A rodent COPD model

Abstract

This disclosure relates to a method of generating a rodent COPD model and use thereof for testing existing and candidate drugs for treating COPD. Disclosed herein is a method of inducing chronic obstructive pulmonary disease (COPD) phenotypes in a rodent, comprising exposing the rodent to cigarette smoke, and administering IL4 and IL13 intranasally to the rodent, thereby inducing the COPD phenotypes in the rodent.

Description

A RODENT COPD MODEL CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefits of priority from U.S. Provisional Application 63 / 554,588, filed February 16, 2024, and U.S. Provisional Application 63 / 554,606, filed February 16, 2024, the entire contents of both of which are incorporated herein by reference. BACKGROUND

[0002] Evidence of type 2 (“T2”) inflammation is present in 20-40% of patients with chronic obstructive pulmonary disease (COPD) and is associated with an increased risk of exacerbations. It is desirable to develop a COPD-like rodent model with a T2 phenotype to better understand the disease biology and mechanisms of existing and candidate therapeutics for treating COPD. SUMMARY

[0003] This disclosure relates to a method of generating a rodent COPD model and use thereof for testing existing and candidate drugs for treating COPD.

[0004] In some embodiments, disclosed herein is a method of inducing chronic obstructive pulmonary disease (COPD) phenotypes in a rodent, comprising exposing the rodent to cigarette smoke, and administering IL4 and IL13 intranasally to the rodent, thereby inducing the COPD phenotypes in the rodent.

[0005] In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for at least 3 or 4 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for up to 1 week. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for up to 2 weeks.

[0006] In some embodiments, the rodent is administered with IL4 and IL13 at 10 g each at leastonce or twice. In some embodiments, the rodent is administered with IL4 and IL13 at least once per day for 2 days. In some embodiments, the rodent is administered with IL4 and IL13 at leastonce per day for 3 days. In some embodiments, IL4 and IL13 are each administered at about10 g each time.

[0007] In some embodiments, the rodent is exposed to cigarette smoke for at least an hour twice each day for 4 days on days 1-4, and is administered intranasally with IL4 and IL13 at about10 g each, once per day, for two days on days 3-4.

[0008] In some embodiments, the COPD phenotypes induced by the present method comprise one or more of the following as compared to a control rodent not exposed to cigarette smoke and not administered IL4 and IL13: (i) increased neutrophils in the lungs, increased eosinophils in the lungs, and reduction in alveolar macrophages, (ii) increased expression of type 2 cytokines and type 1 cytokines in the lungs, and (iii) increased mucus hypersecretion, increased bronchiolitis and alveolitis, increased peribronchiolar inflammatory cells, and / or increased perivascular inflammation.

[0009] In some embodiments, the COPD phenotypes comprise increased neutrophils in the lungs, increased eosinophils in the lungs, and reduction in alveolar macrophages.

[0010] In some embodiments, the COPD phenotypes comprise increased expression of type 2 cytokines and type 1 cytokines in the lungs. In some embodiments, the type 2 cytokines comprise IL5, IL31, IL33, and / or TSLP. In some embodiments, the type 1 cytokines comprise one or more ofMIP-1a, MCP-1, IL1a, IL1b, IL6, GROa, GM-CSF, and TNFa.

[0011] In some embodiments, the COPD phenotypes comprise increased mucus hypersecretion, increased bronchiolitis and alveolitis, increased peribronchiolar inflammatory cells, and / or increased perivascular inflammation.

[0012] In some embodiments, the COPD phenotypes comprise (i) increased eosinophils and reduction in alveolar macrophages in the lungs, (ii) increased expression of IL5, IL33, IL6 and MCP-1, and (iii) increased mucus secretion, each as compared to a rodent exposed to cigarette smoke alone.

[0013] In some embodiments, the COPD phenotypes comprise (i) increased eosinophils and reduction in alveolar macrophages in the lungs, (ii) increased expression of IL6 and MCP1, and (iii) increased mucus secretion, each as compared to a rodent exposed to cigarette smoke alone and to a rodent administered with IL4 / IL13 alone.

[0014] In some embodiments, disclosed herein is a method of testing a candidate drug for treating COPD, comprising inducing COPD phenotypes in a test rodent and in a control rodent, administering the candidate drug to the test rodent before, during, or after said inducing, and assessing the test rodent and the control rodent to determine whether the drug inhibits one or more of the COPD phenotypes.

[0015] In some embodiments, the candidate drug is a small molecule compound, a nucleic acid, a peptide, or an antibody. In some embodiments, the candidate drug is an antibody, e.g., Dupilumab.

[0016] In some embodiments, the rodent is a mouse or a rat. In some embodiments, the rodent is a mouse. In some embodiments, the rodent is a rat.

[0017] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTON OF DRAWINGS

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] The Drawings included herein are for illustration purposes only and do not limit the scope of the methods disclosed herein.

[0020] FIG.1 top depicts an exemplary experimental design for developing a COPD mouse model: Mice are exposed to cigarette smoke for 4 consecutive days, for 1hr twice per day, with a rest period of at least a few hours between the smoking sessions. For cytokine / smoke combination exposure, mice also receive 2 intranasal doses of mouse IL4 and IL13 cytokines (10µg each) on day 3 and day 4. The data at the bottom demonstrate that intranasal delivery of IL4 and IL13 cytokines exacerbates (i) cigarette smoke-induced lung inflammation by driving lung eosinophilia, and (ii) reduction in alveolar macrophages.

