10.14639/0392-100X-N0760
Introduction. Chronic rhinosinusitis (CRS) can be classified as eosinophilic (eCRS) or non-eosinophilic (neCRS) based on infiltration type. The SWI/SNF complex may be involved in the pathophysiology of CRS. Aim. To assess the expression of the SWI/SNF complex in both CRS groups; to correlate blood eosinophil count (BEC), and histopathology eosinophil count (HPEC) with the SWI/ SNF expression level in eCRS and neCRS.Materials and methods. The study population consisted of 96 patients (68 eCRS, 28 neCRS). Immunohistochemical staining was performed on sinonasal mucosa for assessment of SWI/SNF protein expression. Type of tissue infiltration was assessed in samples obtained from examined groups (HPEC). The diagnostic value of eCRS was 10 cells/HPF (high power field). Complete blood count was analysed in order to calculate BEC.Results. BEC and HPEC correlated negatively with all the SWI/SNF subunits. HPEC and BEC correlated positively with clinical findings (L-M and SNOT-22), while SWI/SNF correlated negatively with clinical findings (L-M and SNOT-22).Conclusions. The SWI/SNF was observed in both eCRS and neCRS, with lower expression in former. The meaning of its negative correlation with BEC, HPEC and clinical findings in eCRS group remains to be understood.
application/xml
1827-675X
760
en
Mariola Popko
https://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
eCRS
The SWI/SNF complex in eosinophilic and non eosinophilic chronic rhinosinusitis
Text.Serial.Journal
https://www.actaitalica.it/
41
Rhinology
2021-05-14
2020-03-23
2021-05-17
2021-05-17
159-167
1
Mariola Popko
2021/05/14
10.14639/0392-100X-N0760
159
Department of Otorhinolaryngology, Faculty of Medicine and Dentistry, Medical University of Warsaw, Warsaw, Poland
1827-675X
eCRS
167
The SWI/SNF complex in eosinophilic and non eosinophilic chronic rhinosinusitis
https://www.actaitalica.it/article/download/760/579
41
ACTA OTORHINOLARYNGOLOGICA ITALICA 2021;41:159-167; doi: 10.14639/0392-100X-N0760




Rhinology


The SWI/SNF complex in eosinophilic
and non eosinophilic chronic rhinosinusitis
Il complesso SWI/SNF nella rinosinusite cronica eosinofila e non eosinofila
Katarzyna Kowalik1, Martyna Waniewska-Leczycka1, Elzbieta Sarnowska2, Natalia Rusetska2, Marcin Ligaj3,
Alicja Chrzan3, Mariola Popko1
1
 Department of Otorhinolaryngology, Faculty of Medicine and Dentistry, Medical University of Warsaw, Warsaw, Poland;
2
 Department of Molecular and Translational Oncology, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw,
Poland; 3 Department of Pathology, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland


SUMMARY
Introduction. Chronic rhinosinusitis (CRS) can be classified as eosinophilic (eCRS) or
non-eosinophilic (neCRS) based on infiltration type. The SWI/SNF complex may be in-
volved in the pathophysiology of CRS.
Aim. To assess the expression of the SWI/SNF complex in both CRS groups; to correlate
blood eosinophil count (BEC), and histopathology eosinophil count (HPEC) with the SWI/
SNF expression level in eCRS and neCRS.
Materials and methods. The study population consisted of 96 patients (68 eCRS, 28
neCRS). Immunohistochemical staining was performed on sinonasal mucosa for assess-
ment of SWI/SNF protein expression. Type of tissue infiltration was assessed in samples
obtained from examined groups (HPEC). The diagnostic value of eCRS was 10 cells/HPF
                                                                                                   Received: March 23, 2020
(high power field). Complete blood count was analysed in order to calculate BEC.
                                                                                                   Accepted: November 9, 2020
Results. BEC and HPEC correlated negatively with all the SWI/SNF subunits. HPEC and
BEC correlated positively with clinical findings (L-M and SNOT-22), while SWI/SNF cor-
                                                                                                   Correspondence
related negatively with clinical findings (L-M and SNOT-22).
                                                                                                   Mariola Popko
Conclusions. The SWI/SNF was observed in both eCRS and neCRS, with lower expression                Department of Otorhinolaryngology, Faculty of Me-
in former. The meaning of its negative correlation with BEC, HPEC and clinical findings in         dicine and Dentistry, Medical University of Warsaw,
eCRS group remains to be understood.                                                               Stepinska 19/25. 00-739 Warsaw, Poland
                                                                                                   Tel. +48 694012224. Fax +48 22 31 86 266
KEY WORDS: SWI/SNF, sinusitis, eosinophils, eCRS
                                                                                                   E-mail: popkom@interia.pl

