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\begin{document}

\title{Sputum cytokines associated with raised FeNO after anti-IL5 biologic
therapy in severe asthma.}



\author[1]{Pieter-Paul Hekking}%
\author[1]{Kayla Zhang}%
\author[1]{Carmen Paz Venegas Garrido}%
\author[1]{Raquel Lopez-Rodriguez}%
\author[1]{Melanie Kjarsgaard}%
\author[1]{Manali Mukherjee}%
\author[1]{Parameswaran Nair}%
\affil[1]{St Joseph's Healthcare Hamilton}%


\vspace{-1em}



  
  \date{June 12, 2024}


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\textbf{Sputum cytokines associated with raised FeNO after anti-IL5
biologic therapy in severe asthma.}

To the Editor,

Biomarkers such as circulating absolute eosinophil count, \% of
eosinophils in sputum, and fraction of exhaled nitric oxide (FeNO) are
predictors of response to anti-inflammatory therapy for asthma. Failure
to normalize FeNO with high doses of corticosteroids are likely to be
related to cytokines and chemokines such as IL-5, IL-4, IL-13, eotaxin
and TARC derived from eosinophils and other Th2 cells, and alarmins such
as TSLP and IL-33 from sources such as the airway epithelium(1). All
Anti-IL5 biologics suppress eosinophils in sputum. However benralizumab
(anti-IL5RMab) has greater effect in the severe prednisone-dependent
patients than mepolizumab/reslizumab (anti-IL5 neutralizing Mab)(2,3).
While raised eosinophil count is a predictor of clinical response to
anti-IL5 biologics, raised FeNO is often not(4). However, they reduce
FeNO to variable levels(4,5) suggesting that FeNO is partly regulated by
cytokines derived from eosinophils in the airway(1). The cytokines in
sputum associated with raised FeNO in prednisone-dependent severe
asthmatics treated with effective anti-eosinophil drugs are not known.

In this retrospective observational study, we measured cytokines in
sputum using an automated ELISA reader (Ella\textsuperscript{TM},
Protein Simple, R\&D Systems, BioTechne, Minneapolis) at baseline and
after 4 months of treatment with either benralizumab or
mepolizumab/reslizumab and compared the levels of cytokines in those
whose FeNO remained high after treatment. Raised FeNO was defined by
FeNO \textgreater{}40ppb and an increase of at least 16ppb from
pre-treatment value. The study was approved by Hamilton Integrated
Research Ethics Board (\#11227, 5037), and all patients gave written
informed consent. The cytokines assayed were IL-5, IL-4 and IL-13 (Th2
inflammation) and IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, IL-10, IL-12p70, IL-15 IL-17A, IL-18,
IL-33, IFN\selectlanguage{greek}γ \selectlanguage{english}and TNF\selectlanguage{greek}α \selectlanguage{english}(Th1/Th17 inflammation, Table e3). Details of
baseline demographics, methods and statistics are shown in the online
supplement.

Paired measurements were made in 30 patients who received benralizumab,
and 10 each who received mepolizumab/reslizumab. Overall, as previously
reported(5), FeNO levels were not significantly reduced by anti-IL5
treatment (median FeNO pre-treatment 29 {[}5-156{]} vs FeNO post
treatment 37 {[}6-280{]}; p=0.25; Figure e1). This change in FeNO did
not correlate with a reduction of sputum eosinophils (r=-0.24; p=0.16).

Among 15 patients, FeNO remained raised after treatment (Table 1). On
average, IL-4 and IL-13 were the only cytokines significantly higher in
the sputum of these patients compared to those in whom the FeNO values
normalized (Figure 1, Figure e2). Within this group, there were patients
with raised IL-4 (31\%) and IL-13 (15\%) and those with normal
IL-4/IL-13. A small proportion of those with normal IL4/13 had raised
levels of IL-18 or IL-1\selectlanguage{greek}β \selectlanguage{english}(20\%). Residual eosinophilic airway
inflammation was significantly more present in patients with raised FeNO
(30.8\% vs 8.1\%; P=0.04; Table e2). Patients with raised FeNO remained
to have poor asthma control with an ACQ\textgreater{}1.5, however this
did not significantly differ from those with a normalized FeNO (ACQ
1.7\selectlanguage{ngerman}±0.9 vs 1.4±1.1; P=0.36; Table 1).

