clone TRFK5) were from BioLegend (San Diego, Calif.). PE-labeled anti-mitogen-activated protein-extracellular signal-regulated kinase kinase 1 (MEK1; clone 25/MEK1), peridinin-chlorophyll-protein complex-Cy5.5-labeled mouse anti-signal transducer and activator of transcription (STAT) 3 (pY705) (clone 4/P-STAT3), and Alexa Fluor 488-labeled mouse anti-STAT6 (pY641; clone 18/P-STAT6) were from BD Biosciences (San Jose, Calif.). A rabbit anti-mitogen-activated protein kinase phosphatase 1 (MKP1) antibody was from Santa Cruz Biotechnology (Dallas, Tex.). This was detected with an Alexa Fluor 488-labeled anti-rabbit secondary antibody. The isotype controls were rat IgG1 for the anti-IL-4 antibody, mouse IgG1 for anti-CD4 and anti-IL-17 antibodies, and mouse IgG2a for anti-phosphorylated signal transducer and activator of transcription (pSTAT) 3 and anti-pSTAT6 antibodies. The Fc receptors were blocked by incubating cells first with 10% goat serum and then conducting immunostaining with specific antibodies in 5% goat serum. Flow cytometry was performed with a CyAn ADP Analyzer 9 color flow cytometer (Beckman Coulter, Brea, Calif.), as described previously (Liang Q, et al. IL-2 and IL-4 stimulate MEK1 expression and contribute to T cell resistance against suppression by TGF-beta and IL-10 in asthma. J Immunol 2010; 185:5704-13). Flow cytometric data were analyzed with FlowJo software (Tree Star, Ashland, Ore). Only small and nongranular cells were gated by means of forward and side scatter, respectively, and the large and highly granular BAL cells were excluded from analysis because they tend to bind many antibodies nonspecifically, as reported previously (Thomas S Y, et al. Invariant natural killer T cells in bronchial asthma. N Engl J Med 2006; 354:2613-6). The threshold line for identification of positively stained cells was set conservatively based on the control isotype antibody staining pattern. The emphasis was on exclusion of nonspecifically stained cells. Less than 1% (usually <0.5%) of the cells stained positively by using this control antibody-based thresholding strategy. Specific cell populations were first identified (CD4, CD3, CD163, and CD68) and then the cells were analyzed for the presence of intracellular cytokines. Isotype control antibodies were run in all experiments, and the aforementioned gating and thresholding strategy was applied to all.
Double immunofluorescence staining BAL cells were fixed, and cytospin preparations were immunostained with a combination of mouse monoclonal anti-GATA3 (clone TWAJ; eBioscience, San Diego, Calif.) and a rabbit polyclonal anti-retinoic acid receptor-related orphan receptor gt (RORgt; clone H-190, Santa Cruz Biotechnology) antibody or mouse anti-IL-4 (clone 8D4-8; BD PharMingen, San Jose, Calif.) and rabbit anti-IL-17 (Santa Cruz Biotechnology) antibodies, as described previously (Guo L, et al. Nuclear translocation of MEK1 triggers a complex T cell response through the corepressor silencing mediator of retinoid and thyroid hormone receptor. J Immunol 2013; 190:159-67). Fluorescein isothiocyanate- and PE-labeled secondary antibodies were directed against anti-GATA3 and anti-RORgt antibodies, respectively. The cytospin preparations were counterstained with 49-6-diamidino-2-phenylindole dihydrochloride (DAPI). Z-series images were captured with a Zeiss confocal microscope (Zeiss, Oberkochen, Germany).
IL-17 (IL-17A) was assayed in undiluted BAL fluid by using an ELISA kit from R&D Systems (Minneapolis, Minn.), according to the supplier's instructions.
Comparisons between study groups were done with the Mann-Whitney U test. Comparisons among multiple study groups were performed by using the Kruskal-Wallis test. The Pearson correlation coefficient was used to calculate the correlation.
These results demonstrate the detection of single TH2 and TH17 cells and dual TH2/TH17 cells in BAL fluid from asthmatic patients.