[0021] FIG.2 demonstrates that intranasal delivery of IL4 and IL13 cytokines exacerbates cigarette smoke-induced lung inflammation by driving expression of both type 2 (e.g., IL5, IL31,IL33, and TSLP) and type 1 (e.g., MCP-1, IL1 , IL1 , and IL6) cytokines in the lungs.“LLOQ” stands for lower limit of quantification.

[0022] FIG.3 presents results from histological analysis of lung tissues from control mice, mice administered with IL4 / IL13, mice exposed to smoke, and mice exposed to smoke and administered with IL4 / IL13. A standard scoring scale of 0 (within normal limits) to 3 (severely affected) was applied. A total pathology score was calculated by the sum of all features shown on this slide PLUS pulmonary fibrosis (for a total of 15 [3x5]): (i) peribronchiolar immune cells (using H&E staining), (ii) perivascular inflammatory cells and edema (using H&E staining), (iii) bronchiolitis and alveolitis (using H&E staining), (iv) pulmonary fibrosis (using Masson Trichrome staining) (not shown in this graph because no fibrosis was observed for any of the groups); and (v) mucus cell metaplasia / hyperplasia (using PAS staining). Representative images of PAS (periodic acid-schiff) staining are also shown at the lower right corner of the figure. The results demonstrate that intranasal delivery of IL4 and IL13 cytokines exacerbates cigarette smoke-induced lung inflammation by driving mucus hypersecretion. While smoke drives bronchiolitis and alveolitis, IL4 / IL13 drive mucus hypersecretion.

[0023] FIGS.4A-4B present results of differential gene expression analysis, showing clusters of genes specifically induced by cigarette smoke, IL4 / IL13 exposure, or a combination of cigarette smoke with IL4 / IL13 exposure. Differential Expression Analysis: |FC| > 2, q < 0.05. “FC” stands for fold change. FIG.4A shows a bar graph on top, with bars in red representing the number of genes having increased expression, and bars in blue representing the number of geneshaving decreased expression, under three sets of conditions: saline plus smoke exposure as compared to saline, intranasal delivery of IL4 and IL13 as compared to saline, and smoke exposure combined with intranasal delivery of IL4 and IL13 as compared to saline. FIG.4A shows a Ven Diagram at the bottom, showing the overlap of the differentially expressed genes identified for the three sets of conditions. FIG.4B shows a heat map showing genes expressed under the three sets of conditions as compared to control (no treatment), which illustrates clusters of genes specifically induced by either cigarette smoke or IL4 / IL13 exposure, or a combination of cigarette smoke and IL4 / IL13 exposure. DETAILED DESCRIPTION

[0024] Reference throughout this specification to “one embodiment,” “some embodiments,” “a preferred embodiment(s),” “certain embodiments” or “a certain embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment(s) is included in at least one or more embodiments of the present disclosure. The particular feature(s), structure(s), or characteristic(s) in one embodiment may be combined with those in one or more other embodiments in any suitable manner.

[0025] Unless otherwise specified, “a” or “an” means “one or more.”

[0026] As used herein, the term “about” in connection with a specified number means plus or minus 10%, or plus or minus 5%, or plus or minus 4%, or plus or minus 3%, or plus or minus 2%, or plus or minus 1%, of the specified number, as well as the specified number itself.

[0027] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of” shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps orcompositions (consisting of). Further, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms.

[0028] Type 1 (T1) and type 2 (T2) immunity are distinct immune responses, with T1 responses primarily protecting against intracellular pathogens like viruses and bacteria, while T2 responses are directed against large extracellular parasites like helminths. Dysregulated T1 immune responses contribute to autoimmunity, and excessive T2 immune responses can lead to allergic inflammation. T1 and T2 immune responses are characterized by involvement of specific immune cell types and cytokines; essentially, T1 responses are characterized by neutrophils andT1 cytokines such as IL6 and IL1 , and T2 responses by eosinophils and T2 cytokines such asIL5 and IL31. T1 cytokines include, e.g., GRO , GM-CSF, TNF , MIP1 , IL6, IL1 , IL1 ,MCP1; and T2 cytokines include, e.g., IL5, IL31, TSLP, IL33.

[0029] Disclosed herein is a method of inducing COPD phenotypes in an animal, where the animal is a rodent (e.g., a mouse or a rat).

[0030] In some embodiments, disclosed herein is a method of inducing COPD phenotypes in a rodent, comprising exposing the rodent to cigarette smoke, and administering IL4 and IL13 intranasally to the rodent, thereby inducing the COPD phenotypes in the rodent. COPD phenotypes

[0031] In some embodiments, the COPD phenotypes induced in a rodent comprise one or more of the following, as compared to a control rodent (a rodent not exposed to cigarette smoke and not administered with IL4 or IL13): (i) increased neutrophils in the lungs, increased eosinophils in the lungs, and reduction in alveolar macrophages, (ii) increased expression of type 2 cytokines(e.g., IL5, IL33, TSLP and / or IL31) and type 1 cytokines (e.g., one or more of MIP-1 , MCP-1,IL1 , IL1 , IL6, GRO , GM-CSF, and TNF ) in the lungs, and (iii) increased lunginflammation determined based on histological analysis, e.g., increased mucus hypersecretion, increased bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and / or increased perivascular inflammation.

[0032] In some embodiments, the COPD phenotypes comprise increased neutrophils in the lungs, increased eosinophils in the lungs, and reduction in alveolar macrophages, as compared to a control rodent.