RIASSUNTO                                                                                          Funding
Introduzione. La rinosinusite cronica (CRS) può essere classificata come eosinofila (eCRS)         None.
o non-eosinofila (neCRS), in base al tipo di infiltrato cellulare. Il complesso SWI/SNF po-
trebbe avere un ruolo nella fisiopatologia della CRS.                                              Conflict of interest
Obiettivo. Valutare l’espressione del complesso SWI/SNF in entrambi i gruppi di CRS;               The Authors declare no conflict of interest.
correlare la conta degli eosinofili nel sangue (BEC) e la conta degli eosinofili nel preparato
istopatologico (HPEC) con il livello di espressione SWI/SNF nei gruppi eCRS e neCRS.                 How to cite this article: Kowalik K, Waniews-
Materiali e metodi. La popolazione dello studio è composta da 96 pazienti (68-eCRS,                  ka-Leczycka M, Sarnowska E, et al. The SWI/
28-neCRS), è stata eseguita la colorazione immunoistochimica sulla mucosa rinosinusale               SNF complex in eosinophilic and non eo-
per valutare i livelli di espressione della proteina SWI/SNF, è stato valutato il tipo di infil-     sinophilic chronic rhinosinusitis. Acta Otorhi-
trato cellulare nei campioni ottenuti (HPEC). Valore di eosinofili, diagnostico di eCRS: 10          nolaryngol Ital 2021;41:159-167. https://doi.
cellule / HPF. È stato analizzato l’emocromo completo per calcolare il BEC.                          org/10.14639/0392-100X-N0760
Risultati. BEC e HPEC sono risultati inversamente correlati con tutte le subunità SWI/
SNF e correlati positivamente con i dati clinici (L-M e SNOT-22), mentre SWI/SNF erano             © Società Italiana di Otorinolaringoiatria
correlati negativamente con i risultati clinici (L-M e SNOT-22).                                   e Chirurgia Cervico-Facciale
Conclusioni. Il complesso SWI/SNF è risultato presente sia in eCRS che in neCRS, con
un’espressione inferiore nel primo tipo di rinosinusite cronica. Il significato della sua cor-                    OPEN ACCESS
relazione negativa con BEC, HPEC e risultati clinici nel gruppo eCRS resta da indagare.            This is an open access article distributed in accordance with
                                                                                                   the CC-BY-NC-ND (Creative Commons Attribution-Non-
PAROLE CHIAVE: SWI/SNF, sinusite, eosinofili, eCRS                                                 Commercial-NoDerivatives 4.0 International) license. The
                                                                                                   article can be used by giving appropriate credit and mentio-
                                                                                                   ning the license, but only for non-commercial purposes and
                                                                                                   only in the original version. For further information: https://
                                                                                                   creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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Introduction                                                     The chromatin remodelling in the SWI/SNF complex is
                                                                 involved in transcriptional control, DNA repair, hormonal
Chronic rhinosinusitis (CRS) is a multifactorial inflam-
                                                                 signalling and inflammation 9. Moreover, the SWI/SNF
matory disease of the nasal and paranasal mucosa. CRS            complex may play a role in the development and treat-
patients often experience altered innate immune response         ment of CRS, since it regulates glucocorticoid receptors
in the mucosa and a tendency towards infection by patho-         (GCs), which determine the regulation of glucocorticoid-
genic microorganisms. Until the publication of European          dependent gene expression 10,11. The SWI/SNF also regu-
Position Paper on Rhinosinusitis and Nasal Polyps in 2020        lates the action of vitamin D, and in CRS patients, SWI/
(EPOS 2020), CRS was categorised based on disease phe-           SNF expression correlates with expression of the vitamin D
notype, i.e. presence or absence of nasal polyps (chronic        receptor (VDR) 8. In addition, the combination of activated
rhinosinusitis with (CRSwNP) or without nasal polyps             VDR and the SWI/SNF complex is able to promote anti-