BAL cells from 52 asthmatic patients and 25 disease control subjects were studied. Most of the patients were referred for diagnosis and management of refractory asthma. Others were referred for routine asthma care. The term refractory as used herein indicates uncontrolled asthma, which could be moderate or severe, as determined based on the Expert Panel Report 3 criteria. The clinical characteristics of the study subjects are shown in Table 1. Bronchoscopy and BAL were performed, as described previously (Good J T Jr, et al. Refractory asthma: importance of bronchoscopy to identify phenotypes and direct therapy. Chest 2012; 141:599-606). BAL fluid was processed immediately for flow cytometry or for culture overnight, as described below. After immunostaining, cells were first gated for small nongranular cells by means of forward and side scatter (see
αnumber in the parenthesis indicates median;
In another approach BAL cells for CD4 and CD68 were analyzed (a different macrophage marker) or CD4 and CD3ε(see
Differentiation of Th2 and Th17 cells is controlled by the transcriptional regulators GATA3 and RORγt, respectively. Co-expression of GATA3 and RORgt was examined using immunofluorescence staining and confocal microscopy in BAL lymphocytes from 4 asthmatic patients.
Expression of Dual-Positive IL-4/IL-17 Cells is Associated with Dual-Positive pSTAT6/pSTAT3- and CCR6/CRTH2-Expressing Cells
The phosphorylation and activation of STAT6 by IL-4 and STAT3 by IL-6 and IL-21 are early events during differentiation of TH2 and TH17 cells (Kaplan M H, and Grusby M J. Regulation of T helper cell differentiation by STAT molecules. J Leukoc Biol 1998; 64:2-5; Yang X O, et al. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells. J Biol Chem 2007; 282:9358-63). The expression of activating phosphorylation of STAT3 and STAT6 in BAL CD4 T cells was examined. Significant coexpression of pSTAT3 and pSTAT6 was observed, which followed the pattern of IL-4 and IL-17 coexpression (see
IL-17 Levels are Increased in BAL Fluid from Asthmatic Patients and Negatively Correlate with FEV1
Since expression of IL-17 by Th2/Th17 cells was detected, levels of secreted IL-17 in BAL fluid using ELISA was measured. The IL-17 level was significantly increased in asthmatic patients compared with that seen in disease control subjects (
Dexamethasone does not Inhibit BAL-Derived Dual-Positive Th2/Th17 Cells
Most of the patients were referred for refractory asthma and relative steroid resistance. For this reason, sensitivity of Th2/Th17 to dexamethasone was examined. One of the dexamethasone-responsive genes is MKP1 (Kassel O, et al. Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. EMBO J2001; 20:7108-16). Differential expression of MKP1 in T cells after treatment with dexamethasone was observed. Dexamethasone increased the number of cells that expressed higher levels of MKP1 (
One of the signaling pathways that antagonize the inhibitory action of glucocorticoids is the MEK-extracellular signal-regulated kinase 1/2 pathway (Liang Q, et al. IL-2 and IL-4 stimulate MEK1 expression and contribute to T cell resistance against suppression by TGF-beta and IL-10 in asthma. J Immunol 2010; 185:5704-13; Tsitoura D C, and Rothman P B. Enhancement of MEK/ERK signaling promotes glucocorticoid resistance in CD41 T cells. J Clin Invest 2004; 113:619-27; Adcock I M, et al. Abnormal glucocorticoid receptor-activator protein 1 interaction in steroid-resistant asthma. J Exp Med 1995; 182:1951-8). This pathway induces the activating protein 1 (AP1) transcription factors, which antagonize glucocorticoids. Conversely, glucocorticoids antagonize AP1 by inducing the glucocorticoid-induced leucine zipper (Mittelstadt P R, Ashwell J D. Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem 2001; 276:29603-10). The inventors have previously reported that CD4 T cells from patients with moderate-to-severe asthma have increased expression of MEK1 (Ling et al. 2010). Inhibition of MEK1 reverses T-cell resistance against dexamethasone. BAL CD4 T cells for MEK1 (referred to as MEK) expression and the sensitivity of MEK1 cells to dexamethasone was analyzed. A small CD4 population in BAL fluid that expressed a high level (MFI 133) of MEK was observed (MEKhi;
Based on the frequency of Th2 and Th2/Th17 cells in BAL fluid, the population can be divided into 3 separate subgroups (
Next, clinical and biochemical features of the identified subgroups were examined. Airway hyperreactivity was most severe in the Th2/Th17predominant subgroup (
This example studies the mechanisms of the persistence of asthma in the absence of the inciting allergens. To accomplish this, a mouse model in which asthma persisted for 3 weeks after cessation of repetitive dust mite, ragweed, and Aspergillus species allergen exposure was used (Bullens D M, et al. IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx? Respir Res 2006; 7:135).