[0033] In some embodiments, the increase in neutrophils in the lungs is at least 30 fold. In some embodiments, the increase in neutrophils in the lungs is at least 35 fold. In some embodiments, the increase in neutrophils in the lungs is at least 37 fold. In some embodiments, the increase in neutrophils in the lungs is at least 39 fold. In some embodiments, the increase in neutrophils in the lungs is at least 40 fold. In some embodiments, the increase in eosinophils in the lungs is at least 2 fold.

[0034] In some embodiments, the increase in eosinophils in the lungs is at least 3 fold. In some embodiments, the increase in eosinophils in the lungs is at least 4 fold. In some embodiments, the increase in eosinophils in the lungs is at least 5 fold. In some embodiments, the increase in eosinophils in the lungs is at least 5 to 6 fold.

[0035] In some embodiments, the number of the alveolar macrophages in the lungs is reduced by at least 70%. In some embodiments, the number of the alveolar macrophages in the lungs is reduced by at least 80%. In some embodiments, the number of the alveolar macrophages in the lungs is reduced by at least 90%.

[0036] In some embodiments, the COPD phenotypes comprise increased expression of type 2 cytokines and type 1 cytokines in the lungs, as compared to a control rodent.

[0037] In some embodiments, the COPD phenotypes comprise increased expression of type 2 cytokines that comprise IL5, IL33, TSLP and / or IL31.

[0038] In some embodiments, the increase in the expression of IL5 is at least 1.5 to 3.5 fold. In some embodiments, the increase in the expression of IL5 is 2 to 3 fold. In some embodiments, the increase in the expression of IL5 is 2.4 to 2.5 fold.

[0039] In some embodiments, the increase in the expression of IL33 is at least 1.5 to 3.5 fold. In some embodiments, the increase in the expression of IL33 is at least 2 to 3 fold. In some embodiments, the increase in the expression of IL33 is at least 2.5 to 2.7 fold.

[0040] In some embodiments, the increase in the expression of TSLP is at least 1.5 to 3.5 fold. In some embodiments, the increase in the expression of TSLP is 1.8 to 3 fold. In some embodiments, the increase in the expression of TSLP is 2.0-2.2 fold.

[0041] In some embodiments, the increase in the expression of IL31 is at least 1.5 to 3.5 fold. In some embodiments, the increase in the expression of IL31 is 2 to 3 fold. In some embodiments, the increase in the expression of IL31 is 2.4 to 2.5 fold.

[0042] In some embodiments, the COPD phenotypes comprise increased expression of type 1cytokines that comprise one or more of MIP-1 , MCP-1, IL1 , IL1 , IL6, GRO , GM-CSF,and TNF .

[0043] In some embodiments, the increase in the expression of GRO is at least 1.5 to 3.5 fold.In some embodiments, the increase in the expression of GRO is at least 2 to 3 fold. In someembodiments, the increase in the expression of GRO is at least 2.2 to 2.3 fold.

[0044] In some embodiments, the increase in the expression of GM-CSF is at least 3 to 10 fold. In some embodiments, the increase in the expression of GM-CSF is at least 3 to 7 fold. In some embodiments, the increase in the expression of GM-CSF is at least 4 to 6 fold. In some embodiments, the increase in the expression of GM-CSF is at least 5 fold.

[0045] In some embodiments, the increase in the expression of MIP-1 is at least 3 to 10 fold.In some embodiments, the increase in the expression of MIP-1 is at least 3 to 7 fold. In someembodiments, the increase in the expression of MIP-1 is at least 4 to 6 fold. In someembodiments, the increase in the expression of MIP-1 is at least 5 to 5.2 fold.

[0046] In some embodiments, the increase in the expression of IL6 is at least 10 to 30 fold. In some embodiments, the increase in the expression of IL6 is at least 15 to 30 fold. In some embodiments, the increase in the expression of IL6 is at least 20 to 30 fold. In some embodiments, the increase in the expression of IL6 is at least 22 to 25 fold.

[0047] In some embodiments, the increase in the expression of MCP1 is at least 2 to 5 fold. In some embodiments, the increase in the expression of MCP1 is at least 2 to 4 fold. In someembodiments, the increase in the expression of MCP1 is at least 3 to 4 fold. In some embodiments, the increase in the expression of MCP1 is at least 3.5 fold.

[0048] In some embodiments, the increase in the expression of TNF is at least 1.1 to 2 fold. Insome embodiments, the increase in the expression of TNF is at least 1.2 to 2 fold. In someembodiments, the increase in the expression of TNF is at least 1.3 fold.

[0049] In some embodiments, the increase in the expression of IL1 is at least 1.2 to 2 fold. Insome embodiments, the increase in the expression of IL1 is at least 1.5 to 2 fold. In someembodiments, the increase in the expression of IL1 is at least 1.8 fold.

[0050] In some embodiments, the increase in the expression of IL1 is at least 1.2 to 2 fold. Insome embodiments, the increase in the expression of IL1 is at least 1.5 to 2 fold. In someembodiments, the increase in the expression of IL1 is at least 1.6 fold.

