A Preliminary Investigation of the Differences in

Immune Responses in Different Strains of Mice with

Localized Allergic Rhinitis Tolerance Models
Qidi Zhang
Huazhong University of Science and Technology
Wanting Zhu
Huazhong University of Science and Technology
Zhixin Zou
Huazhong University of Science and Technology
PHD. Wenting Yu
Huazhong University of Science and Technology
PHD. Pei Gao
Huazhong University of Science and Technology
Ziyi Long
Huazhong University of Science and Technology
Ying Wang
The First Affiliated Hospital of Anhui Medical University
Jianjun Chen

Huazhong University of Science and Technology

Research Article

Keywords: allergic rhinitis, animal model, local tolerance, IL-10, TGF-β, RNA-seq, eotaxin, BALB/c,
C57BL/6, IgE

Posted Date: April 24th, 2024

DOI: [Link]

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License

Additional Declarations: No competing interests reported.

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Abstract
Background：

BALB/c and C57BL/6 are two common mice strains for the study of allergic rhinitis (AR). We previously
found that both mice differed in symptoms and immune phenotypes in the local allergy [Link]
study aimed to systematically compare the immune differences between the two mice in a local tolerance
model and explore the underlying mechanisms.

Methods:

We used ovalbumin (OVA) as a topical nasal drop for 15 weeks in BALB/c and C57BL/6 mice. Allergic
symptoms were recorded. Serum-specific OVA antibodies (IgE, IgG1, IgG2a) and cytokines (IL-4, IL-10, and
IFN-γ) in the spleen cell culture supernatants were tested. The expression of eosinophils, goblet cells,
TGF-β, IL-10, eotaxin, and RNA-seq on the nasal mucosa was examined.

Results:

Nasal symptoms of BALB/c worsened first and then relieved, whereas C57BL/6 showed continuously
worsened symptoms. The serum antibodies levels in BALB/c increased over time. IgE and IgG1 levels in
C57BL/6 showed a trend similar to that of BALB/c, while IgG2a levels did not increase throughout the
course. IL-4 first increased and then decreased, while IL-10 remained increased in BALB/c,
whereasC57BL/6 showed no significant changes. In BALB/c, the expression of eosinophils and eotaxin in
the nasal mucosa increased to a certain extent and remained unchanged, whereas TGF-β and IL-10
expression was upregulated. In C57BL/6, the expression of eosinophils and eotaxin was higher than that
in BALB/c at the 15th week. The RNA-sequencing results showed that gene expression in BALB/c was
upregulated in biological processes, the tolerance and negative regulatory pathway at the 15th week.
Gene expression in C57BL/6 is upregulated in cell synthesis- and secretion-related pathways.

Conclusion:

Local tolerance and systemic sensitisation was successfully induced in BALB/c by long-term stimulation
with OVA. However, local tolerance could not be induced in C57BL/6 . The normal function of T regulatory
cells is key to local tolerance.

Introduction
Allergic rhinitis (AR) is a common respiratory disease. Epidemiological studies have shown that the
prevalence of AR is increasing every year and poses a burden to society(1). BALB/c and C57BL/6 mouse
strains are two commonly used animal models for the study of allergic rhinitis. Our previous study
established stable animal models of local sensitizing allergic rhinitis in BALB/c and C57BL/6 mice by
nasal allergen drip and found significant differences in immune responses between the two mice, that is,
BALB/c mice showed relatively weak local allergic inflammatory responses and strong systemic immune
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responses, whereas C57BL/6 mice showed relatively strong local allergic inflammation and relatively
mild systemic immune responses(2).

At the same time, we found that the symptoms of BALB/c mice gradually decreased or even approached
normal status, that is, a state of local tolerance was developed and the systemic immune tolerance
response was also upregulated after a more prolonged nasal drip of local allergen. In contrast, we did not
find local or systemic immune tolerance in C57BL/6 mice subjected to the same procedure. Alan et al.
used enterohemorrhagic Escherichia coli to infect BALB/c and C57BL/6 mice, and found that C57BL/6
mice had a worse prognosis than BALB/c mice, with severe intestinal damage, epithelial barrier
dysfunction, and impaired renal function, leading to an increased percentage of their deaths,
demonstrating that the high survival rate of BALB/c mice is associated with the immune tolerance
mechanism(3).