(CRSsNP). This classification did not inform about differ-       inflammatory processes 12. Vitamin D and its receptors may
ences in pathophysiology and which cells of the immune           play a crucial role in regulating immune mucosal function
system played the biggest role in the inflammation mecha-        in CRS 1. This association of the SWI/SNF with GCs and
nisms 1-3. CRS pathophysiology is multifactorial, including      vitamin D may contribute to the pathogenesis of CRS as
fungi, bacteria, viruses, biofilm, microbiome variability and    reflected in the abundance of the SWI/SNF complex 8,10,12.
superantigens which may activate the immune system 4,5.          The mechanisms underlying eosinophilic inflammation in
One interesting mechanism is the impact of pathogens on          CRS are still largely unknown, and as such, the relationship
T helper lymphocytes and their activation of the different       between CRS and the SWI/SNF must be further analysed.
specialised stages as Th1, Th2 and Th17. Furthermore,            The aims of this study were: 1. to assess expression of
lymphocytes produce cytokines and affect other immune            the SWI/SNF complex in both eCRS and neCRS patient
cells like eosinophils (Th2) and neutrophils (Th1, Th17).        groups; 2. to assess correlations between blood eosinophil
Therefore, given that various inflammatory mediators and         count (BEC) or histopathological eosinophil count (HPEC)
cells are triggered by and influence the course of the dis-      and SWI/SNF expression levels in eCRS and neCRS.
ease, further subclassification of CRS was proposed based
on cytokines and other inflammatory mediators that may
be more important to the mechanisms of the disease 1,5. The      Materials and methods
endotypes of CRS according to the EPOS 2020 are divid-           Study design and population
ed into type 2 of immune response with Th2 lymphocytes           The study population consisted of 96 CRS patients (42
and Type non-type 2 with Th1 or Th17 lymphocytes. Fur-           with CRSsNP and 54 with CRSwNP), all of whom had un-
ther, CRS can be subclassified as either eosinophilic CRS        dergone endoscopic sinus surgery between July 2015 and
(eCRS) or non-eosinophilic CRS (neCRS), based on pre-            February 2017 at the Department of Otorhinolaryngology,
dominant inflammatory cell type that modulates mucosal           Faculty of Medicine and Dentistry, at the Medical Univer-
immunity 1.                                                      sity of Warsaw. The current study group derives from the
The histopathological type of CRS may vary depending             same population of CRS patients we analysed in a previous
on race. Asian populations tend to experience significant        study. Patients were analysed according to the new EPOS
mucosal neutrophilia and associated neutrophilic inflam-         guidelines and extrapolated. All patients had first under-
mation, while Caucasian populations exhibit abundant tis-        gone a three-month course of conservative therapy (Ade-
sue eosinophilia and eosinophilic inflammation in CRS 1.         quate Medical Therapy, or AMT) 1. If AMT was unsuccess-
Moreover, eosinophilic CRS often coexists with asthma            ful, patients then qualified for surgical treatment. The study
and aspirin hypersensitivity. These patients are often re-       group was designed based on the criteria of the EPOS, and
calcitrant and non-responding to CRS therapy and sinus           underwent an interview, physical examination including
inflammation frequently recurs after surgery 6,7. Activated      endoscopic examination of the nasal cavities and computed
eosinophils in the sinonasal mucosa are reported to play a       tomography (CT) scan of the paranasal sinuses 1. Exclusion
role in the inflammatory process through cationic proteins       criteria were: unilateral CRS or nasal polyps, antrochoanal
with the release of granule proteins 1. In our previous study,   polyps, inverted papilloma, nasal or paranasal malignancy,
we showed that lower expression of BRG1 in CRS corre-            allergic fungal rhinosinusitis, systemic disease (such as
lates with higher levels of blood eosinophils. This negative     sarcoidosis, autoimmune disease, or cystic fibrosis) and
correlation of the SWI/SNF complex with eosinophils may          women who may be pregnant.
expound the worse prognosis in this group of patients with       Patients completed a Sino-Nasal Outcome Test (SNOT-22)
eCRS 8.                                                          questionnaire for the evaluation of sinus complaints 1 and