This study, despite its limitation of retrospective design and small
numbers, provide novel information on the cytokine profile in the
airways of severe prednisone-dependent eosinophilic asthma patients
whose FeNO remain high after anti-IL5 treatment. This is a common
clinically encountered situation. Our observations suggest that
IL-4/IL-13 are the cytokines most associated with this phenomenon. This
may be due to the airway eosinophilia being uncontrolled or due to a
non-eosinophilic source of these cytokines. However, there could be
non-IL-4/IL-13 related increase in FeNO that may be due to inflammasome
activation and through non-Th2 cytokine pathways that may raise the
possibility of airway infections or autoimmune activation(6). This has
important clinical implication. These patients may not show adequate
response to switching to anti-IL4R Mab if their asthma remains
uncontrolled. This needs to be evaluated prospectively.

References:

\begin{enumerate}
\tightlist
\item
  Couillard S, Shrimanker R, Chaudhuri R, et al. Fractional Exhaled
  Nitric Oxide Nonsuppression Identifies Corticosteroid-Resistant Type 2
  Signaling in Severe Asthma. Am J Respir Crit Care Med 2021; 204:
  731-734.
\item
  Mukherjee M, Forero DF, Tran S, Boulay ME, et al. Suboptimal treatment
  response to anti-IL-5 monoclonal antibodies in severe eosinophilic
  asthmatics with airway autoimmune phenomena. Eur Respir J 2020 Oct
  8;56(4):2000117. doi: 10.1183/13993003.00117-2020.
\item
  Mukherjee M, Bhalla A, Venegas-Garrido C, et al. Benralizumab
  attenuates blood and airway eosinophilia in severe asthmatics with
  inadequate response to anti-IL-5 neutralizing antibodies
  {[}abstract{]}. Eur Respir J 2022; 60 (suppl 66): 3994; DOI:
  10.1183/13993003.
\item
  Hearn AP, Kavanagh J, d'Ancona G, et al. The relationship between Feno
  and effectiveness of mepolizumab and benralizumab in severe
  eosinophilic asthma. J Allergy Clin Immunol Pract 2021; 9:
  2093-2096.e1.
\item
  Nair P, Kjarsgaard M, Armstrong S, Efthimiadis A, O'Byrne PM,
  Hargreave FE. Nitric oxide in exhaled breath is poorly correlated to
  sputum eosinophils in patients with prednisone-dependent asthma. J
  Allergy Clin Immunol 2010; 126: 404-6.
\item
  Donnelly LE, Barnes PJ. Expression and regulation of inducible nitric
  oxide synthase from human primary airway epithelial cells. Am J Respir
  Cell Mol Biol 2002; 26: 144-51.
\end{enumerate}

Authors

Pieter-Paul Hekking \textsuperscript{1,2}, Kayla
Zhang\textsuperscript{1}, Carmen Paz Venegas Garrido
\textsuperscript{1}, Raquel Lopez-Rodriguez \textsuperscript{1,3},
Melanie Kjarsgaard\textsuperscript{1}, Manali Mukherjee
\textsuperscript{1}, Parameswaran Nair \textsuperscript{1}

1. Division of Respirology, Department of Medicine, St. Joseph's
Healthcare Hamilton, McMaster University, Hamilton, Ontario, Canada.

2. Department of Respiratory Diseases, STZ Centre of Excellence for
Asthma \& COPD, Franciscus Gasthuis \& Vlietland Hospital, Rotterdam,
the Netherlands.

3. Department of Allergy, Lucus Augusti Hospital, Lugo, Spain

Correspondence

Dr Parameswaran Nair

Firestone Institute for Respiratory Health

St Joseph's Healthcare Hamilton

50 Charlton Avenue East

Hamilton, Ontario, L8N4A6, Canada

Tel: 905-522-1155 x 35044

Fax: 905-521-6183

E-mail: parames@mcmaster.ca

\textbf{Acknowledgements:}

Author contribution statement: All authors met the criteria for
authorship

Financial statement: No financial support was received for the submitted
work

Conflict of Interest Statement

MM reports research grants from AstraZeneca, Sanofi, Methapharm
Specialty Pharamceuticals and Mirimus, consulting fees from AstraZeneca,
Sanofi, Respiplus, GSK, Mirimus. PN reports research grants from
AstraZeneca, Teva, Sanofi, Foresee and consulting fees from AstraZeneca,
Teva, Sanofi, Equillium, Arrowhead pharma. All other authors have no
conflict of interest within the scope of the submitted work.

