Effects of IL-4R-alpha Inhibition (Dupixent) Inhibition On The Respiratory Microbiome And Immunologic Correlates In Patients With Severe Asthma
Phase: PHASE4
Conditions: Asthma
Sponsor: University of Michigan

Study Title:  Effects of Interleukin (IL)- 4R-alpha Inhibition on Respiratory Microbiome and 
Immunologic Correlates in Severe Asthma
PI: Yvonne Huang, MD
NCT:  NCT05036733
Protocol Version IRB Approval Date:  9/8/2021

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Effects of Il-4r𝛼𝛼 Inhibition On The Respiratory Microbiome And Immunologic 
Correlates In Patients With Severe Asthma: A Pilot Study 
 
Principal Investigator:   
Yvonne Huang, MD; UM Division of Pulmonary/Critical Care Med 
Co-Principal Investigator:  Alan Baptist MD; UM Division of Allergy/Clinical Immunology 
Co-Investigator:   
 
 
Njira Lugogo; UM Division of Pulmonary/Critical Care Med 
 
I. Overview 
This prospective pilot study will address the following main research question: “Does IL-4Rα 
(dupilumab) inhibition of Type 2-mediated immune responses alter the respiratory microbiome 
within a 4 month period of treatment initiation and follow up, and if so, what are the specific 
microbiological changes and immune response correlates of these changes?” We will compare 
pre- and post-dupilumab effects on the microbiomes, immune responses and metabolome of 
severe asthma patients who are clinically eligible for dupilumab treatment. Eligible patients will be 
screened and enrolled prior to start of therapy and return for visits at 1 month and 4 months after 
initiation of treatment. The overall goal of this study is to understand biological responses related to 
dupilumab treatment among severe asthma patients who have clinical indication to be treated.  We 
will collect biospecimens (nasal, sputum, stool, blood) to examine dupilumab-related changes in 
the microbiome, measured immune responses, and clinical outcome measures. Research methods 
will include microbiome profiling of respiratory and stool samples, cytokine measurements from 
nasal epithelial lining fluid, and metabolite analysis of stool samples.   
 
II. Background 
Inhibition of the IL-4 receptor α (IL-4Rα) by dupilumab has been shown to significantly improve 
clinical outcomes and disease severity measures in Type 2 (T2)-driven phenotypes of severe 
asthma, as well as atopic dermatitis (AD), and chronic rhinosinusitis with nasal polyposis 
(CRSwNP) [1-4]. These findings are accompanied by demonstration of dupilumab-induced 
suppression of T2-related allergic responses, both in the blood and in organ-specific compartments 
such as lesional AD skin and nasal samples in CRSwNP. These include measures of total IgE, 
TARC, and eotaxin-3 [5,6], and of nasal polyp tissue levels of the same plus eosinophilic cationic 
protein and PARC (CCL18) [4]. Gene expression analysis of lesional AD skin identified congruent 
findings with Th2-associated mediators, but also decreased expression of genes involved in other 
immune cell pathways, such as dendritic cells and Th17 cells [6]. Thus, biomarkers of effective 
suppression of T2-related immune responses are evident both systemically and in affected tissue 
compartments. Further analyses of the latter may reveal localized interactions that contribute to 
differences between severe asthma patients in disease phenotype and therapeutic response. 
Indeed, despite recent advancements in anti-T2 directed therapies, not all severe asthma patients 
clinically respond to such treatments. Thus, more research to understand this is needed, as was 
noted in a recent NEJM editorial [7]. 
 
 
Published data on changes in airway immune responses with dupilumab treatment are sparse, 
but similar suppression of T2-mediated inflammation would be expected.  Although T2-high 
inflammation plays a key role in many with poorly controlled asthma, at the same time the potential 
ramifications of long-term suppression of T2-related immune responses are uncertain, particularly 

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from a microbiological perspective. Increased presence of fungi in the airways is associated with 
Th2 responses in individuals with allergic airway disease [8], suggestive of a causal role, and 
eosinophils serve important antimicrobial and other regulatory functions [9,10]. Further, a wealth of 
evidence exists implicating perturbed human microbiota patterns (gut, nasopharyngeal; both 
bacterial and fungal) as a risk factor for allergy and asthma [11]. Relevant to this proposal, we have 
found in recent multi-center investigations of the airway microbiome in asthmatic adults that 
attributes of the airway microbiota differ significantly between those with T2-high versus T2-low 
asthma [12,13].  Specifically, airway bacterial burden is significantly lower in those with higher T2 
airway inflammation. This finding translates into differences in the compositional structure of airway 
microbiota, including differences in the proportional abundance of specific bacteria, when 
compared to T2-low asthmatic subjects. Conversely, we have found that T2-low asthmatic subjects 
harbor higher airway bacterial burden and enrichment in a greater number of different bacteria. 
Similar trends were observed in recent analysis of induced sputum data from the same subjects in 
this NHLBI AsthmaNet study [14]. Separately, studies in mice have found that levels of lung 
inflammatory cytokines (IL-4, IL1-α) correlate with variation in lung microbiota composition and that 
such interactions likely have bidirectional influence [15].  
 
 
Given this evidence, we propose a pilot study, leveraging the inherent value in using a specific 
immunologic intervention, to explore the question: “Does IL-4Rα inhibition of T2-mediated immune 
responses alter the respiratory microbiome within a 4 month period of treatment initiation and 
follow up, and if so, what are the specific microbiological changes and immune response correlates 
of these changes?” We will compare pre- and post-dupilumab effects on the respiratory 
microbiome and determine potential associations between dupilumab-related changes on the 
microbiome, measured immune responses, and clinical outcome measures. Since dupilumab is 
systemically administered, we will similarly explore gut microbiome characteristics related to 
dupilumab-associated changes in clinical and immunologic measures. 
 