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                                                                                         The SWI/SNF complex in eosinophilic CRS




disease severity was categorised using CT scans, according     mucosal tissue with the highest degree of eosinophilic in-
to the Lund-Mackay scoring system 13. During ENT exami-        filtration and examined those samples under x400 magni-
nation, patients were assessed endoscopically according        fication with a 10x10 mm reticulate in the eyepiece. The
to the Lund-Kennedy (L-K) scale 14. None of the patients       total number of eosinophils was determined as the count
was treated with antibiotics, systemic or topical corticos-    per high-power field (HPF). Eosinophilic polyps were de-
teroids, or other immune-modulating drugs for at least one     fined as those where the eosinophil count was more than 10
month before surgery. Aspirin intolerance was defined as       cells/HPF; non-eosinophilic polyps were defined as those
the exacerbation of nasal symptoms triggered by aspirin or     where the eosinophil count was less than 10 cells/HPF 1,6.
nonsteroidal anti-inflammatory drug (NSAID) exposure 15.
Data on allergy status were collected in medical interviews,   Statistical analysis
and confirmed with skin prick tests and by assessing total     The results were subjected to statistical analysis using Sta-
immunoglobulin E (IgE) levels in blood. Bronchial asth-        tistica 13.1. Normality distribution and uniformity of va-
ma was determined according to GINA 2015 criteria 16.          lence were determined. Basic statistics were analysed due
Complete blood count was analysed to calculate blood cell      to the antibodies being evaluated. A Mann-Whitney U test
counts, especially eosinophil counts, for both groups (AL-     and chi-square test were used to determine whether the dif-
AB laboratory).                                                ferences between groups were significant. A Spearman test
The study was conducted in accordance with the ethical         was used to analyse correlations between variables in the
standards of the Local Ethics Committees of Warsaw Medi-       groups and protein expression levels of the SWI/SNF com-
cal University in Poland, No. KB/209/2016, and with the
                                                               plex. Differences were considered to be significant when
Helsinki Declaration. All participants provided informed
                                                               p < 0.05 19.
written consent.

Immunohistochemical staining                                   Results
Sinonasal mucosa were surgically removed from the osteo-       Clinical characteristics of the study groups
meatal complex and examined by immunohistochemistry.           Forty-two patients with CRSsNP and 54 patients with
Samples were fixed in 10% formalin solution and embed-         CRSwNP were enrolled in the study (a total of 96 CRS
ded in paraffin. Staining was performed on 3.5 μm sections     patients) (Tab. I). Participants were categorised as either
of tissue sheared from the paraffin blocks. Immunohisto-       eCRS or neCRS based on routine histopathology exami-
chemistry was performed using the method detailed in our
                                                               nation of H&E specimens. The eCRS group consisted of
previous study 17. The sections were treated with optimal
                                                               68 patients (42 males and 26 females, ranging in age from
antibody dilutions: anti-SMARCC1/BAF-155 (1:200 for 1
                                                               18 to 83 and with a mean age of 48.8 years). The neCRS
hour at 25oC) monoclonal antibodies (D7F8S) (Cell Signal-
                                                               group constituted of 28 patients (18 males and 10 females,
ling Technology), BRG1 (1:100 for 12 hours at 5oC) (G-7)
                                                               ranging in age from 18 to 81 and with a mean age of 41.7
(Santa Cruz Biotechnology), and BRM (1:200 for 1 hour
                                                               years). In the CRSwNP group (n = 54), seven patients had
at 25oC) (D9E8B) XP (Santa Cruz Biotechnology) subu-
nits of the chromatin remodelling complex the SWI/SNF          neCRS and 47 had eCRS. In the CRSsNP group (n = 42),
type. Colour was developed using 3.3’-diaminobenzidine         21 patients had neCRS and 21 had eCRS. Patients with
tetrahydrochloride (Dako). Haematoxylin staining was per-      eCRS accounted for 87% of the CRSwNP group (n = 47)
formed for 1 min and sections were embedded in balsam.         and 50% of the CRSsNP group (n = 21). The distribution
Protein expression levels for each the SWI/SNF subunit         of the CRSsNP and CRSwNP patients was different be-
were analysed by light microscopy and based on the H-          tween eCRS and neCRS groups and influenced the clinical
score system.                                                  features of patients. Patient characteristics are presented in
                                                               Table I. We found no differences in age and sex between
Quantification of eosinophils in tissue samples                groups (p > 0.05). Patients in the eCRS group presented
To evaluate the degree of cell infiltration, we used quan-     with a significantly higher incidence of asthma, allergies
tification techniques described by Bhattacharyya et al. to     and Lund-Mackay CT scores or Lund-Kennedy scores
count the number of eosinophils determined by haema-           compared with neCRS patients (p < 0.05). We observed
toxylin and eosin (H&E) staining 18. First, we performed       differences in subjects with aspirin-exacerbated respiratory
a low-power survey of each slide at x20 and x100 magni-        disease (AERD) and in SNOT-22 results when comparing
fication to identify sinus mucosa. Polyps and mucosa with      eCRS and neCRS groups, but these were not statistically
glandular elements were excluded. Next, we identified the      significant (p > 0.05).