C57Bl/6 CD45.1, C57Bl/6 CD45.2, BALB/c, Rag1−/−, Rag2−/−: γc−/− and Erk1−/− mice were used in this study.
Allergens used included extracts of dust mite (Dermatophagoides farinae), ragweed (Ambrosia artemisiifolia), and Aspergillus fumigatus (Greer Laboratories, Lenoir, N.C.). Intranasal allergens (dust mite, 5 μg; ragweed, 15 μg; Aspergillus species, 5 μg [DRA]) or Aspergillus species (5 μg) were delivered in 15-mL quantities in saline. Acute asthma was produced by means of immunization of 8- to 12-weekold BALB/c mice twice 1 week apart with Aspergillus species (5 μg) in adjuvant (1:1 vol/vol). Adjuvant was aluminum and magnesium hydroxide (Pierce, Rockford, Ill.). Asthma was initiated by using 3 consecutive intranasal exposures to Aspergillus species (5 μg in 15 mL of saline), and asthma was evaluated 72 hours after the final exposure.
Chronic asthma was produced by intranasal delivery of the DRA mixture twice a week for 6 consecutive weeks in female mice (C57Bl/6 CD45.2 or BALB/c mice, as appropriate) 8 to 12 weeks of age. For characterization of asthma chronicity experiments, mice were analyzed at the indicated time points (1, 2, or 6 months after cessation of allergen exposure). A timeline of manipulations and interventions for the chronic asthma protocol with irradiation is shown in
For IL-33 blockade experiments, intraperitoneal anti-IL-33 (15 μg/200 mL injection in saline; R&D Systems, Minneapolis, Minn.) was delivered 3, 5, and 7 days before analysis in week 15 (6 weeks after irradiation). All non-irradiated control mice with asthma were rested an equivalent amount of time before analyses. For CD3 and IL-13 blockade, hamster anti-mouse CD3ε (200 μg per dose, clone 145 2C11; eBioscience, San Diego, Calif.) (Haile S, et al. Mucous-cell metaplasia and inflammatory-cell recruitment are dissociated in allergic mice after antibody- and drug-dependent cell depletion in a murine model of asthma. Am J Respir CellMol Biol. 1999; 20:891-902) and anti-IL-13 (50 μg per dose; Calbiochem, San Diego, Calif.) antibodies and hamster IgG or rat IgG were administered intraperitoneally on 3 consecutive days in week 10, and outcomes were examined 3 days later.
Spleen CD4 T cells were negatively selected by using antibody-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and transferred intravenously (4×106 cells) to naive mice. ILC2s (lin-CD45+CD25+) were sorted on MoFlow XDP (Beckman Coulter, Fullerton, Calif.) and delivered intravenously (2×105 cells) to naive mice 24 hours after sorting. Intranasal DRA (5 μg, 15 μg, and 5 μg, respectively, per 15-mL dose in saline) was performed on 3 consecutive days after intravenous transfer of CD4 T cells. CD4 T cell-recipient mice were studied on days 6 and 21. ILC recipient mice were analyzed on day 21.
Measurement methodologies have been explained in depth in Goplen et al. (Goplen N, et al. Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma. J Allergy Clin Immunol 2009; 123:925-32.e11). Briefly, mice were anesthetized with ketamine (180 mg/kg), xylazine (9 mg/kg), and acepromazine (4 mg/kg). After loss of footpad pinch reflex, a tracheotomy was performed, and the mouse was attached with an 18-gauge cannula to a small-animal ventilator with a computer-controlled piston (FlexiVent; SCIREQ, Montreal, Quebec, Canada). Mice were ventilated at a frequency of 90 breaths/min with a tidal volume of 20 mL/kg during nebulization and otherwise with a frequency of 150 breaths/min with a tidal volume of 20 mL/kg while breathing against an artificial positive endexpiratory pressure of 2.5 to 3 cm H2O. Lungs were inflated to total lung capacity twice to standardize volume history. Resistance measurements were then taken to establish baselines for total lung resistance and at each methacholine dose. Group averages were expressed as fold increases in baseline resistance (means+SEMs).