III. Objectives 
 
Primary objective  
• 
To determine changes in the respiratory microbiome after inhibition of IL-4Rα in patients 
with asthma at 1 and 4 months after initiation of treatment   
 
Primary Endpoint (Biological) Measures  
• 
Changes in measured features of respiratory microbiota composition (bacteria, fungi), 
profiled from induced sputum and nasal samples. Such features include alpha- and beta-
diversity ecologic measures, which reflect microbiota composition within and between-
samples, respectively. Examples include Shannon and phylogenetic diversity indices, 
richness and evenness metrics (alpha-diversity), and Unifrac or Bray-Curtis distance 
measures to compare across samples (beta-diversity). Additional readouts of the 
microbiome to be analyzed include bacterial burden by quantitative PCR, abundance 
profiles of microbial community members, and microbial gene functions annotated from 
metagenomic sequence data.  
• 
Changes in similarly calculated measures of microbiota composition in stool samples 
collected before and after dupilumab initiation (baseline and 4 months) 

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Secondary Objectives  
• 
To examine correlations between changes in measured features of the respiratory 
microbiome and changes in blood and airway immune responses with dupilumab treatment 
• 
To determine relationships between changes in measured features of the respiratory 
microbiome and changes in clinical outcome measures including lung function and asthma 
control scores with dupilumab treatment 
• 
To determine if combinations of correlated immune markers and microbiota patterns predict 
improvement or lack of improvement in clinical outcome measures related to dupilumab 
treatment 
• 
To explore associations between gut microbiome composition, aeroallergen sensitization, 
severe asthma features and response to dupilumab treatment. 
• 
To explore the relationship between gut microbiota compositional, the intestinal 
metabolome, and response to dupilumab. 
• 
To explore and compare dupilumab-related, compartment-specific changes in the 
respiratory microbiome to that observed in the gut microbiome 
 
Secondary Endpoint (Clinical) Measures  
 Clinical outcome measures (all visits) 
• 
Spirometry: FEV1/FVC ratio, FEV1, and FVC pre- and post-bronchodilator.  
• 
Fractional exhaled nitric oxide (FeNO) 
• 
Asthma Control Test [16] 
• 
Asthma quality of life questionnaire (mini-AQLQ) [17] 
• 
SNOT-22 [18, 19] 
• 
Asthma medication use and dosage 
• 
Asthma exacerbations requiring at least 3 days of oral corticosteroids 
 
Immune and Other Microbial Biomarkers - blood, induced sputum, nasal epithelial lining fluid 
(NELF), stool, exhaled air 
• 
Serum total IgE and specific IgE to aeroallergen panel (ImmunoCap) 
• 
Complete blood count with cell differential  
• 
Sputum cell differential counts  
• 
Sputum, NELF cytokines (Mesoscale Discovery; 30-plex panel: Eotaxin, Eotaxin-3, GM-
CSF, IFN-γ, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-1α, IL-1β, IL-2, 
IL-4, IL-5, IL-6, IL-7, IL-8, IL-8 (HA), IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC, 
TNF-α, TNF-β, VEGF-A) 
• 
Sputum antimicrobial peptide expression (RT-PCR): defensins, lysozyme, lactoferrin, LL-
37, and secretory leukocyte protease inhibitor  
• 
Total bacterial burden in sputum samples by 16S rRNA Q-PCR or droplet digital PCR; with 
determination of both relative and absolute abundances of taxa. 
• 
Respiratory microbial functional gene annotations from metagenomic sequencing 
• 
Fecal metabolites by global/untargeted and targeted short-chain fatty acid assays 
 
Primary hypotheses: 

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Hypothesis 1. Dupilumab-induced suppression of T2 inflammation in severe asthma is associated 
with changes in respiratory tract bacterial burden at 1 month and 4 months after treatment initiation 
(16S rRNA qPCR of bacterial DNA in induced sputum)  
Hypothesis 2. Dupilumab-induced suppression of T2 inflammation in severe asthma is associated 
with changes in the compositional structure of respiratory and/or gut microbiota, detectable at 1 
month and 4 months after initiation of treatment. (bacterial and fungal microbiota profiles and 
related measures of microbial diversity in induced sputum and stool) 
Hypothesis 3. Dupilumab-induced changes in specific biomarkers (e.g. T2 and non-T2-related 
cytokines; targeted gene expression) correlate with changes in the respiratory and/or gut 
microbiome (phylogenetic diversity, specific compositional and functional profiles) 
Hypothesis 4. Dupilumab-related improvement or lack of improvement in clinical outcomes (lung 
function, asthma control scores) over a 4-month period are associated with changes in specific 
characteristics of the respiratory and/or gut microbiome.    
 
IV. STUDY OVERVIEW, INCLUSION/EXCLUSION CRITERIA, PROCEDURES 
Dupilumab-eligible severe asthma patients will be identified and recruited from asthma and allergy 
clinics at the University of Michigan. Severe asthma patients are seen and cared for by Pulmonary 
and Allergy faculty at two main outpatient clinic locations. All severe asthma patients who meet 
clinical prescribing criteria for dupilumab are considered eligible and will be recruited for this study. 
The study coordinator and/or physician investigator will meet with subjects to provide information 
about the study and invite them to participate in this 3-visit study occurring over a 4-month period. 
We will enroll 15 severe asthma patients who complete all 3 visits. The funder of the grant for this 
project (Regeneron) will provide dupilumab for this study. 
 
Inclusion criteria:   
1. Adults ages 18 or older with physician-diagnosed/-managed severe asthma who are clinically 
eligible for dupilumab (clinical criteria below). 
 
2. Current treatment with a medium-to-high-dose inhaled glucocorticoid (fluticasone propionate at 
a total daily dose of ≥440 μg or equipotent equivalent) plus up to at least one additional 
controller (e.g., a long-acting β2-agonist or leukotriene receptor antagonist) 
 
3. Eosinophilic asthma phenotype (blood eosinophil level >300) or asthma requiring daily oral 
corticosteroids 
 
4. Asthma that is uncontrolled, as defined by a score on the Asthma Control Test of 19 or lower, 
OR a worsening of asthma in the past year that led to an asthma hospitalization, ED visit, or 3 
days of oral corticosteroids 
 
5. Severity of asthma that, in the opinion of the subject’s asthma care specialist, requires 
dupilumab for control 
 
Exclusion Criteria:  
1. Patients with diagnosis of other chronic lung diseases (e.g. COPD, idiopathic pulmonary 
fibrosis, Churg-Strauss syndrome, ABPA, etc.)  
 