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Table I. Clinicopathological characteristics of CRS patients.
 Group                           CRSsNP                CRSwNP                      p-value                 eCRS                              neCRS                       p-value
                                 (n = 42)               (n = 54)                                         (n = 68)                           (n = 28)
                                                                                                 (47 CRSwNP; 21 CRSsNP)              (7 CRSwNP; 21 CRSsNP)
 Age range                             18-77                    22-83                0.66                    18-83                              18-81                     0.68
    Average                             43.0                     49.6                                         48.8                               41.7
 Sex
    M                                   25                        35                  0.7                      42                                 18                      0.85
    F                                   17                        19                                           26                                 10
 Asthma                                  1                        18                0.009                      16                                  3                      0.03
 Allergy                                16                        23                 0.66                      30                                  9                      0.04
 AERD                                    0                         6               < 0.03                       4                                  2                      0.07
 SNOT-22 score                         34.2                      38.2                0.29                     36.1                               35.7                     0.78
 Lund-Kennedy score                     5.1                       8.7              < 0.001                     7.7                                5.5                    0.001
 Lund-Mackay CT score                   7.3                      16.3              < 0.001                    13.9                                8.5                    0.001
 BEC (cells/µL) range               0.02-0.87                 0.04-1.59            < 0.01                  0.02-1.59                           0.02-0.8                 < 0.001
    Average                            0.22                      0.43                                         0.41                               0.15
 HPEC (cells/HPF) range               0-550                     2-405               < 0.01                  10-550                                0-9
    Average                            66.8                      97.7                                        117.4                                3.4
CRSsNP: patients with chronic rhinosinusitis without nasal polyps; CRSwNP: patients with chronic rhinosinusitis with nasal polyps; eCRS: patients with eosinophilic chronic
rhinosinusitis; neCRS: patients with non-eosinophilic chronic rhinosinusitis; M: male; F: female; AERD: aspirin-exacerbated respiratory disease; BEC: blood eosinophil count; HPEC:
histopathological eosinophil count.



Eosinophils in blood and tissue                                                              relates positively with SNOT-22 (r = 0.31; p = 0.026), and
The type of tissue infiltration was assessed using histo-                                    Lund-Mackay CT scores (r = 0.43; p = 0.0001) (Fig. 3B).
pathology samples obtained from both examined groups
(Fig. 1). Eosinophilic infiltration was defined by an eo-                                    SWI/SNF protein expression levels in CRS
sinophil count of more than 10 cells/HPF for the eCRS                                        In both groups, SWI/SNF expression was confirmed.
group and fewer than 10 cells/HPF for the neCRS group                                        Mucosal nuclear staining results are presented in Fig-
(Fig. 1). Patients in the eCRS group exhibited higher his-                                   ure 4A. In the eCRS group, we observed a significant
topathology eosinophil counts (HPEC) (mean 117.4 cells/                                      correlation between BEC and the SWI/SNF subunits.
HPF, range 10-550 cells/HPF) than those in the neCRS                                         Figure 4B presents the negative correlation between
group (mean 3.4 cells/HPF, range 0-9 cells/HPF). The dif-                                    BEC and BRM protein expression levels (r = -0.34;
ferences between the two groups were statistically signifi-                                  p = 0.04), BRG 1 protein expression levels (r = -0.45;
cant (p < 0.001) (Fig. 2A).                                                                  p=0.006) and BAF 155 protein expression levels
Furthermore, significant differences were observed
                                                                                             (r = -0.43; p = 0.01). Furthermore, significant correla-
when measuring blood eosinophil count (BEC) for both
                                                                                             tions between HPEC and the SWI/SNF subunits were
the eCRS and neCRS groups (p < 0.004). Subjects in
                                                                                             observed in eCRS patients. Figure 4C shows the cor-
the eCRS group exhibited higher BEC than those in the
                                                                                             relation between HPEC and BRM protein expression
neCRS group (Fig. 2B). The mean BEC was 0.41 cells/µL
for eCRS patients and 0.15 cells/µL for neCRS patients                                       levels (r = -0.40; p = 0.015), BRG 1 protein expression
(Tab. I). Statistical analysis showed that the differences                                   levels (r = -0.34; p = 0.045) and BAF 155 protein ex-
between groups were significant. Furthermore, there was                                      pression level (r = -0.32; p = 0.01).
a strong positive correlation between BEC and HPEC                                           The SWI/SNF subunits correlated negatively with clini-
(r = 0.59; p = 0.000) (Fig. 2C).                                                             cal findings. Expression of BRM (r = -0.26; p = 0.03) and
The analysis of the other clinical data showed that histo-                                   BAF 155 (r = -0.35; p = 0.004) correlated negatively with
pathological eosinophil count (HPEC) correlated positive-                                    L-M CT scores (Fig. 5A). Moreover, all examined subunits
ly with Lund-Mackay CT score (r = 0.32; p = 0.01) (Fig.                                      (BRM: r = -0.25; p = 0.05, BRG 1: r = -0.26; p = 0.04,
3A). The other indicator of the CRS severity is SNOT-22,                                     BAF 155: r = -0.26; p = 0.04) correlated negatively with
and we observed that blood eosinophil count (BEC) cor-                                       SNOT-22 (Fig. 5B).