Studies in humans and mice suggest that regulatory T cells (Tregs), regulatory antigen-presenting cells
(APC), other suppressor cells, and cytokines make important contributions to the induction and
maintenance of immune tolerance(4–6). For example, dendritic cells (DCs), which are specialized
antigen-presenting cells, are the initial cells in asthma and other types of allergic inflammation. DCs can
induce both allergic responses and immune tolerance(7, 8). Treg cells play an important role in immune
tolerance by producing cytokines such as IL-10 and TGF-β and using suppressive surface molecules such
as CTLA4 and PD1 to exert their function(9–13). However, the mechanisms underlying immune tolerance
remain relatively unknown.

In this study, we used a certain dose of ovalbumin (OVA) for long-term continuous nasal drip in both
mouse strains to systematically compare the immunological differences, especially the differences
related to immune tolerance, to provide more evidence on the mechanisms of local and systemic immune
tolerance.

Materials and Methods

Experimental animals
48 female mice (24 BALB/c and 24 C57BL/6), aged 5 weeks, SPF grade, were purchased from Beijing
Vital River Laboratory Animal Technology Co., Ltd. This study protocol was reviewed and approved by the
Committee on the Ethics of AnimalExperiments of Huazhong University of Science andTechnology,
Wuhan, China, approval number 2844.

Model construction
BALB/c and C57BL/6 mice were randomly divided into four groups: BALB/c PBS group, BALB/c OVA
group, C57BL/6 PBS group, C57BL/6 OVA group. Each group contained 12 animals. The mice were
dripped with phosphate buffered solution (PBS) or 25 mg/ml ovalbumin (OVA) daily, 10ul on each side of
the nasal cavity. Half of the mice in each group were sacrificed on 8th week, and the remaining mice were

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sacrificed on 15th week (Fig. 1). Finally, eight subgroups of mice were formed, i.e. 8th BALB/c PBS group,
8th BALB/c OVA group, 8th C57BL/6 PBS, 8th C57BL/6 OVA, 15th BALB/c PBS, 15th BALB/c OVA, 15th
C57BL/6 PBS, and 15th C57BL/6 PBS groups.

According to a previous study, the symptoms of BALB/c mice peak at the 8th week, and the symptoms of
C57BL/6 mice also peak at 8th ~ 9th week. Serum-specific antibodies and localized nasal mucosal
eosinophils in both mice also increased significantly(2). At the 15th week, the symptoms of the BALB/c
mice were greatly relieved and approached normal. However, the symptoms in C57BL/6 mice were still
severe, and the risk of death in C57BL/6 mice increases with time. Therefore, we chose the time points at
which the mice were sacrificed at the 8th week and 15th week.

Allergic symptoms
Sneezing was the most prominent symptom; therefore, we counted the number of sneezes to reflect the
severity of nasal symptoms. We recorded the number of sneezes within 10 min after nasal drip on the
first day at 0th, 4th, 8th, 12th and 15th week.
Detection of serum OVA-specific antibody levels
Blood was collected from the tail vein of the mice every three weeks and within 24h after the last nasal
drip at the 15th week. The blood was centrifuged at 2500 rpm for 5 min by ultracentrifugation, and the
upper layer of yellowish serum was stored at -20°C for examination. The OD values of OVA-specific IgE,
IgG1, and IgG2a were determined using ELISA (eBioscience, San Diego, CA, USA).