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2. Current smoker or reported smoking within 1 month of the screening visit (tobacco or any 
inhaled recreational product) 
 
3. Greater than 10 total pack-year of cigarette smoking history 
 
4. Treatment with oral corticosteroids for an asthma exacerbation 1 month prior to screening or 
during the screening period 
 
5. Use of any biologic therapy for asthma within the past 3 months 
 
6. Respiratory or GI illness within 1 month prior to screening or during the screening period 
 
7. Treatment with antibiotics for acute infections within six weeks prior to screening or during 
the screening period.   
 
8. Pregnancy at enrollment or during the study 
 
9. Known hypersensitivity to dupilumab or its excipients 
 
Procedures 
The following lists the study procedures subjects will be asked to perform. Some are indicated only 
for visit-specific criteria as detailed in subsequent sections and summarized in Table 1. 
1. Full and abbreviated medical history intake and physical exam by study physician 
2. Review of inclusion/exclusion criteria at each visit to ensure criteria still met. 
3. Questionnaires 
• 
Asthma Control Test (ACT; 5 items; Appendix 1; [16])  
• 
Mini-Asthma Quality of Life Questionnaire (mAQLQ; 15 items; Appendix 2 [17])  
• 
SNOT-22 (22 items; Appendix 3 [18,19])  
3. Blood draw (for clinical lab tests and plasma tube for Huang lab processing) 
4. Nasal epithelial lining fluid (NELF) collection (Appendix 4) 
5. Spirometry pre- and post-albuterol administration 
6. Oral rinse/tongue scraping prior to sputum induction 
7. Sputum induction 
8. Stool collection (two samples: one for metabolite analysis- EasySampler kit, and one for 
microbial using Zymo; Appendix 5)  
 

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Table 1. Schedule of visits and procedures (V1-V3 conducted at Medical Clinical Research Unit)1 
Visit 
Screening 
V1 
TC14 
V2 
TC24 
V3 
 
 
2-4 wks  
post-screen 
Brief 
Phone call 
1 month 
post-V1 
Brief 
phone call 
4 months  
post-V1 
Dupilumab treatment status 
No drug 
Start drug 
On drug 
On drug 
On drug 
On drug 
Procedures 
 
 
 
 
 
 
COVID-19 testing per Michigan Medicine policies2 
 
X 
 
X 
 
X 
Review of inclusion/exclusion criteria3  
X 
X 
 
X 
 
X 
Full or short history/exam3 
 
X 
 
X 
 
X 
ACT questionnaire 
 
X 
 
X 
 
X 
SNOT-22 questionnaire 
 
X 
 
X 
 
X 
Mini-AQLQ 
 
X 
 
X 
 
X 
Exhaled nitric oxide (FeNO)3 
 
X 
 
X 
 
X 
Nasal epithelial lining fluid (NELF) collection4 
 
X 
 
X 
 
X 
Spirometry pre- and post-bronchodilator 
 
X 
 
X 
 
X 
Blood draw/Phlebotomy lab 
 
X 
 
X 
 
X 
Complete blood count with differential 
 
X 
 
X 
 
X 
Serum total IgE 
 
X 
 
 
 
 
Respiratory Allergen panel (specific IgE tests) 
 
X 
 
 
 
 
Plasma collection for research lab assays 
 
X 
 
 
 
X 
Oral saline rinse/tongue scraping 
 
X 
 
X 
 
X 
Sputum induction  
 
X 
 
X 
 
X 
Stool collection materials (distribution, instruction) 
 
X 
 
 
 
X 
Administer/provide dupilumab doses at study visit  
 
X 
 
X 
 
X 
1 
Subjects should not eat for at least 4 hours prior to visits to limit intolerance of spirometry or sputum induction due to a full stomach. Subjects should not have 
caffeine (as is in coffee, teas, colas, and chocolate) for at least 6 hours prior to visits, for accuracy of measurements in lung function tests.  
2 
Per current policy (as of Jan 2021) at Michigan Medicine, subjects must undergo COVID-19 testing (nasal swab) and have a negative test within 96 hrs prior to 
aerosol-generating procedures. Testing will be arranged by study team for each visit. If the test is positive for COVID-19 or test result is not available, the study 
visit will need to be rescheduled.  
3 
A physician must see the subject at each visit. 
4 
FeNO measurement and NELF collection should be performed before spirometry and sputum induction. 
5 
 Brief telephone call by study coordinator to (a) ensure subjects are self-administering dupilumab doses between study visits, (b) address any interim questions, 
and (c) remind them of upcoming in-person study visit. The TC encounters are important to maintain contact with subjects and ensure adherence and follow 
through during the course of the study 

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V.  VISIT-SPECIFIC DETAILS 
 
SCREENING. 
This is the first encounter with eligible patient at which time information about the study is provided 
by the study coordinator. This may occur by phone or email. A short list of questions will be asked 
to confirm eligibility. If on the basis of this screen, an individual is eligible to participate the study, a 
copy of the informed consent will be provided for subject review prior to Visit 1. Signed informed 
consent will be collected at Visit 1. (Anticipated total time by study coordinator: 10 minutes.) 
 
VISIT 1.  
Preparations 
1. V1 should be scheduled within 2-4 weeks after screening. Subjects will be reminded by study 
coordinator to take their usual asthma treatments as prescribed including on the day of the 
visit. Coordinate V1 scheduling with physician investigator availability to perform history and 
physical exam.  
 
2. Coordinate with Research Pharmacy preparation of dupilumab syringe for administration to 
subjects after completing all visit testing. Ensure appropriate orders are submitted in advance 
of V1 date. Also ensure subject will have subsequent doses of dupilimab prepared for self-
administration at home between V1 and V2. 
 
3. Notify Lesa Begley in Huang Lab (674-9414) the day before the visit so that the lab can 
prepare necessary materials to collect and process samples. Ensure a bucket of ice is 
available to place collected samples for transport to Huang lab. 
 
Visit procedures 
4. Study coordinator must don appropriate protective personal equipment (PPE) for the study visit 
and keep on during the duration of the visit (gloves, masks, safety goggles or face shield for 
eye protection; corrective glasses do not suffice).  
 
5. If not previously collected, obtain signed informed consent from the subject. Confirm no 
changes in continued eligibility of subject since screening, review inclusion/exclusion criteria 
(study coordinator or physician time: 5-10 min).  
 