162
                                                                                                                       The SWI/SNF complex in eosinophilic CRS




Figure 1. Eosinophilic infiltration of CRS tissue in histopathology samples. (A) Non-eosinophilic chronic rhinosinusitis (neCRS, < 10 eos/HPF) (p < 0.05). (B) Eo-
sinophilic chronic rhinosinusitis (eCRS, > 10 eos/HPF). Magnification: 400x. Arrows indicate the position of eosinophils.




Figure 2. Eosinophils in blood and tissue of patients with chronic rhinosinusitis (CRS). (A) Higher histopathological eosinophil counts (HPEC) in patients with eo-
sinophilic chronic rhinosinusitis (eCRS) than in patients with non-eosinophilic chronic rhinosinusitis (neCRS) (p < 0.05). (B) Higher blood eosinophil counts (BEC) in
patients with eCRS than in patients with neCRS (p < 0.05). (C) Correlation between BEC and HPEC in patients with eCRS. In this group, BEC and HPEC presented
a positive correlation (p < 0.05).



Discussion                                                                           studied (BRM, BRG1, and BAF155), histopathology eo-
                                                                                     sinophil count (HPEC) correlates negatively with the SWI/
Recent publications have sought to better understand the                             SNF subunits, HPEC and BEC correlate positively with
pathophysiology of CRS, and the division of CRS patients                             clinical findings (L-M CT scores and SNOT-22) and SWI/
into groups by eosinophilic or non-eosinophilic inflamma-                            SNF correlates negatively with clinical findings (L-M CT
tion reflects this tendency. While eCRS is histologically                            scores and SNOT-22).
characterised by notable levels of eosinophil infiltration of                        Worse prognoses and more frequent relapse characterise
sinonasal tissue, it has been suggested that the degree of                           eCRS 1, and Fokkens et al. have proposed alternate ways
eosinophilic inflammation is also associated with the clini-                         for patients to manage the disease 1. The inflammatory
cal manifestation and recurrence of the disease, as well as                          mechanisms and molecular processes for eCRS are still be-
greater symptomatology and higher rates of CRS treatment                             ing analysed, and only recently was a relationship between
failure 1,20,21. Our study makes an important contribution to                        eosinophils and the SWI/SNF chromatin remodelling com-
studying the pathophysiology of CRS. Our main findings                               plex documented 8. The SWI/SNF subunits are engaged in
are: expression of SWI/SNF occurs in the sinonasal mu-                               controlling transcription, hormonal signalling, DNA repair
cosa of both CRS groups, blood eosinophil count (BEC)                                and inflammatory processes 9, but the complex itself also
correlates negatively with all SWI/SNF subunits that were                            regulates GC receptors and vitamin D function 10,12. The



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K. Kowalik et al.




Figure 3. The Spearman correlation for histopathology eosinophil count (HPEC) and clinical findings. (A) Correlation between HPEC and Lund-Mackay (L-M) CT
scores in patients with eosinophilic chronic rhinosinusitis (eCRS). In this group, HPEC presented a positive correlation with L-M CT scores (p < 0.05). (B) Correla-
tion between blood eosinophil count (BEC) and L-M CT scores/SNOT-22 in patients with eCRS. In this group, BEC correlated positively with both L-M CT scores
and SNOT-22 (p < 0.05).