Splenocyte culture and cytokine level assay

After deep anesthesia with isoflurane, the mice were treated with cervical dislocation and disinfected with
75% alcohol. The spleens were removed from mice on an ultra-clean bench (Aintech, Suzhou, China) and
ground into centrifuge tubes using RPMI-1640 (Hyclone, Beijing, China) with a 200 mesh filter. After
centrifugation for 10 min, the supernatant was discarded, the cell precipitate was resuspended in RPMI-
1640, and lymphocytes were isolated using a Mouse Spleen Lymphocyte Isolation Kit (TBDscience,
Tianjin, China). Lymphocytes (5*106/ml lymphocytes were cultured in culture medium containing 20%
serum, 1% double antibody, 30ug/ml OVA). Splenocyte supernatants were collected after 72h in an
incubator and stored at -20°C. IL-4, IFN-γ, and IL-10 levels in splenocyte supernatants were measured
using ELISA (R&D Systems, Minneapolis, MN, USA). The OD values at 450 nm were measured using an
automated enzyme marker (BioTek, Beijing, China) and the concentrations were analyzed.
Histology of the nasal mucosa
The nasal mucosa was extracted according to the Dunston method(14).The skin was removed. The
muscles and jaws around the nose were cut off with scissors, and the nasal bone on one side was cut
slowly until the nasal mucosa was completely exposed. The nasal mucosa was quickly placed in a sterile
centrifuge tube and stored at -80°C for RNA sequencing. The unexposed nasal mucosa on the other side
was fixed in paraformaldehyde overnight. The nasal mucosa was removed the following day for

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decalcification in 10% EDTA for one week. The nasal mucosa was embedded in paraffin wax. HE and PAS
staining was performed after parallel sectioning and roasting. The morphological structure of the nasal
mucosa tissue and eosinophil and goblet cell infiltration were observed under a light microscope (1000×).
Goblet cell cells were graded according to Judith A(15): grade 0, no PAS staining; grade 1, PAS staining ≤
25%; grade 2, PAS staining 26–50%; grade 3, 51–75%; and grade 4, PAS staining > 75%.

Immunohistochemical staining
Paraffin sections of the nasal mucosa were sequentially dewaxed, rehydrated, and antigen-repaired. The
sections were then treated with a blocking solution containing 3% bovine serum albumin for 45 min.
Sections were incubated overnight at 4°C with TGF-β antibody (Abcam, Shanghai, China), IL-10 antibody
(R&D Systems, Minneapolis, MN, USA), and eotaxin antibody (Proteintech, Wuhan, China) and then
incubated with anti-rabbit IgG-HRP (Boster, Wuhan, China), followed by DAB color development.
Subsequently, hematoxylin was re-stained, dehydrated, made transparent, sealed, and placed under a
light microscope for examination.

Gene sequencing
Lyophilized nasal mucosa was subjected to RNA sequencing analysis. Total RNA was extracted using
TRIzol reagent and subjected to DNA digestion. RNA quality was determined using a NanodropTM OneC
spectrophotometer (Thermo Fisher Science Inc., USA), and RNA integrity was confirmed using 1.5%
agarose gel electrophoresis. Qualified RNA was quantified using the Qubit 3.0. RNA sequencing libraries
were prepared using a Ribooff rRNA Depletion Kit (human/mouse/rat). Sequencing was performed using
a DNBSEQ-T7 sequencer (MGI Tech Co., Ltd. China).

After the raw data were filtered, genes differentially expressed between groups were identified using the
edgeR package (3.12.1), and P < 0.05 and logFC cutoff values were set to 2 to determine the statistical
significance of gene expression differences. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes
and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) of differentially expressed
genes were performed using KOBAS software (version 2.1.1). Differentially expressed genes were entered
into the search tool for the retrieval of interacting genes (STRING) database to construct a protein-protein
interaction (PPI) network of differentially expressed genes. The CytoHubba plug-in in the Cytoscape
software was used to calculate the connectivity of protein nodes and to screen for significantly different
proteomic modules. The top 10 genes were identified as core genes that were considered statistically
significant.

Statistical Analysis
GraphPad Prism 9.0 and SPSS 22 (IBM, Armonk, NY) were used for statistical analysis. Allergic
symptoms, serum -specific antibodies, cytokines in the splenic culture supernatant, and eosinophils
numbers were expressed as mean ± standard deviation. Tukey's multiple comparison method in one-way
ANOVA was used to assess the statistical significance of symptoms, serum antibodies, cytokine

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concentrations, eosinophil and goblet cell numbers and immunohistochemical indices. P < 0.05 was
considered statistically significant.