6. Perform procedures as listed in Table 1. Plan the order of procedures accordingly with the 
following performed by the study coordinator. Questionnaires should be completed first (time: 
10 minutes). FeNO measurement (time: 3-5 minutes) and NELF collection (time: 10 minutes) 
should be performed next, before spirometry pre- and post-bronchodilator (time: 45 minutes 
including 15min wait pre-/post-bronchodilator), oral rinse/tongue scraping collection (time: 2 
minutes), and sputum induction (time: 15 minutes).  Blood draw at phlebotomy lab (time: 5 min) 
can be done anytime during the visit. 
 
• 
FeNO measurement will be performed using NiOx Vero machine per device instructions.   
 

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 
NELF collection (instructions and schematic of sample collection provided by UNC Alexis 
Lab/Spirovation; Appendix 4) 
 
• 
The NELF procedure involves sitting the patient comfortably upright, and the head 
extended backwards 
• 
Place the absorbent end of the nasosorption strip device on the lateral wall of the nasal 
cavity, onto the lateral/inferior surface of the inferior turbinate 
• 
Hold the strip in place by pressing a finger against the nostril for 60 seconds to absorb 
nasal secretions 
• 
Remove the device (moist with nasal mucosal lining fluid) from the nostril and place it on 
wet ice until storage 
• 
Repeat these steps in the other nostril using a fresh nasosorption strip 
 
7. Perform spirometery pre- and post-bronchodilator. Collect oral rinse/tongue scraping sample, 
then perform sputum induction assuming the subject’s post-bronchodilator FEV1 meets safety 
criteria per Sputum Induction MOP. Record lung function (FEV1) every 2 minutes per protocol 
during sputum induction. Notify Huang lab personnel when specimens are ready for pick-up.  
 
8. After the above procedures are completed, 1st dose of dupilumab (600 mg subcutaneously, 
loading dose) should be administered to the subject in MCRU. Subjects will be monitored post-
injection for 30 minutes after their first injection. For high-risk patients, defined as those with 
prior history of anaphylaxis to any medication, very poor asthma control or low lung function, 
this will be extended for up to 60 minutes post-injection.  
 
9. Prior to end of visit: 
• 
Ensure subjects have received subsequent doses of dupilumab (300 mg 
subcutaneously) and instruction on how to self-inject dupilumab every 2 weeks. The 2nd 
injection and subsequent injections between study visits should be administered at 
home, per usual prescribing criteria. (time: 10 min) 
• 
Schedule V2 which should be at least 4 weeks after Visit 1.  
• 
Ensure subjects know how to contact study team in case of interim medical events or 
changes in health status before Visit 2  
 
After-visit tasks by study coordinator and physician PI (Dr. Huang). 
Data entry into REDCAP by coordinator with integrity review, plus physician interpretation: 
 
Questionnaire items/results (ACT, SNOT-22, miniAQLQ):  15 minutes/5-10 minutes 
 
Spirometry pre-/post-bronchodilator and calculation of change: 10 minutes/5 minutes 
 
FENO result: 2 minutes/1 minute 
 
All individual blood test results: 20 minutes/5-10 minutes 
 
TELEPHONE CALL 1.  
1. The purpose of TC1 is to check in with the subjects around 2 weeks after Visit 1 and review the 
following:   
 

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• 
Coordinator should inquire about any interim health issues and ensure subject knows 
and has self-administered 2nd dose of dupilumab (every 2 weeks).  
• 
Remind subjects to continue to take all their other usual asthma treatments as 
prescribed including on the day of upcoming Visit 2. 
• 
Arrangements for COVID-19 testing should be made so that results are available prior to 
Visit 2 (within 96 hrs).    
 
VISIT 2.  
Preparations 
1. Visit 2 should take place approximately 4 weeks after Visit 1.  
 
2. Coordinate with Research Pharmacy for preparation of dupilumab injection at visit. This 
should include dose to be administered at Visit 2 and subsequent doses that the subject 
will self-administer at home every 2 weeks, until Visit 3. Therefore, enough doses should 
be ensured to cover the 3-month period until the last visit. 
 
3. Notify Lesa Begley the day before the visit so that the lab can prepare necessary materials to 
collect and process samples. If a stool specimen is brought in by the subject, the sample will be 
transported to Dr. Huang’s lab. 
 
Visit procedures 
 
4. Perform Visit 2 procedures as indicated Table 1. Plan the order of procedures accordingly, as 
before. All are performed by the study coordinator. Anticipated order and estimated times: 
Questionnaires first (time: 10 minutes). FeNO measurement (time: 3-5 minutes) and NELF 
collection (time: 10 minutes) next, followed by  spirometry pre- and post-bronchodilator (time: 
45 minutes including 15 min required wait pre-/post-bronchodilator measurement), oral 
rinse/tongue scraping collection (time: 2 minutes), and sputum induction (time: 15 minutes).   
 
5. Prior to discharging the subject: 
• 
Remind subject to continue home dupilumab, every 2 weeks until Visit 3.  
• 
Ensure that the subject has all doses of dupilumab needed for the next 3 months.  
• 
Provide subjects with a stool collection packet and instruct them not to collect specimen 
until the day of Visit 3 to be returned in person. 
• 
Schedule Visit 3, which should occur 3 months after Visit 2.  
• 
Arrange COVID-19 pre-procedural testing per institutional policies. 
 
After-visit tasks by study coordinator and physician PI (Dr. Huang). 
Data entry into REDCAP by coordinator with integrity review, plus physician interpretation: 
 
Questionnaire items/results (ACT, SNOT-22, miniAQLQ):  15 minutes/5-10 minutes 
 
Spirometry pre-/post-bronchodilator and calculation of change: 10 minutes/5 minutes 
 
FENO result: 2 minutes/1 minute 
 

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TELEPHONE CALL 2.  
 
1. Like TC1, the purpose of TC2 is to check in with the subject at around 6 weeks post-Visit 2 
(halfway between V2 and V3) 
 
• 
Coordinator inquires about any interim health issues and ensure subject is continuing 
home administration of dupilumab every 2 weeks.  
• 
Remind subjects to continue to take all other usual asthma treatments as prescribed per 
their primary physician.  
 