[0051] In some embodiments, the COPD phenotypes comprise increased lung inflammation determined based on histological analysis, e.g., increased mucus hypersecretion, increased bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and / or increased perivascular inflammation, as compared to a control rodent. For example, lung tissues can be collected from mice and are fixed in 10% formalin. Slides of the lung are then stained with Periodic acid-Schiff (PAS) and hematoxylin and eosin (H&E) and scored. A 0-3 scoring scale is used: 0-0-within normal limits, 1-minimal, 2-moderatly affected, 3-severely affected. A total pathology score is calculated for each rodent animal by adding the individual histopathological feature scores (for a total of 15).

[0052] In some embodiments, the histological score for mucus hypersecretion is increased by at least 100%. In some embodiments, the histological score for mucus hypersecretion is increased by at least 150%. In some embodiments, the histological score for mucus hypersecretion is increased by at least 200%.

[0053] In some embodiments, the histological score for peribronchiolar inflammatory cells is increased by at least 300% to 700%. In some embodiments, the histological score forperibronchiolar inflammatory cells is increased by at least 400%. In some embodiments, the histological score for peribronchiolar inflammatory cells is increased by at least 500%.

[0054] In some embodiments, the histological score for bronchiolitis and alveolitis is increased from negligible (i.e., a score of 0) in a control rodent to at least 0.8. In some embodiments, the histological score for bronchiolitis and alveolitis is increased from negligible (i.e., a score of 0) in a control rodent to at least 0.9. In some embodiments, the histological score for bronchiolitis and alveolitis is increased from negligible (i.e., a score of 0) in a control rodent to at least 1.0.

[0055] In some embodiments, the histological score for perivascular inflammation is increased from negligible (i.e., a score of 0) in a control rodent to at least 1.0. In some embodiments, the histological score for perivascular inflammation is increased from negligible (i.e., a score of 0) in a control rodent to at least 1.5. In some embodiments, the histological score for perivascular inflammation is increased from negligible (i.e., a score of 0) in a control rodent to at least 1.8. In some embodiments, the histological score for perivascular inflammation is increased from negligible (i.e., a score of 0) in a control rodent to at least 1.9.

[0056] In some embodiments, the total histological score based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by at least 200%. In some embodiments, the total histological score based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by at least 300%. In some embodiments, the total histological score based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by at least 400%. In some embodiments, the total histological score based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by at least 450%. In some embodiments, the total histological score based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by at least 500%.

[0057] In some embodiments, an intranasal administration of IL4 and IL13 exacerbates the COPD phenotypes induced by cigarette smoke alone.

[0058] In some embodiments, an intranasal administration of IL4 and IL13 exacerbates the COPD phenotypes induced by cigarette smoke alone by driving lung eosinophilia, i.e., exhibiting increased eosinophils in the lungs as compared to a rodent exposed to cigarette smoke alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 100% relative to a rodent subjected to cigarette smoke alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 125% relative to a rodent subjected to cigarette smoke alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 150% relative to a rodent subjected to cigarette smoke alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and smoke exposure are increased by 165% relative to a rodent subjected to cigarette smoke alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 200% relative to a rodent subjected to cigarette smoke alone.

[0059] Furthermore, the combination of cigarette smoke and IL4 / IL13 administration exhibits synergistic effect in that eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are also increased relative to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 20% relative to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 30% relative to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 40% relative to a rodent subjected to IL4 / IL13 administration o alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 50% relative to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, eosinophils in the lungs as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke are increased by 60% relative to a rodent subjected to IL4 / IL13 administration alone.

[0060] In some embodiments, an intranasal administration of IL4 and IL13 exacerbates the COPD phenotypes induced by cigarette smoke alone by driving expression of type 2 cytokines(e.g., IL5, IL33, TSLP and / or IL31) and type 1 cytokines (e.g., one or more of MCP-1, IL1 ,IL1 , and IL6) in the lungs, i.e., increased expression of type 2 cytokines and type 1 cytokines inthe lungs as compared to a rodent exposed to cigarette smoke alone.

[0061] In some embodiments, the expression of IL5 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 50% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL5 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 60% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL5 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 70% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL5 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 80% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL5 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 90% as compared to a rodent subjected to cigarette smoke alone.

[0062] In some embodiments, the expression of IL31 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 30% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL31 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 40% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL31 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 50% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL31 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 60% as compared to a rodent subjected to cigarette smoke alone.

[0063] In some embodiments, the expression of TSLP in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 60% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of TSLP in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 70% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of TSLP in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 80% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of TSLP in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 90% as compared to a rodent subjected to cigarette smoke alone.

[0064] In some embodiments, the expression of IL33 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 100% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL33 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 125% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL33 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 150% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL33 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 175% as compared to a rodent subjected to cigarette smoke alone.

[0065] In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 250% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 350% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 400% as compared to a rodent subjected to cigarettesmoke alone. In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 450% as compared to a rodent subjected to smoke exposure alone.

[0066] In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 40% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 50% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 60% as compared to a rodent subjected to cigarette smoke alone. In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 70% as compared to a rodent subjected to cigarette smoke alone.

[0067] In some embodiments, the expression of IL1 in the lungs of a rodent as a result of beingsubjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 15% as compared to a rodent subjected to cigarette smoke alone. In some embodiments,the expression of IL1 in the lungs of a rodent as a result of being subjected to a combination ofIL4 / IL13 administration and cigarette smoke is increased by at least 20% as compared to arodent subjected to cigarette smoke alone. In some embodiments, the expression of IL1 in thelungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 25% as compared to a rodent subjected to cigarette smoke alone.