Table 1: Patient characteristics of severe asthma patients with
normalized FeNO low versus raised FeNO after treatment with anti-IL5
biologic.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{Normalized FeNO} & \textbf{Raised FeNO} &
\textbf{p-value}\tabularnewline
\midrule
\endhead
\textbf{n} & 35 & 15 &\tabularnewline
\textbf{Age} & 57.2 \selectlanguage{ngerman}± 12.5 & 46.7 \selectlanguage{ngerman}± 15.5 & 0.15\tabularnewline
\textbf{Gender (Female)} & 16 (45.7\%) & 7 (46.7\%) &
0.95\tabularnewline
\textbf{BMI (Kg/m2)} & 30.7 \selectlanguage{ngerman}± 4.6 & 32.5 \selectlanguage{ngerman}± 6.2 & 0.15\tabularnewline
\textbf{Atopy - yes (\%)} & 12 (40.0\%) & 13 (92.9\%) &
\textbf{\textless{}0.01}\tabularnewline
\textbf{Chronic Rhinosinusitis - yes (\%)} & 20 (66.7\%) & 8 (57.1\%) &
0.54\tabularnewline
\textbf{Nasal polyps - yes (\%)} & 14 (46.7\%) & 7 (50.0\%) &
0.84\tabularnewline
\textbf{Bronchiectasis - yes (\%)} & 2 (6.7\%) & 0 (0.0\%) &
0.32\tabularnewline
\textbf{ACQ post treatment} & 1.4 \selectlanguage{ngerman}± 1.1 & 1.7 \selectlanguage{ngerman}± 0.9 &
0.36\tabularnewline
\textbf{ACQ decrease post treatment} & -0.7 \selectlanguage{ngerman}± 1.2 & - 0.6 \selectlanguage{ngerman}± 1.1 &
0.68\tabularnewline
\textbf{Blood eosinophils pre treatment} & 0.4 {[}0.2 - 0.7{]} & 0.3
{[}0.1 - 0.8{]} & 0.70\tabularnewline
\textbf{Blood eosinophils post treatment} & 0.0 {[}0.0 - 0.02{]} & 0.0
{[}0.0 - 0.15{]} & 0.10\tabularnewline
\textbf{Sputum eosinophils pre treatment} & 7.5 {[}1.5 - 31.1{]} & 18.3
{[}6.7 - 58.8{]} & 0.17\tabularnewline
\textbf{Sputum eosinophils post treatment} & 0.0 {[}0.0 - 0.5{]} & 0.0
{[}0.0 - 9.2{]} & 0.08\tabularnewline
\textbf{ICS dose post treatment (mcg/day)} & 1250 {[}1000 - 2000{]} &
1000 {[}625 - 1750{]} & 0.32\tabularnewline
\textbf{OCS dose post treatment (mg/day)} & 5.0 {[}0.0 - 10.0{]} & 6.3
{[}0.0 - 10.6{]} & 0.79\tabularnewline
\textbf{FEV1 increase post treatment} & 0.16 \selectlanguage{ngerman}± 0.41 & 0.13 \selectlanguage{ngerman}± 0.54 &
0.30\tabularnewline
\bottomrule
\end{longtable}

BMI: body mass index; ACQ: asthma control questionnaire; ICS: Inhaled
corticosteroids; OCS: Oral corticosteroids; FEV\textsubscript{1}: Forced
expiratory volume in first second

Figure 1: Sputum cytokines in patients with raised FeNO
(\textgreater{}40ppb and \textgreater{}16ppb increase after treatment)
versus normal FeNO ([?]40) after treatment with anti-IL5, coloured by
residual airway inflammation.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Plots showing difference in levels of sputum cytokines between severe
asthma patients with FeNO [?]40 versus \textgreater{}40 after treatment
with anti-IL-5 biologic. Dotted lines are the normal values based on the
90\textsuperscript{th} percentile of 22 non-atopic healthy donors (mean
age 49+/-15) with no evidence of airway eosinophilia. IL-4 and IL-13 are
significantly higher in the raised FeNO group. The pink asterisk is an
outlier and details on this patient are shown in the online repository.
IL interleukin; FeNO Fraction of Nitric Oxid.

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