2.  Within 1-2 weeks before Visit 3, coordinator should: 
• 
Arrange for COVID-19 pre-procedural testing per current institution policies 
 
VISIT 3.  
Preparations 
1. 
Visit 3 occurs ~ 6 weeks after TC2 and 3 months after Visit 2  
 
2. 
Notify Lesa Begley the day before the visit so that the lab can prepare necessary materials to 
collect and process samples. If a stool specimen is brought in by the subject, the sample will 
be transported to Dr. Huang’s lab.  
 
Visit procedures 
 
3. 
Perform Visit 3 procedures as indicated Table 1. Plan the order of procedures accordingly as 
before. Questionnaires first (time: 10 minutes). FeNO measurement (time: 3-5 minutes) and 
NELF collection (time: 10 minutes) next, followed by spirometry pre- and post-bronchodilator 
(time: 45 minutes including 15 min required wait pre-/post-bronchodilator), oral rinse/tongue 
scraping collection (time: 2 minutes), and sputum induction (time: 15 minutes).  Blood draw 
at phlebotomy lab (time: 5 min) can be done anytime during the visit. 
 
4. 
Prior to discharging the subject: 
• 
Remind subject to discuss/coordinate with their primary physician if prescription of 
dupilumab to be continued for clinical reasons.  
• 
If subject has not yet returned stool sample for Visit 3 timepoint, remind them to do so 
within 1-3 days of Visit 3.  
 
After-visit tasks by study coordinator and physician PI (Dr. Huang). 
Data entry into REDCAP by coordinator with integrity review, plus physician interpretation: 
 
Questionnaire items/results (ACT, SNOT-22, miniAQLQ):  15 minutes/5-10 minutes 
 
Spirometry pre-/post-bronchodilator and calculation of change: 10 minutes/5 minutes 
 
FENO result: 2 minutes/1 minute 
 
All individual blood test results: 20 minutes/5-10 minutes 
 

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VI.  Rationale for Data Collection and Procedures  
1. Medical History and Exam: Medical history intake and physical exam will be conducted by the 
study physician at each visit. A full H&P will be performed at Visit 1, with shorter H&P 
appropriate at Visits 2 and 3.  
2. Inclusion/Exclusion criteria: Reviewed in full at Visit 1 and re-checked at each visit to ensure no 
new exclusionary criteria met. 
3. Questionnaires will be used in this study to collect information about asthma severity/control 
(ACT), asthma-related quality of life (mAQLQ), and sinus symptomatology (SNOT-22). These 
data will allow for analysis of clinical symptoms and asthma control in conjunction with 
molecular data generated on the airway and gut microbiomes, and immune mediators 
measured before and after initiating dupilumab. These validated instruments have been used 
extensively in asthma clinical trials and outcomes research. 
4. Exhaled nitric oxide measurement (FeNO).  FENO is a recognized biomarker for monitoring 
Type 2 airway inflammation in asthma. Its use in the clinical management of asthma is 
supported by clinical asthma guidelines.  
5. NELF sample collection. The purpose of collecting nasal epithelial lining fluid is that it is a 
simple non-invasive way of sampling the respiratory mucosa to measure immune cytokines.  
NELF has been used in a number of research investigations to profile mucosal immune 
mediators relevant to asthma. 
6. Spirometry pre- and post- bronchodilator. A standard clinical test for the presence of airflow 
obstruction and assessment for reversibility. FEV1 and FVC will be determined before and after 
4 puffs of albuterol. This will be performed by experienced study coordinators using existing 
equipment for research use. 
7. Blood draw: This will be performed by a clinical phlebotomist or nursing staff  
8. Complete blood count (CBC) with differential. Results will be used to determine peripheral blood 
eosinophil counts, to gauge biological response to treatment.  
9. Total and specific IgE. Clinical tests of allergy-related immune responses. We will measure both 
total serum IgE and respiratory allergen specific IgE based on ImmunoCap assays 
10. 
Plasma will be stored at -80°C for future analysis of other immune or metabolic response 
markers. 
11. 
Oral saline rinse/tongue scraping: This involves having the subject swish and gargle 10mL of 
sterile saline than spit into a collection container. This is followed by applying a commercially 
available plastic tongue scraper to remove any film on the tongue into the same collection 
container. This is performed prior to sputum induction and will be processed for microbiome 
analyses as biological controls. 
12. 
Sputum induction: This will follow a standardized manual of procedures involving inhalation 
of 3% saline mist for 12 minutes. Subjects will be instructed to periodically cough up sputum into 
a specimen cup and FEV1 is monitored every 2 minutes using a handheld PiKo-6 meter per a 
standardized protocol. If the FEV1 declines ≥20% of baseline, the procedure would be stopped 
and albuterol administered with follow-up monitoring. The sputum will be portioned into aliquots 
to be processed for different analytic purposes, including cell count/differential analysis, 
microbial community profiling, mediator analyses.  
13. 
Stool sample collection.  This will be processed for analysis of gut microbiome composition 
and intestinal metabolites and their relationships to clinical measures of treatment response.  
 

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VII. Recruitment 
Subject with severe asthma who are clinically eligible for potential treatment with dupilumab will be 
recruited from pulmonary asthma and allergy clinics at the University of Michigan. Identified eligible 
patients will be screened by the study coordinator to confirm eligibility and interest in participating. 
 
VIII. Human Subjects and Risks 
A. Subjects: 
1. General Description: inclusion/exclusion criteria are as described earlier in this document.  
2. Gender/Minority Inclusion: No subject will be excluded based on gender or ethnicity.  
3. Exclusion of children: Children (<18 years of age) are excluded from this study as the 
research aim relate to hypotheses about the effects in adult asthmatics of dupilumab 
treatment on host-microbiome interactions and clinical outcomes. 
 
B. Potential Risks and Procedures for Minimizing Risks: Potential risks can be categorized as 
“Likely”, “Infrequent”, or “Rare”.  
 