subunits of the chromatin SWI/SNF remodelling complex                               levels of BRM, BRG1 and BAF155 protein expression,
interact directly with the glucocorticoid receptor (GR) and                         while patients with high HPEC had lower expression of all
regulate the expression of the GC response genes through                            the SWI/SNF subunits examined. This might be because
binding to repeating DNA sequences called hormonal re-                              impaired SWI/SNF expression influences the inflammatory
sponse elements (HREs) 10,11. Therefore, proper GR func-                            processes in eCRS and creates resistance to GC treatment.
tion is dependent on the SWI/SNF complex, which de-                                 There is evidence that the SWI/SNF complex is involved
termines the regulation of glucocorticoid-dependent gene                            in GC function and that it may control inflammatory pro-
expression. The latest studies indicate that the SWI/SNF                            cesses through macrophages that influence the expression
complex is able to promote anti-inflammatory processes 12                           of many important cytokines, e.g., IL-6 10-12,22.
and, as such, the SWI/SNF complex may also be involved                              When diagnosing CRS, it is also important to determine
in the pathophysiology of CRS. In this study, we demon-                             disease stage. The severity of general CRS symptoms can
strated that protein expression of the SWI/SNF occurs in                            be estimated using a variety of tools. One is by using CT
the sinonasal mucosa of eCRS and neCRS patients.                                    scans of the paranasal sinuses to assess gradation of shad-
We also found a negative correlation between the SWI/SNF                            owing. The Lund-Mackay CT score is a widely used meth-
subunits with blood and histopathology eosinophil count                             od for radiologic staging of CRS 13. Despite its simplicity,
(BEC and HPEC). Patients with high BEC presented lower                              it correlates well with disease severity and enables medical



164
                                                                                                                  The SWI/SNF complex in eosinophilic CRS




Figure 4. The SWI/SNF complex in patients with eosinophilic chronic rhinosinusitis (eCRS) and correlations with eosinophil counts. (A) Immunostaining (IHC)
of sinonasal epithelial cells with BRM, BRG1, and BAF155 antibodies. Antibody dilutions: 1:200 BRM; 1:100 BRG1; 1:200 BAF155. Magnification: 400x. Arrows
indicate the nuclei after IHC staining. (B) Correlation between blood eosinophil count (BEC) and H-scores for BRM, BRG1, and BAF155 in eCRS patients. In this
group, BEC correlated negatively with SWI/SNF subunit expression levels (p < 0.05). (C) Correlation between histopathology eosinophil count (HPEC) and H-scores
for BRM, BRG1, and BAF155 in patients with eCRS. In this group, HPEC correlated negatively with all analysed SWI/SNF subunit expression levels (p < 0.05).


professionals to plan surgical next steps 23. Bhattacharyya                       the eCRS group who presented with high Lund-Mackay
et al. found that Lund-Mackay CT scores correlate with the                        CT scores showed higher HPEC and BEC than patients
degree of tissue eosinophilia 18. Additionally, Kountakis et                      with low Lund-Mackay scores. Furthermore, we found a
al. proposed different subtypes of CRS based on CT im-                            significant negative correlation between SWI/SNF expres-
aging of the paranasal sinuses and other parameters (nasal                        sion and Lund-Mackay CT scores. Patients with lower
endoscopy, HPEC, BEC) 24. Similarly, Tokunaga et al. used                         protein expression levels for BRM and BAF 155 presented
CT scans of the paranasal sinuses, nasal endoscopy results                        worse CRS changes in CT scans.
and blood eosinophilia to distinguish eCRS from neCRS 25.                         In order to assess CRS severity, sinonasal symptoms are
Our study contributes to this burgeoning field by showing                         determined using the SNOT-22 scale 26. Wang et al. no-
the positive correlations between the blood eosinophils                           ticed that eCRS patients had significantly worse preopera-
count (BEC) and histopathology eosinophilic tissue infil-                         tive and postoperative SNOT-22 scores than neCRS pa-
tration (HPEC) with Lund-Mackay CT scores. Patients in                            tients 20. Likewise, we found a positive correlation between



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K. Kowalik et al.




Figure 5. The Spearman correlation for expression of SWI/SNF subunits with SNOT-22 and Lund-Mackay (L-M) CT scores in patients with eosinophilic chronic
rhinosinusitis (eCRS). (A) Correlation between H-scores for BRM and BAF155 and L-M CT scores in patients with eCRS. In this group, protein expression levels
of SWI/SNF subunits correlated negatively with L-M CT scores (p < 0.05). (B) Correlation of H-scores for BRM, BRG, and BAF155 with SNOT-22 in patients with
eCRS. In this group, protein expression levels of SWI/SNF subunits correlated negatively with SNOT-22 (p < 0.05).