Paraffin-embedded lungs were sectioned and stained with hematoxylin and eosin (H&E) for morphometric analysis or mucin staining or toluidine blue for mast cell staining. Sections for immunofluorescence staining were permeabilized with 0.01% saponin in PBS, blocked with 10% goat serum, and stained with a primary antibody against human IL-33 and visualized with Alexa Fluor 488-conjugated secondary antibody, as described in Gorska et al. (Gorska M M, et al. MK2 controls the level of negative feedback in the NF-kappaB pathway and is essential for vascular permeability and airway inflammation. J Exp Med 2007; 204:1637-1652) and counterstained with 49-6-diamidino-2-phenylindole dihydrochloride for nuclear staining.
Inflammation was quantified by using Metamorph image acquisition and analysis software on H&E-stained lung sections at 20× magnification, as described in Goplen et al. 2009. Airway epithelial hypertrophy and peribronchial smoothmuscle hypertrophyweremeasured as the area of epithelial or smoothmuscle per circumference of airway basement membrane. Airway inflammation was measured as the area of inflammatory infiltrates as a percentage of the total field. A minimum of 5 airways per mouse and 5 to 9 mice per group were quantified. Images were acquired on a Nikon Eclipse TE2000-U microscope by using 320 dry lenses at room temperature through a Diagnostics Instruments camera model #4.2 with Spot software 5.0 (Diagnostic Instruments, Inc, Sterling Heights, Mich.). H&E sections were mounted with Permount medium (Thermo Fisher Scientific, Inc, Waltham, Mass.). Images were adjusted for brightness and contrast to improve viewing.
Lungs were perfused with saline, and single-cell suspensions of lung cells were acquired by using mechanical mincing of lungs followed by digestion at 37° C. for 45 minutes in RPMI with 10% FBS, 1% penicillin/streptomycin, and collagenaseA (1 mg/mL). Cell suspensions were agitated at room temperature for 10 minutes in RPMI with 100 U/mL DNAse I before filtration through 40-mm filters and red blood cell lysis. Single-cell suspensions were subsequently fixed in 4% paraformaldehyde for flow cytometric analysis.
Mouse ILC2s. For mouse flow cytometric staining, all conjugate antibodies were purchased from BioLegend (San Diego, Calif.), unless otherwise stated. Mouse lung digest cells were fixed with 4% paraformaldehyde and incubated with 10% donkey serum and 1% 2.4G2. ILCs were stained with PerCp/Cy5.5-labeled CD45.2 (clone 104), phycoerythrin/Cy7-labeled CD45.1 (clone A20), or both, as appropriate; Pacific Blue-labeled lineage marker antibodies (CD3, Ly-6G/Ly-6C, CD1 b, CD45R/B220, TER-119/erythroid cells); Alexa Fluor 488-conjugated CD25 (clone PC61); allophycocyanin-labeled IL-5 (TRFK5); and phycoerythrin-labeled IL-13 (clone eBio13A, eBioscience). ILC2s were initially characterized with the addition of the primary antibody anti-IL-33R (ST-2; clone AF1004, R&D Systems), followed by allophycocyanin-labeled secondary antibody against the primary antibody and allophycocyanin-eFluor 780-conjugated IL-7Ra (clone eBioRDR5, eBioscience), allophycocyanin-labeled CD117 (ckit; clone ACK2), phycoerythrin-labeled Ly-6A/E (Sca-1; clone D7), phycoerythrin-Cy7-labeled NK1.1 (clone PK136), and allophycocyaninlabeled FcεRI (clone MAR-1).
Lung IL-331 cells. IL-33 levels and localizations were characterized with the primary antibody against IL-33 (clone Poly5165), followed by fluorescein isothiocyanate-labeled secondary and primary antibodies against pro-surfactant protein C (AB3786; Millipore, Temecula, Calif.), followed by allophycocyanin-labeled secondary or phycoerythrin/Cy7-labeled CD11b (clone M1/70) or the primary antibody against E-cadherin (clone H108; Santa Crux Biotechnology, Dallas, Tex.), followed by a Pacific Blue-labeled secondary antibody or allophycocyanin-labeled FcεRI (clone MAR-1).
Human BAL Fluid ILC2s.