 
Likely Risks 
• Venipuncture risks: Drawing blood may cause temporary discomfort from the needle stick, 
bruising, and infection. Dizziness may occur and subjects may be asked to lie down for 
venipuncture if they indicate this history. 
• Spirometry/peak flow risks: Breathing fast and hard into a spirometer or peak flow device can be 
associated with cough or lightheadedness. Such symptoms are usually temporary and resolve 
within a few minutes without intervention.  
• Albuterol use risks: Albuterol is an approved drug that broncho-dilates the airways and widely 
used to treat asthma symptoms. Albuterol use may cause muscle tremors, palpitations, rapid 
heart rate, and headache. If these symptoms do occur, they are temporary and usually 
disappear within 1 hour.  
• Sputum induction risks: The sputum induction manual of procedures for this study is adapted 
from those used in prior NHLBI asthma clinical research studies. The risks associated with this 
test include an unpleasant taste in the mouth, asthma-like symptoms (chest tightness, 
wheezing, shortness of breath), and occasionally nausea. Infrequently, the procedure may 
result in an asthma exacerbation requiring treatment.  To prevent asthma-like symptoms, all 
subjects will have received 4 puffs of albuterol prior to undergoing induction. FEV1 will be 
monitored during the procedure which may be terminated for sustained decline in FEV1. 
Additional albuterol can be administered per to reverse bronchoconstriction which is rarely 
refractory to additional albuterol. 
 
 
Infrequent Risks 
• Sputum induction resulting in asthma exacerbation: Subjects will be pre-treated with albuterol 
and their lung function closely monitored during the procedure, as outlined in the sputum 
induction MOP.  Sterile ampoules of 3% saline will be used for the procedure. If a decrease in 
FEV1 of ≥ 20% occurs during the procedure, subjects will be given additional albuterol and the 
FEV1 must improve to ≥ 90% of their baseline value prior to being discharged from the visit. If 

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the FEV1 does not improve to this value, further evaluation and treatments may be required, as 
determined by the study physician.   
 
 
Rare  
• Sputum induction: There is a theoretic possibility that the coughing associated with sputum 
induction could cause a failure of any prior hernia surgeries a subject may have had. Such 
surgeries include repair of hiatal hernia, other surgery for gastro-esophageal reflux 
(“heartburn”), or surgery to repair hernias elsewhere in the abdomen, groin, or pelvis. If a 
subject has such history on evaluation, they will be given the opportunity to discuss the study 
related procedures with their surgeon prior to proceeding.  
• 
Breach of confidentiality risks: Participation in any research carries the risk of breach of 
confidentiality because participants provide personal information. Measures implemented in 
this study to protect subject privacy in collected research data and samples include the 
following: Use of servers and electronic database repositories to record clinical and 
demographic data that are HIPAA-compliant as approved by the University of Michigan (e.g. 
REDCAP, MBox). Study-related electronic and paper data files are made accessible only to 
essential study research staff involved in particular aspects of the study. Master key linking 
subject identifiers and assigned subject ID codes are accessible only to the PI and lead study 
coordinator(s). For sample storage, processing and data analyses, only coded subject 
identifiers will be used.  
 
C. Asthma Exacerbations  
If a subject notifies the study team of symptoms that suggest an asthma exacerbation, they will be 
referred to their primary asthma provider (or directed to urgent care if necessary) for clinical 
evaluation and determination of any treatments. A clinically adjudicated asthma exacerbation that 
required treatment intervention will be documented in the study record when the study team is 
made aware of this information.  
 
IX. Adverse Events and Data Safety Monitoring Plan 
An adverse event shall be defined as any detrimental change in the subject’s condition, whether it 
is related to asthma or to an unrelated illness or condition. Adverse events related to asthma 
exacerbations will be managed as noted above.  An adverse event is deemed serious if it presents 
a significant hazard, contraindication, or side effect. Serious adverse events include any 
experience that is fatal or life-threatening, is permanently disabling, requires or prolongs inpatient 
hospitalization, or is a congenital anomaly, cancer, or overdose. Serious adverse events deemed 
potentially related to study procedures will be reported to the University of Michigan’s Institutional 
Review Board and to study funders according to respective policies or reporting guidelines. 
Adverse events due to therapy or concurrent illnesses other than asthma may be grounds for 
withdrawal as determined by study PIs if the illness is considered significant enough to affect study 
findings or if the subject is no longer able to effectively participate in the study.  
 
It is not considered an adverse event if subjects experience minor intercurrent illnesses during the 
study. Examples of minor intercurrent illnesses of relevance include acute rhinitis, sinusitis, upper 
respiratory infections, or accompanying gastrointestinal symptoms. Documentation recorded will 
include information on the nature, severity, duration of the illness and any new medications 

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required to treat the illness. Medications are allowed for treatment of these conditions as per 
clinical guidance of the subject’s personal physician.  
 
Documentation of adverse events will be recorded on a Clinical Adverse Event Report Form and 
will include the following information: dates and description of the event/illness, assessment 
performed by the study team, any necessary treatments administered or prescribed for the 
event/illness (medications, doses, and dose frequency/duration), need for emergency transfer or 
hospitalization was required, and outcome. 
 
Adverse Events and their reporting are further defined as follows: 
 
Definition and Reporting 
Adverse events include the following:  
• Clinical Adverse Events: Any unintended worsening in structure or function of the body; any 
illness that occurs during the trial. These events are documented on a Clinical Adverse Event 
Report form.  
• Laboratory Adverse Events: Occurrences of abnormal laboratory tests or other test results. These 
events are documented on the Laboratory Adverse Event Report form. The study physician will 
offer to communicate the test results, if desired by the subject, to their primary care physician. 
• Serious Adverse Events:  Any experience that poses a significant hazard to a participant is 
considered a serious adverse event. With respect to human clinical experience, a serious adverse 
event includes any experience that meets at least one of the following criteria:  
1. Results in death  
2. Is life threatening (places the participant at immediate risk of death from the event as it occurred)  
3. Results in a significant or persistent disability/incapacity  
4. Requires inpatient hospitalization or prolongation of an existing hospitalization  
5. Results in a congenital anomaly/birth defect  
6. Any other adverse event that, based upon appropriate medical judgment, may jeopardize the 
participant’s health and may require medical or surgical intervention to prevent one of the other 
outcomes listed in this definition. Examples include allergic bronchospasm requiring intensive 
treatment in an emergency department or at home, or the identification of other blood test 
abnormalities that do not require immediate inpatient management. 
 