[0068] In some embodiments, the expression of IL1 in the lungs of a rodent as a result of beingsubjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 15% as compared to a rodent subjected to cigarette smoke alone. In some embodiments,the expression of IL1 in the lungs of a rodent as a result of being subjected to a combination ofIL4 / IL13 administration and cigarette smoke is increased by at least 20% as compared to arodent subjected to cigarette smoke alone. In some embodiments, the expression of IL1 in thelungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 30% as compared to a rodent subjected to cigarette smoke alone.

[0069] Furthermore, the combination of cigarette smoke and IL4 / IL13 administration exhibitssynergistic effect in that the expression of IL6, MCP1, IL1 and IL1 in the lungs of a rodent asa result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is also increased relative to a rodent subjected to IL4 / IL13 administration alone.

[0070] In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 90% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 100% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 120% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the expression of IL6 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 140% as compared to a rodent subjected to IL4 / IL13 administration alone.

[0071] In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 30% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 40% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 50% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the expression of MCP1 in the lungs of a rodent as a result of being subjected to a combination of IL4 / IL13administration and cigarette smoke is increased by at least 60% as compared to a rodent subjected to IL4 / IL13 administration alone.

[0072] In some embodiments, the expression of IL1 in the lungs of a rodent as a result of beingsubjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 8% as compared to a rodent subjected to IL4 / IL13 administration alone. In someembodiments, the expression of IL1 in the lungs of a rodent as a result of being subjected to acombination of IL4 / IL13 administration and cigarette smoke is increased by at least 10% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, theexpression of IL1 in the lungs of a rodent as a result of being subjected to a combination ofIL4 / IL13 administration and cigarette smoke is increased by at least 12%-13% as compared to a rodent subjected to IL4 / IL13 administration alone.

[0073] In some embodiments, the expression of IL1 in the lungs of a rodent as a result of beingsubjected to a combination of IL4 / IL13 administration and cigarette smoke is increased by at least 10% as compared to a rodent subjected to IL4 / IL13 administration alone. In someembodiments, the expression of IL1 in the lungs of a rodent as a result of being subjected to acombination of IL4 / IL13 administration and cigarette smoke is increased by at least 15% as compared to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, theexpression of IL1 in the lungs of a rodent as a result of being subjected to a combination ofIL4 / IL13 administration and cigarette smoke is increased by at least 20%-23% as compared to a rodent subjected to IL4 / IL13 administration alone.

[0074] In some embodiments, an intranasal administration of IL4 and IL13 exacerbates the COPD phenotypes induced by cigarette smoke alone by driving mucus hypersecretion, i.e., increased mucus secretion as compared to a rodent exposed to cigarette smoke alone.

[0075] In some embodiments, the histological score for mucus hypersecretion in a rodent subjected to both IL4 / IL13 administration and cigarette smoke is increased by 150% relative to a rodent subjected to smoke exposure alone. In some embodiments, the histological score for mucus hypersecretion in a rodent subjected to both IL4 / IL13 administration and cigarette smoke is increased by 200% relative to a rodent subjected to cigarette smoke alone. In someembodiments, the histological score for mucus hypersecretion in a rodent subjected to both IL4 / IL13 administration and cigarette smoke is increased by 250% relative to a rodent subjected to cigarette smoke alone. In some embodiments, the histological score for mucus hypersecretion in a rodent subjected to both IL4 / IL13 administration and cigarette smoke is increased by 300% relative to a rodent subjected to cigarette smoke alone.

[0076] In some embodiments, the total histological score in a rodent subjected to both IL4 / IL13 administration and cigarette smoke based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by 30% relative to a rodent subjected to cigarette smoke alone. In some embodiments, the total histological score in a rodent subjected to both IL4 / IL13 administration and cigarette smoke based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by 40% relative to a rodent subjected to cigarette smoke alone. In some embodiments, the total histological score in a rodent subjected to both IL4 / IL13 administration and cigarette smoke based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by 50% relative to a rodent subjected to cigarette smoke alone.

[0077] Furthermore, a combination of IL4 / IL13 administration and cigarette smoke exhibits a synergistic effect in that the total histological score in a rodent subjected to both IL4 / IL13 administration and cigarette smoke is also increased relative to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the total histological score in a rodent subjected to both IL4 / IL13 administration and cigarette smoke based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by 30% relative to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the total histological score in a rodent subjected to both IL4 / IL13 administration and cigarette smoke based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by 40% relative to a rodent subjected to IL4 / IL13 administration alone. In some embodiments, the total histological score in a rodent subjected to both IL4 / IL13 administration and cigarette smoke based on mucus hypersecretion, bronchiolitis and alveolitis, peribronchiolar inflammatory cells, and perivascular inflammation, is increased by 50% relative to a rodent subjected to IL4 / IL13 administration alone.

[0078] In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 2 days to 2 weeks. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 3 days to 2 weeks. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 4 days to 2 weeks. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 5 days to 2 weeks. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 6 days to 2 weeks days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 7 days to 2 weeks.

[0079] In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 2 to 7 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 2 to 6 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 2 to 5 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 2 to 4 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 2 to 3 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 3 to 7 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 4 to 6 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for between 3 to 5 days. In some embodiments, the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for 3 or 4 days.

[0080] In some embodiments, the rodent is administered with IL4 and IL13 at between 2 - 20 geach at least once or twice. In some embodiments, the rodent is administered with IL4 and IL13at between 5 - 15 g each at least once or twice. In some embodiments, the rodent isadministered with IL4 and IL13 at between 5 - 10 g each at least once or twice. In someembodiments, the rodent is administered with IL4 and IL13 at about 10 g each at least once ortwice. In some embodiments, the rodent is administered with IL4 and IL13 at between 2 - 20 geach once per day for 2 days. In some embodiments, the rodent is administered with IL4 andIL13 at between 2 - 20 g each once per day for 3 days.