BAL fluid cells were fixed in 4% paraformaldehyde and incubated in 10% goat serum. ILC2s were stained with Pacific Blue-labeled lineage marker antibodies (CD3, CD14, CD16, CD19, CD20, and CD56), fluorescein isothiocyanate-labeled IL-7Ra (clone A019D5), PerCp/Cy5.5-labeled IL-13 (clone JES10-5A2), or phycoerythrin-labeled IL-5 (clone JES1-39D10). IL-33R was identified with the addition of a primary antibody against IL-33R (ST-2; clone HB12; MBL International, Woburn, Mass.), followed by allophycocyanin-labeled secondary antibody against the primary antibody. After washing, cells were analyzed by using flow cytometry with the CyAn ADP cytometer (Beckman Coulter). Data were analyzed with FlowJo version 7.6.5 software (TreeStar, Ashland, Ore).
Splenocytes and mediastinal lymph node cells (2×106/mL) were cultured for 96 hours on anti-CD3 and anti-CD28 (1 μg/mL)-coated 48-well plates or stimulated separately with the following allergens: dust mite (10 μg/mL), ragweed (50 μg/mL), and Aspergillus species (10 μg/mL) (Gorska et al. 2007). In one set of cultures, cells were labeled with 1 μmol/L carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, Carlsbad, Calif.). For measurement of cytokines (IL-2 and IL-4), monensin was added to another culture set 6 hours before the conclusion. T cells were detected by addition of allophycocyanin-labeled anti-CD4 (clone GK1.5, BioLegend) before flow cytometric analysis on the CyAn ADP cytometer (Beckman Coulter) for proliferation and intracellular cytokine expression.
Immediately after flexiVent analysis, lung sections were collected in RIPA buffer (50 mmol/L Tris [pH 7.4], 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L NaF, 1 mmol/L Na3VO4, and 0.1 mmol/L phenylmethylsulfonyl fluoride) containing protease and phosphatase inhibitors and homogenized. Lysed samples were subjected to 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and then immunoblotted with primary antibodies (IL-33 [R&D Systems], tubulin [H-235], and b-actin [Santa Cruz Biotechnology, Dallas, Tex]). After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody. After additional washing, the membranes were developed with ECL reagent, as previously described (Gorska et al. 2007). A549 cell culture A549 cells were cultured in RPMI in 10% FBS (HyClone; Thermo Scientific, Waltham, Mass.) and 1% penicillin/streptomycin (Gibco, Carlsbad, Calif.) and maintained at 378C in a humidified 5% CO2 incubator. All cytokines were purchased from PeproTech (Rocky Hill, N.J.). In one experiment cells were treated with medium, IL-1b (2 ng/mL), IL-4 (10 ng/mL), IL-13 (20 ng/mL), IL-17 (10 ng/mL), IL-33 (20 ng/mL), TNF (2 ng/mL), IFN-g (10 ng/mL), and TNF (2 ng/mL) plus IFN-g (10 ng/mL) for 72 hours. Cellular RNA was analyzed for mRNA for IL-33 and IL-33R. In a second experiment cells were pretreated for 24 hours with medium, IL-1b (2 ng/mL), IL-4 (10 ng/mL), IL-13 (20 ng/mL), IL-17 (10 ng/mL), TNF (2 ng/mL), or IFN-g (10 ng/mL) before stimulation for 72 hours with IL-33 (20 ng/mL). Cellular RNAwas analyzed for mRNA for IL-33. In a third experiment cells were treated for 24 hours with DRA (5 μg/mL dust mite, 15 μg/mL ragweed, and 5 μg/mL Aspergillus species). Cellular RNA was analyzed for mRNA for IL-33. In a fourth experiment cells were treated for 24 hours with IL-13 (20 ng/mL) and ATP, and the supernatant was collected and analyzed for IL-33 levels by means of ELISA, according to the manufacturer's instructions (R&D systems).
Total RNA was isolated from frozen tissues by using Trizol (Invitrogen) or from cells by using a kit (Purelink Mini RNA kit, Life Technologies), and cDNA was synthesized with an ImProm-II cDNA synthesis kit (Promega, Madison, Wis.), according to the manufacturer's instructions, as described previously (Gorska et al. 2007). Gene-specific PCR products were amplified with SYBR Green (Thermo Scientific) and primers outlined in Table 3 by using an Applied Biosystems 7000 Sequence Detection System (Applied Biosystems, Foster City, Calif.). The levels of target gene expression were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression by using the 2−ΔΔCt method.