Results
Nasal symptoms
Both BALB/c OVA and C57BL/6 PBS groups started to show symptoms at the 4th week. Nasal symptoms
of BALB/c mice peaked at 7th-8th week, then gradually resolved and remained at a low level from 10th
week to 15th week. Nasal symptoms of C57BL/6 mice reached their peak at 8th-9th week and then
fluctuated at peak levels without any reduction with time (Fig. 2).

Serum OVA-specific antibodies

From the 0th week to the 15th week, OVA-specific IgE, IgG1, and IgG2a in the BALB/c OVA group showed
an increasing trend over time. In the C57BL/6 OVA group, OVA-specific IgE tended to increase with longer
nasal drip time, sIgG1 peaked at 12th week and remained stable at 15th week, while IgG2a did not show
significant changes throughout the course. Serum antibody concentrations were higher in the BALB/c
OVA group than in the C57BL/6 OVA group at all time points (Fig. 3).

Cytokines in splenic culture supernatants

In the BALB/c OVA group, the concentrations of IL-4 and IL-10 were significantly higher in splenic culture
supernatants at 8th week compared than in the BALB/c PBS group. The IL-4 concentration was lower and
the IL-10 concentration was higher in the 15th BALB/c OVA group than in the 8th BALB/c OVA group. The
concentrations of IL-4, IL-10, and IFN-γ were not changed in the C57BL/6 OVA group at 8th and 15th week
compared to in the C57BL/6 PBS group (Fig. 4).

HE and PAS staining of the nasal mucosa

Nasal eosinophil infiltration was observed in both 8th BALB/c OVA group and 8th C57BL/6 OVA groups.
There was no significant difference in eosinophil numbers between 8th BALB/c OVA group and 15th
BALB/c OVA groups. A significant increase in eosinophil number was observed in the 15th C57BL/6 OVA
group compared to that in the 8th C57BL/6 OVA group.

PAS results showed a significant increase in the number of nasal goblet cells in both the 8th BALB/c OVA
group and the 8th C57BL/6 OVA group. There was a significant decrease in the goblet cell number in the
15th BALB/c OVA group compared with the 8th BALB/c OVA group, and a continued increase in goblet
cells in the 15th C57BL/6 OVA group compared to the 8th C57BL/6 OVA group. At both time points of 8th
week and 15th week, the number of goblet cells in the BALB/c OVA group was lower than that in the
C57BL/6 OVA group (Fig. 5).

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Immunohistochemical staining of the nasal mucosa
The expression of eotaxin was higher in the C57BL/6 OVA group than in the control group at both the 8th
week and 15th week, and was even higher at the 15th week than at the 8th week. There was an increasing
trend in eotaxin expression in the 8th BALB/c OVA group, but the difference was not statistically
significant. Eotaxin staining intensity was higher in C57BL/6 nasal mucosa than that in BALB/c mice at
all time points (Fig. 6A, 6D).

No significant changes in TGF-β1 and IL-10 were observed in the nasal mucosa of the BALB/c or
C57BL/6 OVA groups at 8th week. TGF-β1 and IL-10 expression was significantly increased in the 15th
BALB/c OVA group. In contrast, there was no significant increase in TGF-β1 and IL-10 expression in the
C57BL/6 OVA group at any time point. (Fig. 6B,6C,6E,6F).

Nasal mucosal gene sequencing

We have previously studied the genetic alterations in the nasal mucosa of BALB/c and C57BL/6 mice in
the 8th week(2). In this study, we focused on nasal mucosal gene alterations at 15th week. We found
3903 differentially expressed genes in the 15th BALB/c OVA group compared with the control, including
2063 upregulated and 1840 downregulated genes. GO-KEGG analysis showed significant enrichment in
intrinsic immune response, immune system response, and inflammatory response. There were 225
differential genes in the 15th C57BL/6 OVA group compared to the control group, including 83
upregulated genes and 142 downregulated genes. GO-KEGG analysis showed significant enrichment in
the acute inflammatory response and B-cell proliferation.