[0081] In some embodiments, the rodent is exposed to cigarette smoke for at least an hour twice each day for 4 days on days 1-4, and is administered intranasally with IL4 and IL13 at about 10 g each once per day for two days on days 3-4.

[0082] In some embodiments, disclosed herein is a method of testing a candidate drug for treating COPD, comprising inducing COPD phenotypes in a test rodent and in a control rodent according to a method disclosed herein, administering the candidate drug to the test rodent before, during, or after said inducing, and assessing the test rodent and the control rodent to determine whether the drug inhibits one or more of the COPD phenotypes.

[0083] In some embodiments, drugs that can be evaluated using the rodents disclosed include candidate inhibitors of the IL4-IL4Ra signaling, for example, but not limited to, a small molecule inhibitor compound, a nucleic acid-based inhibitor (e.g., siRNA, ribozyme, antisense construct, etc.), an antigen-binding protein (e.g., antibody or antigen-binding fragment thereof), or a blocking peptide / peptide inhibitor.

[0084] In some embodiments, a candidate inhibitor is an antibody or antigen-binding fragment thereof. Both monoclonal and polyclonal antibodies are suitable to be tested in a rodent disclosed herein. In some embodiments, an antibody specifically binds to IL4 or IL4 receptor. In some embodiments, the candidate drug is dupilumab, a human monoclonal antibody directed to human IL4Ra that inhibits induced biological activities from IL4 and IL13.

[0085] Candidate drugs can be evaluated by determining whether a candidate drug can inhibit the one or more COPD phenotypes in a rodent as described hereinabove. The term "inhibiting" includes ameliorating the severity, slowing down the progression, eliminating, delaying or preventing the onset of one or more COPD phenotypes, or a combination thereof, as compared to a rodent not administered with the candidate drug. A drug is considered to be effective in inhibiting a COPD phenotype if, for example, the extent of changes in the immune cells (e.g.,increase in neutrophils, increase in eosinophils, reduction in alveolar macrophages) is reduced by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more; the increase in the expression of one or more type 2 cytokines and type 1 cytokines in the lungs described above is reduced by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more; and / or the extent of lung inflammation as determined from an increased histological score based on mucus hypersecretion, increased bronchiolitis and alveolitis, increased peribronchiolar inflammatory cells, and / or increased perivascular inflammation is reduced by at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0086] In some embodiments, a rodent is administered with a candidate drug prior to, during, or after the COPD phenotypes are induced, i.e., prior to, together with, and / or after the administration of IL4 / IL13 or cigarette smoke. In some embodiments, a candidate drug is administered at least once when a rodent is exposed to cigarette smoke and before the IL4 / IL13 administration and at least once after before the IL4 / IL13 administration. In some embodiments, a candidate drug is administered after a rodent has been exposed to cigarette smoke and has been administered with IL4 / IL13.

[0087] Candidate drugs may be dosed via any desired route of administration including parenteral and non-parenteral routes of administration. Parenteral routes include, e.g., intravenous, intraarterial, intraportal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intracerebro ventricular, intracranial, intrapleural or other routes of injection. Non- parenteral routes include, e.g., oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, ocular. Administration may also be by continuous infusion, local administration, sustained release from implants (gels, membranes or the like), and / or intravenous injection.

[0088] In some embodiments, the rodent is a mouse. In some embodiments, the mouse is an Il4rahu / huIl4hu / hudouble humanized mice, as described in U.S. Patent 9,565,841 (Regeneron Pharmaceuticals), incorporated herein by reference in its entirety. In some embodiments, the mouse is an Il4rahu / huIl4hu / huIl33hu / hutriply humanized mice, as described in U.S. Patent 9,565,841 (Regeneron Pharmaceuticals), incorporated herein by reference in its entirety.

[0089] The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literaturereferences, issued patents, and published patent applications as cited throughout this application) are hereby expressly incorporated by reference in their entireties. EXAMPLE 1

[0090] This Example describes an exemplary, non-limiting embodiment of the present method for inducing chronic obstructive pulmonary disease (COPD) phenotypes in a rodent such as a mouse. Experimental Design

[0091] Mice were exposed to cigarette smoke for 4 consecutive days, for 1hr 2x / day, with a rest period of at least a few hours between the smoking sessions. Briefly, mice were placed in smoke chambers (inExpose compact inhalation exposure system from SCIREQ) and exposed to research 3R4F reference cigarette smoke (research cigarettes from University of Kentucky) at a rate of 2 puffs / min (~18 cigarettes / hr). Particulate concentration was monitored and targeted at a peak level of 4000mg / m3. For cytokine / smoke combination exposure, mice also received 2 intranasal doses of mouse IL4 and IL13 cytokines (10µg each from R&D Systems) on day 3 and 4. Lung tissues were collected on day 5 for the following analyses. See top of FIG.1.

[0092] Flow cytometry analysis of immune cell populations: lung tissues were dissociated (with Dnase and Liberase TH from Roche) using a gentle MACS dissociator (from Myltenyi Biotec), followed by red blood cell lysis (with lysis buffer from Sigma Aldrich). Single-cell suspensions were stained with LIVE / DEAD® Fixable Blue Dead Cell Stain (from Invitrogen) and labelled with a cocktail of antibodies (from BD, Biolegend, Invitrogen and eBioscience) to identify eosinophils, neutrophils, monocytes and alveolar macrophages. Sample data were acquired on a Cytek Aurora cell analyzer (from Cytek Biosciences).