The OneArray microarray service from Phalanx Biotech (Palo Alto, Calif.) was used in which 29,922 mouse genome probes and 1,880 experimental control probes were used for the array. Each sample was studied in triplicates. The following analyses of the microarray results were performed and provided with the service: (1) Rosetta profile error model calculation; (2) normalized intensities (excluding flagged and control data) with median scaling; (3) basic statistic plot and Pearson correlation coefficient; (4) pairwise ratio calculation; (5) principal component analysis; and (6) gene ontology analysis.
BAL fluid cells were studied from asthmatic patients and disease control subjects. Asthma was diagnosed based on the presence of reversible airway obstruction, positive methacholine test results (PC20, <8 mg/mL), or both, per the Expert Panel Report 3 criteria. The clinical characteristics of the study subjects are shown in Table 4. Bronchoscopy, BAL, and endobronchial biopsy were performed, as described in Good et al. (Good J T, et al. Refractory asthma: importance of bronchoscopy to identify phenotypes and direct therapy. Chest 2012; 141:599-606). BAL fluid was processed immediately for flow cytometry. IL-33 was analyzed in BAL fluid by means of ELISA, according to the manufacturer's directions (R&D Systems).
Data are presented as means 6 SEMs. For comparison of airway hyperreactivity, 2-way ANOVA for repeated measures with a Bonferroni post hoc test was used. For pairwise comparisons, a Student t test was used. Data from human subjects were analyzed by using nonparametric tests (Mann-Whitney U test and Kruskal-Wallis test). A P value less than 0.05 was considered significant.
Repetitive Allergen Exposure Induces Asthma that Persists Longer than 6 Months after Cessation of Allergen Exposure
To establish experimental asthma in mice, 3 representative allergens were administered intranasally (ie, dust mite, ragweed, and Aspergillus species) without adjuvant twice a week for 6 weeks, as described in Goplen et al. (Goplen N, et al. Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma. J Allergy Clin Immunol 2009; 123:925-32.e11). In this model airway hyperreactivity persisted for longer than 6 months (
To examine the mechanism of asthma persistence, the following timeline was designed for various interventions (
Second, the inventors adoptively transferred splenic CD4 T cells from chronic saline-treated mice or from the chronic asthma model (isolated in week 10) to naive mice and subsequently challenged the recipient mice intranasally with the sensitizing allergens on 3 consecutive days. Adoptively transferred CD4 T cells from mice with chronic asthma potently induced airway hyperreactivity in naive mice 6 days after the transfer (
Third, mice with chronic asthma were subjected to lethal irradiation at the end of week 9 and transplanted bone marrow from naive mice to sustain mouse viability. Splenic and mediastinal lymph node-derived T cells collected 6 weeks after irradiation proliferated in response to anti-CD3/CD28 antibodies but did not respond to the recall antigens dust mite (see
Airway inflammation was monitored at weeks 11, 13, and 15 (2, 4, and 6 weeks after immune ablation). Consistent with the absence of allergen-specific T cells, airway inflammation was nearly absent at week 11 but gradually increased, reaching statistical significance at week 15 (
Gene expression in lung tissue from the chronic asthma model was compared using a microarray with 2 different controls, acute asthma and saline controls (Goplen N, et al. Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma. J Allergy Clin Immunol 2009; 123:925-32.e11). Approximately 1000 genes were observed upregulated and downregulated in the acute model by using a cutoff of a 3-fold difference with the saline control and nearly 3 times more genes upregulated than downregulated in mice with chronic asthma compared with saline control animals (
Previous studies have demonstrated a crucial role for ILC2s in the development of lung inflammation from Alternaria species (Doherty T A, et al. 2012) and papain (Halim T Y, et al. Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation. Immunity 2012; 36:451-63) airway hyperreactivity caused by influenza (Chang Y J, et al. Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity. Nat Immunol 2011; 12:631-8), and allergic sensitization from dust mite and peanut (Chu D K, et al. IL-33, but not thymic stromal lymphopoietin or IL-25, is central to mite and peanut allergic sensitization. J Allergy Clin Immunol 2013; 131:187-200, e181-8). The inventors microarray data suggest a role for ILC2s in the maintenance of chronic asthma. ILC2s was studied in the lung digest, as described previously (Neill D R, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010; 464:1367-70; Saenz S A, et al. IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses. Nature 2010; 464:1362-6; Halim T Y, et al. Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation. Immunity 2012; 36:451-63). First, the inventors gated for live CD45+ cells (see