When comparing the 15th BALB/c OVA group and the 15th C57BL/6 OVA group, there were 4299
differential genes, including 2354 upregulated genes and 1945 downregulated genes. GO-KEGG analysis
showed that pathways of BALB/c mice were significantly enriched in the T-cell receptor signaling
pathway, B-cell receptor signaling pathway, cytokine receptor interaction, TGF-β signaling pathway, Fc
epsilon RI signaling pathway, TH1 and TH2 differentiation, and antigen presentation pathways. Pathways
of C57BL/6 mice were significantly enriched in the HIF-1 signaling pathway, PI3K-Akt signaling pathway,
calcium signaling pathway, ABC transporter protein, cAMP signaling pathway, protein processing in the
endoplasmic reticulum, amino acid biosynthesis, protein export, and RNA transport response pathway.
GSEA results showed that BALB/c mice were significantly enriched in the TGF-β pathway and negatively
regulated gene expression.

We constructed a PPI network of differentially expressed genes between the 15th BALB/c OVA group and
the 15th C57BL/6 OVA group, which generated 8848 nodes and 99525 edges, and obtained the top 10
HUB nodes: Rps27a, Uba52, Ubc, Kng2, Gnal, C3, Gng7, Gnb3, Reep5, and Reep6 (Fig. 7).

Discussion

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In this study, we compared local immune tolerance in BALB/c and C57BL/6 mice, as well as systemic
immune differences, through the use of long-term nasal OVA drop stimulation. BALB/c mice were found
to develop local tolerance: nasal symptoms were aggravated at first and then relieved with longer nasal
drip; the systemic TH2 response was enhanced at first and weakened with time, and the Treg response
was continuously enhanced. In contrast, C57BL/6 mice did not develop a local tolerance response even
with the prolongation of nasal drip time, and the local symptoms and inflammatory response continued
to worsen and remain at a high level; the systemic immune response only maintained a certain degree of
TH2 response and the Treg response was not enhanced.

BALB/c and C57BL/6 mice are two inbred strains commonly used in immunological studies. Based on
the cytokine production tendency, BALB/c mice are thought to favor TH2-type responses (e.g., IL-4, IL-5,
IL-10, and IL-13), whereas C57BL/6 mice favor TH1-type responses (TNF-α and IFN-γ)(16). BALB/c mice
are susceptible to viral infections and exhibit lower cytotoxic responses. When stimulated by allergens,
the systemic humoral response is stronger, consistent with the cytokine profile. In contrast, C57BL/6 mice
are more reactive to viral infection, exhibiting a higher cytotoxic response and a relatively weaker
systemic allergic response(17, 18). We observed a local nasal drip AR model in BALB/c and C57BL/6
mice and found that there were significant differences between their immune responses; i.e. BALB/c mice
showed relatively mild local allergic inflammatory responses and strong systemic allergic responses. In
contrast, C57BL/6 mice show a stronger local allergic inflammatory response and milder systemic
allergic response(2).

We observed that the concentrations of OVA-specific IgE, IgG1, and IgG2a in BALB/c mice increased as
nasal drip time increased. However, we found that OVA-specific IgE increased with time, sIgG1 increased
first and then leveled off at 12th week, and IgG2a did not change throughout the whole course in
C57BL/6 mice. Meanwhile, in BALB/c mice, the splenic culture supernatant results showed that IL-4
concentration increased at 8th week then decreased at 15th week, and IL-10 concentration continued to
increase with longer nasal drip. In contrast, C57BL/6 mice showed no change in IL-4, IFN-γ, and IL-10
levels during the entire course of nasal OVA drip. These results suggest that when BALB/c mice were
stimulated with a certain dose of allergen for a long time, their allergic reaction (TH2 function) was
aggravated, but their TH1 and Treg functions also tended to increase with time and eventually
suppressed TH2 function. In contrast, although the increase in serum sIgE (TH2 function) in C57BL/6
mice was lower than that in BALB/c mice, Treg function was not significantly enhanced with time, so the
overall response was allergic. Some reports have focused on the immunity and tolerance between
BALB/c and C57BL/6 mice. For example, Alan et al. found that BALB/c mice produced more specific
antibodies than C57BL/6 mice and showed more significant tolerance to enterohemorrhagic Escherichia
coli(3). Gera et al. demonstrated that BALB/c mice were more tolerant to colitis than C57BL/6 mice,
possibly because of enhanced BALB/c retinoic acid signaling and the consequent increased ability to
fight mucosa-associated lesions(19–24). Chen et al. found that BALB/c thymus and peripheral lymphoid
organs had more Treg cells and showed a more potent suppressive effect compared to C57BL/6
mice(25). In a study by Ki-Il et al., it was also found that IL-4, IFN-γ, and IL-10 were not significantly
increased in the spleen cell culture supernatant of C57BL/6 mice sensitized by dust mites, and only IL-6
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was mildly increased. In contrast, all of the above cytokines were significantly increased in BALB/c mice,
showing active TH1 responses, TH2 responses, and Treg responses(26).