Serious adverse events are reported on the Serious Clinical Adverse Event Report form and noted 
also on the regular Clinical Adverse Events form. 
 
Each time the study team has contact with a participant, whether for a scheduled visit or phone 
contact, impromptu visit, or unexpected phone call, the possibility of adverse events should be 
reviewed. This would include reviewing prior to the contact (if applicable), the participant's file for 
any ongoing adverse events at the last visit/contact, updating a reported adverse event if an 
ending date/outcome becomes available, or identifying any new adverse events to report. 
 
If there have been no adverse events since the prior visit or last subject contact, then indicate ‘No’ 
in the appropriate section of the respective visit/encounter form. 

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Events that are ongoing at the time a participant completes or leaves the study should be left open 
for stop dates (i.e. report as ‘ongoing’). The participant should be probed for any stop dates that 
are now known to close out previously recorded events. All adverse event report forms will be 
reviewed quarterly (or sooner as necessary) per the Data Safety Monitoring Plan below and 
reported as indicated. 
 
Data Safety Monitoring Plan: 
Adverse events will be reviewed every 3 months as needed by the study team physicians (Huang, 
Baptist, Lugogo) and study coordinators. In case of uncertainty or disagreements among the above 
individuals regarding adjudication of possible study-related AES, or serious AEs, the team will seek 
additional input from non-study clinicians (e.g. Dr. Michael Coffey of the Pulmonary Division) 
 
Potential Benefits Gained from Data  
The described risks of the study represent minor increases over minimal risk. Spirometry and 
sputum induction are used in clinical care setting, where the incidence of serious adverse events 
related to these procedures is very low. The anticipated scientific benefits of this study are 
increased knowledge of the biological effects of dupilumab on host-microbiome interactions, how 
such effects may impact clinical responses to the treatment and identify potential new 
mechanisms.  We believe the potential benefits of the study outweigh the minor increase over 
minimal risk of the proposed testing procedures.  
 
X.  Sample Processing/Analysis (Overview). Respiratory samples will be processed for 
microbiota analysis by 16S rRNA and shotgun DNA sequencing, measurement of airway bacterial 
burden by quantitative PCR, cytokine measurements, sputum inflammatory cell counts/differential 
and airway gene expression studies (targeted). Blood will be analyzed for complete blood count 
with differential analysis, total IgE, respiratory allergen specific IgE, and blood cytokine mediators.  
Stool will be processed for gut microbiome and metabolomic analyses (global and targeted for 
short chain fatty acids). These data will be generated using University of Michigan Clinical Lab and 
Research Core facilities (Microbiome Core, Advanced Genomics Core, Metabolomics Core), as 
well as subcontract collaboration with UNC-Chapel Hill (Spirovation).  
 
XI. Data Management and Analysis.  
A HIPAA-compliant, passcode-secured REDCAP database for the study will be created. The study 
team (coordinator, PI) will enter all clinical test results. This database will reside on University of 
Michigan servers approved to store HIPAA-related research information. Access is given only to 
study-specific research team members for whom the PI provides permission.  Subjects will be 
assigned a code using a study-specific identifier for downstream sample processing and data 
analyses. All samples for molecular sequencing/cytokine analysis will be identified only by a unique 
code.  
 
 
All sequencing data (large data files) will be processed/stored on platforms maintained by 
University of Michigan Advanced Research Computing (https://arc.umich.edu/systems-services), 
which is part of UM Information and Technology Services.  Specifically, we will store coded data 

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(no HIPAA identifiers) on Turbo and utilize the Great Lakes high-performance cluster to process 
the raw sequence data and perform analyses of the metagenomic sequence data.  Several of the 
analysis pipelines to be used are already or can be installed for microbial metagenomic analysis. 
UM-DropBox (HIPAA-compliant) will be used to store intermediary and final result files that need to 
be easily accessed for non-computationally intensive analyses, discussions and write-ups by the 
research team. 
 
XII. Data Analysis 
This is a pilot, proof-of-concept study. There is no available published data (at the time of writing) 
that addresses the same research questions to perform a sample size calculation based on 
changes in microbiome readouts (primary objective/endpoint) related to dupilumab intervention. 
However, we have previously reported significant changes in the airway microbiome, related to a 
prospective treatment intervention (fluticasone propionate), in comparisons that also involved small 
numbers of subjects.  Specifically, in an earlier NHLBI AsthmaNet Proof-of-concept study, we 
examined the effects of randomized, placebo-controlled fluticasone intervention (6 weeks) on the 
airway microbiome in subjects with mild asthma. We identified fluticasone-associated changes in 
the airway microbiome between steroid-responders vs. non-responders, in an initial analysis of 
paired protected bronchial brushings from 16 subjects [13]. In an analysis of induced sputum from 
the same study, we recently reported changes in the airway microbiome related to fluticasone 
intervention, which differed from placebo [14]. Notably, we observed a significantly greater change 
in airway microbial community structure in ICS non-responders (n=8) compared to responders 
(n=12). 
 
To address our exploratory hypotheses in this pilot study, we will first generate the following 
readouts of microbial community composition: (1) read counts or relative abundance data for each 
microbial species determined from sequence alignments to reference databases. These readouts 
can be at the level of ASVs (amplicon sequence variant) or OTUs (operational taxonomic unit), 
which can also be collapsed into higher taxonomic levels for analysis, e.g. genus or family-level 
comparisons; (2) within-sample and between-sample measures of microbial diversity, i.e. alpha- or 
beta-diversity. Alpha-diversity measures will include Shannon, inverse Simpson, richness, and the 
phylogeny-based Faith index.  Beta-diversity measures will include calculation of Bray-Curtis and 
Unifrac distance measures, the latter being a phylogeny-based calculation of between-sample 
differences in overall microbial composition.  We will also determine 16S rRNA gene copy numbers 
in induced sputum by quantitative PCR, which serves as a proxy measure of ‘bacterial burden’ 
often used in the airway/lung microbiome literature.  Other generated molecular data will be used 
to explore secondary questions related to whether dupilumab intervention effects change in 
predicted microbial functions (KEGG orthologue modules and pathways), intestinal metabolites, 
and whether changes in the airway or gut microbiome correlate with changes in inflammatory 
markers and immune mediators measured in sputum, blood and NELF.  
 