The total lin-CD25+ ILC population was increased in the chronic asthma versus saline control groups (
To determine the role of ILC2s in the persistence of asthma, bone marrow was transferred from recombination-activating gene (Rag1)−/−, Rag2−/− and common gc chain of the IL-2 receptor (yc)−/−, and wild-type mice to irradiated mice with chronic asthma. Rag1−/− mice are deficient in T and B cells but have normal ILC numbers, whereas Rag2−/−: γc−/− mice are additionally deficient in ILCs (Halim T Y, et al. 2012). Mice that received marrow from Rag1−/− and wild-type mice maintained airway hyperreactivity (
Persistent production of IL-33 after immune ablation (
The foregoing experiments demonstrated that airway hyperreactivity could not be sustained in the absence of IL-33 or ILC2s. Lung CD45+ lin-CD25+ cells were sorted from the chronic asthma and saline control groups (both CD45.11) and adoptively transferred (2×105 cells) to naive congenic CD45.2+ mice to demonstrate whether activated ILC2s from asthmatic mice were sufficient to sustain airway hyperreactivity. Donor-derived ILCs were detected in the recipient lung 21 days after transfer (see
Self-sustenance of biological processes can be facilitated by the development of a positive feedback circuit or circuits. This putative mechanism was tested by examining the effect of IL-13, a major product of ILC2s, on epithelial production of IL-33 in the human lung epithelial cell line A549. IL-13 was the most potent inducer of IL-33 mRNA compared with IL-33, IL-1b, IL-4, IL-17, TNF, and IFN-γ (
Previous studies have shown that IL-33 induces acute asthma in mice when examined 24 hours after the ultimate dose (Bartemes K R, et al. IL-33-responsive lineage-CD251 CD44(hi) lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs. J Immunol 2012; 88:1503-13; Kondo Y, et al. Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system. Int Immunol 2008; 20:791-800; Kurowska-Stolarska M, et al. IL-33 induces antigen-specific IL-51 T cells and promotes allergic-induced airway inflammation independent of IL-4. J Immunol 2008; 181:4780-90; Hardman C S, et al. IL-33 citrine reporter mice reveal the temporal and spatial expression of IL-33 during allergic lung inflammation. Eur J Immunol 2013; 43:488-98). To extend these findings to chronic asthma, the inventors examined the persistence of asthma 15 days after intranasal IL-33 administration. IL-33 induced airway hyperreactivity (
Airway epithelial cells express low basal levels of mRNA for IL-33R (Yagami A, et al. IL-33 mediates inflammatory responses in human lung tissue cells. J Immunol 2010; 185:5743-50), thus permitting IL-33 autoinduction. IL-13 and IL-13, but not IL-4 or IL-17, strongly induced IL-33R (
The inventors previously reported that a single administration of an anti-IL-13 antibody inhibited acute but not chronic asthma (Goplen N, et al. Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma. J Allergy Clin Immunol 2009; 123:925-32.e11). Because this study uncovered that IL-13 is likely to be involved in establishing a positive feedback mechanism, the inventors reasoned that repetitive IL-13 blockade would be required to override the feedback mechanism. To test this, 3 doses of an anti-IL-13 antibody were administered intraperitoneally to mice with chronic asthma in week 10. The airway hyperreactivity resolved 3 days after the anti-IL-13 antibody treatment (
Next, the clinical relevance of IL-33 and ILC2s was examined. IL-33 levels were significantly increased in BAL fluid from asthmatic patients compared with disease control subjects (median, 560 pg/mL in asthmatic patients vs 295 pg/mL in disease control subjects;
While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following exemplary claims. Each publication and reference cited herein is incorporated herein by reference in its entirety.
This application is a divisional application of U.S. application Ser. No. 15/080,972, filed Mar. 25, 2016 which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/138,262, filed Mar. 25, 2015. The entire disclosure of U.S. application Ser. No. 15/080,972 and U.S. Provisional Patent Application No. 62/138,262 are incorporated herein by reference.
This invention was made with government support under grant number R01 AI091614A received from the National Institute of Allergic and Infectious Diseases (NIAID). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62138262 | Mar 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15080972 | Mar 2016 | US |
| Child | 16165402 | US |