[0093] Analysis of tissue cytokines was performed using a ProcartaPlex 25-Plex Mouse Immune Response Panel (from Thermo Fisher Scientific). Fluorescence was read on a Luminex FLEXMAP 3D instrument (from Thermo Fisher Scientific).

[0094] Histology analysis: lung tissues collected from mice were fixed in 10% formalin. Paraffin embedding, sectioning, and stainings were performed by Histoserv. Slides of the lungswere stained with Periodic acid-Schiff (PAS), Masson trichrome (MT) and hematoxylin and eosin (H&E) and scored by a board-certified veterinary pathologist. A standard scoring scale of 0 (within normal limits) to 3 (severely affected) was applied. The following semi-quantitative features were recorded: peribronchiolar immune cells (H&E), perivascular inflammatory cells and edema (H&E), bronchiolitis and alveolitis (H&E), pulmonary fibrosis (MT), and mucus cell metaplasia / hyperplasia (PAS). A total pathology score was calculated by the sum of all features (for a total of 15).

[0095] Bulk RNAseq analysis: After RNA extraction from lung tissues and sequencing library preparation, RNA sequencing (RNA-seq) libraries were prepared from 500 ng of RNA using the KAPA Stranded mRNA-Seq Kit (from KAPA Biosystems). The libraries were amplified by a 12-cycle polymerase chain reaction (PCR). Sequencing was performed on Illumina HiSeq2000 (from Illumina) by multiplexed single read run with 33 cycles. The resulting FASTQ files were analyzed using FastQC to ensure sufficient data quality. Results

[0096] In aiming at developing a COPD-like mouse model with a T2 phenotype, mice were acutely exposed to cigarette smoke and intranasally received the T2 cytokines IL4 and IL13. Cigarette smoke exposure alone was found to drive neutrophilic lung inflammation. Intranasal delivery of IL4 / 13 cytokines was found to exacerbate cigarette smoke-induced lung inflammation by driving lung eosinophilia and potentiating alveolar macrophage destruction. IL4 / 13 was also found to drive mucus hypersecretion and expression of both type 1 (T1) and T2 cytokines, which remained elevated in the presence of smoke. Interestingly, some T1 cytokines were exacerbated by the combination. The results depicted in FIGS.1-3 are also presented and summarized in Table 1. Finally, RNAseq analysis identified clusters of genes specifically induced by either cigarette smoke or IL4 / 13 exposure. This analysis also emphasized combinatorial effects of cigarette smoke and IL4 / 13 on gene expression, with increased expression of inflammatory genes in the context of combined cigarette smoke and T2 cytokine exposure. Overall, these findings highlight that the T2 cytokines IL4 and IL13 exacerbate cigarette smoke-induced lung inflammation in mice by driving a T2 phenotype and exacerbatingoverall inflammation. This suggests that IL4 and IL13 are key drivers of COPD with T2 inflammation.d n r a ur r rero n ito n n n e ne e e itoitoitoits , ,e nususususek a,ek a,ek a,ek a,ek a oe ek oito o o oe e e eonib onib onib onionir ror r r rb ben4.05.00.10.86.77.20.42.14.74.18.0.4.1.7.2.3.0.0.9.1.4.li3 6 2 73 7 0 3 0 0 0 00 1 a 2 1 2 S yr )ot3 / )a()n 3 m o mit / (3 / ss( insa aitioorlfmloit ibnim rafe e vlrcfo)5 laina eN 1ss soi r / ssir– / te)(til lialp 3 h hethc )uily / (edrp p oy scF n 3cs iohse o u o oca S aod nr / hsisv csaiesrto u nMe o R o E N Mvl51333Pa L OC- aF1- 1 PIP b(savcnucor6 a1 b1 CirllirourelsatALILILIS R M T G G N T MLILILIM e Pece PrB MbiF b o o T y g rtein)n nu) xe y esusg)we uleuninisi log e lom Fotmnissett otnu usymsitm ci(llogis l( sie utc L rp(n luHEXAMPLE 2

[0097] This Example describes an experiment in which the effect of dupilumab was tested in a COPD rodent model generated as described in Example 1 above.

[0098] Dupilumab is an antagonistic fully human monoclonal antibody against human IL-4Rthat inhibits induced biological activities from IL-4 and IL-13. Dupilumab blocks IL-4 signal transduction by preventing its binding to receptor subunits, whereas the inhibitory effect on IL- 13 signaling is likely mediated through interfering with the dimeric receptor interaction.

[0099] Il4rahu / huIl4hu / huIl33hu / hutriply humanized mice, as described in U.S. Patent 9,565,841 (Regeneron Pharmaceuticals), incorporated herein by reference in its entirety, were acutely exposed to cigarette smoke for 4 consecutive days and received 2 intranasal administrations of IL4 and IL13 cytokines at days 3 and 4. Mice received 2 subcutaneous injections of either dupilumab or an isotype control antibody (one dose 3 days before beginning of smoke exposure, and another dose at day 2). Lung tissues were collected at day 5 for analysis of lung inflammation by assessing lung immune cells by flow cytometry and lung cytokines by Luminex (ProcartaPlex Mouse Immune Response panel from Thermo Fisher Scientific and Milliplex MAP Human TH17 panel from Millipore).