For Hypothesis 1, we will compare sputum bacterial burden (normalized 16S rRNA gene copy 
number) before and after treatment (baseline vs month 1; baseline vs. month 4; all three 
timepoints), using paired t-test if normally distributed (Wilcoxon sign-rank test if non-normal 
distribution) and repeated measures ANOVA for all three timepoints.  

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For Hypothesis 2, we will compare alpha-diversity measures before and after treatment (baseline 
vs month 1; baseline vs. month 4; all three timepoints), using paired t-test, Wilcoxon sign-rank test 
or rm-ANOVA.  For beta-diversity -based measures of overall microbial composition, we will use 
Bray-Curtis or Unifrac distance matrices to perform (i) pairwise PERMANOVA tests (R package 
vegan; adonis2 or in QIIME2 (q2-longitudinal) [20,21]; (ii) linear mixed-effects models with a 
random intercept and slope for each individual; (iii) Procrustes analysis with Mantel test. We will 
also explore other available tools for time-series microbiome analysis such as MDSINE [22].  
Differences in specific species (ASVs, OTUs) between time points will be analyzed using DESeq2 
which applies a variance-stabilizing transformation to the data. Other methods using log-ratio 
transformation (e.g., CLR) of species counts will also be explored.   
For Hypothesis 3, we will examine correlations between change in biomarkers (e.g., cytokines, 
inflammatory cell counts, targeted gene expression) and changes in the airway or gut microbiome. 
The latter based on either specific compositional readouts (ASV, OTUs) or in terms of overall 
microbial community structure.  The following approaches will be used: (i) initial correlation 
analyses performed between paired data at each time point (e.g., specific biomarker dataset vs. 
microbial ASVs), using Spearman correlations, with adjustments for multiple comparisons by q-
values; (ii) linear mixed-effects models, e.g. levels of immune markers as predictors of post-
treatment microbiota diversity or abundances of each microbiota member; fixed effects would 
include age, sex, BMI, and chronic treatments including corticosteroids or macrolides (type, dose, 
route); (iii) tests for biomarker association with overall microbial community structure, using 
distance-based PERMANOVA (adonis2) at each time point or in a paired design; (iv) additional 
computational approaches based on methods for high-dimensional dataset comparisons, as 
described below.  
For Hypothesis 4, which focuses on microbial correlations with clinical outcome measures rather 
than biomarkers, we will apply same approaches as described for Hypothesis 3. In addition, we will 
explore the following analysis methods (also applicable to Hypothesis 3): (i) feature-volatility action 
algorithm, which performs longitudinal feature selection based on random forest regression, 
implemented in q2-longitudinal in QIIME2; (ii) tools in the R mixOMICS package which bundles 
access to several sparse multivariate modeling methods that aim to identify key features that 
explain a biological or clinical outcome of interest.  
 
Related to this, and to leverage tools for examining microbiome data with other -omics/high-
dimensional data we will apply tools such as DIABLO (available in mixOMICS package). DIABLO 
performs data integration coupled with discriminant analysis approach to identify combinatorial 
features predictive of a binary outcome (e.g., is there a combination of microbial and metabolite 
features that predict improved asthma control or not? or that associate with an immune biomarker 
of interest?) As an example, we have recently used DIABLO to explore microbial and metabolite 
features associated with COPD outcome measures in a multicenter study. In addition, we will 
determine co-occurrence relationships between microbiota (SparCC [24] or SPIEC-EASI [25]) to 
identify highly correlated species and determine if ‘hubs’ of microbiota associate with particular 
cytokines or metabolites, following by analysis for clustering patterns of microbial-immune 
relationships [26]. Given the likely heterogeneity of measures in the molecular data types and the 
small number of subjects, we will apply corrections for multiple comparisons where indicated.  
 

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APPENDICES for IRB. 
 
Appendix 1.  Asthma Control Test (ACT) 
The Asthma Control Test (ACT) is a patient self-administered tool for identifying those with poorly 
controlled asthma. It is based on a 4-week recall of symptoms and daily functioning captured in 5 items 
on the questionnaire. These include assessment of the frequency of shortness of breath, general 
asthma symptoms, use of rescue medications, the effect of asthma on daily functioning, and overall 
self-assessment of asthma control.  Each item is scored on a 5-point scale, with higher scale values 
indicative of, depending on the question, infrequent/absent symptoms or better asthma control.  
 
The ACT score is the sum of the values for all 5 items. The scores range from 5 (poor control of 
asthma) to 25 (complete control of asthma), with higher scores reflecting greater asthma control. 
 
An ACT score >19 indicates well-controlled asthma; the minimal clinically important difference in score 
is 3. 
 
Appendix 2.  Mini-Asthma Quality of Life Questionnaire Control (mAQLQ) 
The shorter and simpler mAQLQ consists of 15 questions and was developed to meet the need for 
greater efficiency in large clinical trials and group patient monitoring. The mAQLQ is a patient self-
administered test with questions covering 4 domains: Symptoms (5 questions), Activity Limitations (4 
questions), Emotional Function (3 questions), and Environmental Stimuli (3 questions). The recall time 
for the mAQLQ is 2 weeks, so patients must base their answers on their experiences during the 
previous 2 weeks.    
 
To score the mAQLQ, there is an overall score and a mean score for each domain. 
Overall score (average of 15 questions): add up responses to all questions, and divide by 15. 
Mean for each domain: add up responses to each item and take the average for each domain. 
 
Symptoms:   
 
 
 
items 1, 4, 6, 8, 10   (sum & divide by 5; variable label- mAQLQ-S) 
Activity Limitations:   
items 12, 13, 14, 15  (sum & divide by 4; variable label- mAQLQ-AL) 
Emotional Function:  
items 3, 5, 9  
 
 
(sum & divide by 3; variable label- mAQLQ-EF) 
Environmental Stimuli: items 2, 7, 11   
 
(sum & divide by 3; variable label- mAQLQ-ES) 
 
Appendix 3. SNOT-22 Questionnaire.   
 
Appendix 4. Nasal Epithelial Lining Fluid (NELF) collection (diagram, instructions).  
 
Appendix 5. Stool Collection Instructions.