SNOT-22 scores and BEC, as well as a significant negative                       differentiation of CRS patients in favour of eCRS diagno-
correlation between expression of the all SWI/SNF subunits                      sis (87% in CRSwNP and 50% in CRSsNP), and according
and SNOT-22 scores. Due to the fact that the SWI/SNF is                         to our results, the mean BEC for eCRS and neCRS patients
involved in infectious diseases and inflammation, patients’                     was 0.41 cells/µL and 0.15 cells/µL, respectively. Our results
quality of life may be affected by SWI/SNF abundance 9,22.                      showed high positive correlation BEC and HPEC (r = 0.59;
When diagnosing eCRS or neCRS, histopathological ex-                            p = 0.0001). Our findings contribute to the knowledge that
amination of the sinonasal tissue and calculation of HPEC                       the gold standard for diagnosing of eCRS is histopathologi-
are crucial. Some authors have also suggested that BEC                          cal examination of sinonasal tissue and calculation of HPEC.
is a good parameter because it correlates with tissue in-                       A drawback of this study is the lack of detailed molecu-
filtration and may be a marker of eCRS 24,27-29. Ho et al.                      lar mechanisms explaining how SWI/SNF plays role in the
indicated that BEC was a moderately specific predictor of                       pathophysiology of CRS. However, the study describes po-
eCRS, with mean BEC values for eCRS and neCRS pa-                               tential functional aspects related to the expression of the
tients being 0.42 ± 0.34x109/L and 0.17 ± 0.13x109/L, re-                       SWI/SNF in eCRS and resistance to steroids. The strength
                                                                                of our study is that we analysed the clinical aspects of
spectively. They distinguished eCRS based on HPEC (> 10
                                                                                eCRS in relation to its pathophysiology.
cells/HPF) and proposed a cut-off value for BEC for eCRS
patients as ≥ 0.24 x 109/L27. Like Ho et al. 27, and according
to the EPOS 2020, we established an HPEC cut-off value                          Conclusions
of > 10 cells/HPF as a marker for distinguishing eCRS                           Our study demonstrated that the SWI/SNF subunits are ex-
from neCRS. HPEC indicates a specific local eosinophilic                        pressed in both eCRS and neCRS and that the SWI/SNF
infiltration in the paranasal sinuses, while BEC defines sys-                   subunits show a strong correlation between blood and tissue
temic eosinophilia, which may be related to allergies, para-                    eosinophil counts (BEC and HPEC). There is not only mo-
sites, viruses, bacteria, or even fungus 30,31. For this reason,                lecular but also clinical relevance (Lund-Mackay scoring sys-
we believe HPEC may be a more reliable mean for distin-                         tem, SNOT-22, comorbidities) to these findings. Lower levels
guishing eCRS from neCRS that BEC. Our HPEC cut-off                             of SWI/SNF expression and more eosinophilic infiltration
value of > 10 cells/HPF for eCRS patients influenced the                        are more advanced clinical symptoms of eCRS. We propose



166
                                                                                                                   The SWI/SNF complex in eosinophilic CRS




that the SWI/SNF complex may play an important role in the                       14
                                                                                      Psaltis AJ, Li G, Vaezeafshar R, et al. Modification of the Lund-
                                                                                      Kennedy endoscopic scoring system improves its reliability and
pathophysiology of CRS and may be of particular importance
                                                                                      correlation with patient-reported outcome measures. Laryngoscope
in cases of eCRS. The negative correlation of BEC and HPEC                            2014;124:2216-2223. https://doi.org/10.1002/lary.24654
with the SWI/SNF subunits may also be a contributor to less                      15
                                                                                      Sakalar EG, Muluk NB, Kar M, et al. Aspirin-exacerbated respiratory
successful treatment of eCRS. Patients with the lower SWI/                            disease and current treatment modalities. Eur Arch Otorhinolaryngol
SNF expression levels may be resistant to steroids and not                            2017;274:1291-1300. https://doi.org/10.1007/s00405-016-4273-1
respond to typical CRS therapy. Finally, the positive correla-
                                                                                 16
                                                                                      Horak F, Doberer D, Eber E, et al. Diagnosis and management of asth-
                                                                                      ma - Statement on the 2015 GINA Guidelines. Wien Klin Wochenschr
tions between eosinophil counts and clinical findings (Lund-                          2016;128:541-554. https://doi.org/10.1007/s00508-016-1019-4
Mackay and SNOT-22 scores) may suggest that there are op-                        17
                                                                                      Tomaszewska M, Sarnowska E, Rusetska N, et al. Role of vitamin D
portunities to better estimate the severity and prognosis for                         and its receptors in the pathophysiology of chronic rhinosinusitis. J
eCRS patients, as well as treatment response.                                         Am Coll Nutr 2019;38:108-118. https://doi.org/10.1080/07315724.2
                                                                                      018.1503102
                                                                                 18
                                                                                      Bhattacharyya N, Vyas DK, Fechner FP, et al. Tissue eosinophilia in
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