[0100] Cigarette smoke exposure alone was found to drive neutrophilic lung inflammation. Intranasal delivery of IL4 / 13 cytokines was found to exacerbate cigarette smoke-induced lung inflammation by driving lung eosinophilia and potentiating alveolar macrophage destruction. Dual IL4 / 13 inhibition with dupilumab prevented the increase of lung eosinophils in both groups exposed to the T2 cytokines IL4 and IL13 alone, and to the combination of cigarette smoke and T2 cytokines. However, as expected, dupilumab treatment did not impact reductions in alveolar macrophages and increases in lung neutrophils observed upon exposure to cigarette smoke alone or in combination with T2 cytokines.

[0101] Consistent with previous observations, the T2 cytokines IL4 and IL13 drove expression of several T1 and T2 cytokines in the lungs, which was reduced upon dual IL4 / 13 blockade with dupilumab in mice exposed to either IL4 / 13 cytokines alone or a combination of IL4 / 13 cytokines and cigarette smoke.

[0102] Overall, the results demonstrate that blockade of the T2 cytokines IL4 and IL13 with dupilumab broadly reduced lung inflammation in a COPD-like mouse model with a T2 phenotype, by preventing infiltration of eosinophils into the lungs and tissue induction / exacerbation of both T1 and T2 cytokines.

[0103] Various publications, including patents, patent applications, published patent applications, accession numbers, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference, in its entirety and for all purposes, in this document.

Claims

WHAT IS CLAIMED:

1. A method of inducing chronic obstructive pulmonary disease (COPD) phenotypes in a rodent, comprising: exposing the rodent to cigarette smoke, and administering IL4 and IL13 intranasally to the rodent, thereby inducing the COPD phenotypes in the rodent.

2. The method of claim 1, wherein the COPD phenotypes comprise one or more of the following as compared to a control rodent not exposed to cigarette smoke and not administered IL4 and IL13: (i) increased neutrophils in the lungs, increased eosinophils in the lungs, and reduction in alveolar macrophages, (ii) increased expression of type 2 cytokines and type 1 cytokines in the lungs, and (iii) increased mucus hypersecretion, increased bronchiolitis and alveolitis, increased peribronchiolar inflammatory cells, and / or increased perivascular inflammation.

3. The method of claim 1 or 2, wherein the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for at least 3 or 4 days.

4. The method of claim 3, wherein the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for up to 1 week.

5. The method of claim 3, wherein the rodent is exposed to cigarette smoke for at least an hour each time, at least once or twice per day, for up to 2 weeks.

6. The method of any one of claims 1-5, wherein the rodent is administered with IL4 and IL13 at 10 g each at least once or twice.

7. The method of claim 6, wherein the rodent is administered with IL4 and IL13 at least once per day for 2 days.

8. The method of claim 6, wherein the rodent is administered with IL4 and IL13 at least once per day for 3 days.

9. The method of any one of claims 6-8, wherein IL4 and IL13 are each administered at about 10 g each time.

10. The method of claim 1, wherein the rodent: is exposed to cigarette smoke for at least an hour twice each day for 4 days on days 1-4, and is administered intranasally with IL4 and IL13 at about 10 g each, once per day, for two days on days 3-4.

11. The method of any one of claims 1-10, wherein the COPD phenotypes comprise increased neutrophils in the lungs, increased eosinophils in the lungs, and reduction in alveolar macrophages.

12. The method of any one of claims 1-11, wherein the COPD phenotypes comprise increased expression of type 2 cytokines and type 1 cytokines in the lungs.

13. The method of claim 12, wherein the type 2 cytokines comprise IL5, IL33, TSLP and / or IL31.

14. The method of claim 12, wherein the type 1 cytokines comprise one or more ofMIP-1 , MCP-1, IL1 , IL1 , IL6, GRO , GM-CSF, and TNF15. The method of any one of claims 1-14, wherein the COPD phenotypes comprise increased mucus hypersecretion, increased bronchiolitis and alveolitis, increased peribronchiolar inflammatory cells, and / or increased perivascular inflammation.

16. The method of any one of claims 1-15, wherein the COPD phenotypes comprise (i) increased eosinophils and reduction in alveolar macrophages in the lungs, (ii) increased expression of IL5, IL33, IL6 and MCP1, and (iii) increased mucus secretion, each as compared to a rodent exposed to cigarette smoke alone.

17. The method of any one of claims 1-16, wherein the COPD phenotypes comprise (i) increased eosinophils and reduction in alveolar macrophages in the lungs, (ii) increased expression of IL6 and MCP1, and (iii) increased mucus secretion, each as compared to a rodent exposed to cigarette smoke alone and to a rodent administered with IL4 / IL13 alone.

18. A method of testing a candidate drug for treating COPD, comprising inducing COPD phenotypes in a test rodent and in a control rodent according to a method of any one of claims 1-17, administering the candidate drug to the test rodent before, during, or after said inducing, and assessing the test rodent and the control rodent to determine whether the drug inhibits one or more of the COPD phenotypes.

19. The method of claim 18, wherein the candidate drug is a small molecule compound, a nucleic acid, a peptide, or an antibody.

20. The method of claim 19, wherein the antibody is dupilumab.

21. The method of any one of claims 1-20, wherein the rodent is a mouse.

22. The method of any one of claims 1-20, wherein the rodent is a rat.

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