In this study, we found that eosinophils and goblet cells increased in the nasal mucosa of BALB/c mice at
8th week, while the number of goblet cells decreased and the number of eosinophils stopped increasing
at 15th week. The expression of local IL-10 and TGF-β also increased over time, showing the
characteristics of local tolerance. In C57BL/6 mice, local eosinophils and goblet cells increased
continuously from 8th week to 15th week, and eotaxin expression increased significantly. However, the
local IL-10 and TGF-β levels did not change throughout the course. As IL-10 and TGF-β are important
cytokines for immune tolerance, the lack of IL-10 and TGF-β may influence the formation of local
tolerance(27).

There were 3903 activated genes locally at 15th week in the BALB/c OVA group, indicating that the local
immune responses were active in BALB/c mice. It has also been suggested that the formation of local
tolerance in BALB/c mice is an active and positive immunoregulatory process. There were 225 activated
genes in the C57BL/6 OVA group at 15th week, much lower than that in the BALB/c OVA group. Although
the TH2 response was weaker in C57BL/6 mice than in BALB/c mice at 8th week, the lack of regulatory
and inhibitory mechanisms led to an increase in symptomatic and inflammatory responses. We
compared the genetic alterations in BALB/c and C57BL/6 at 15th week and found that BALB/c mice and
C57BL/6 mice differed significantly in the classical Wnt signaling pathway, which has a negative
regulatory effect on Treg cells(28).There was also an elevated negative regulation of JUN kinase activity
in BALB/c mice. The JUN kinase pathway has a positive regulatory effect on allergic rhinitis. Therefore,
the negative regulation of Wnt signaling pathway and JUN kinase pathway may be related to immune
tolerance in BALB/c [Link] constructing the PPI network for the BALB/c OVA group with C57BL/6 OVA
group DEGS at 15th week, we derived the top 10 HUB nodes: Rps27a, Uba52, Ubc, Kng2, Gnal, C3, Gng7,
Gnb3, Reep5, Reep6.

Recent reports have found that Rps27a is involved in protein synthesis, post-translational modification of
proteins, and transcription of genes, and is highly expressed in tumor tissues such as breast fibroids,
colorectal cancer and kidney(29, 30). Ubiquitin ribosomal fusion protein (UBA52) is sheared into ubiquitin
molecules and ribosomal protein L40 by the action of cell-intrinsic enzymes, where ubiquitin molecules
are mainly involved in ubiquitination processes in vivo, such as the degradation of proteins, regulation of
the TGF-β pathway and immune response(31, 32). UBC genes play key roles in maintaining ubiquitin (Ub)
homeostasis(33). CAMP signaling induces kininogen 2 (Kng2) production in brown adipose tissue, which
inhibits brown adipose tissue thermogenesis and may be related to local metabolic function(34). Gnal
encodes the stimulatory G protein Gαolf, which is essential for activating the cAMP pathway in striatal
projection neurons and is associated with dystonia and hyperactivity disorders(35, 36). G protein subunit
γ7 (Gng7), a member of the G protein family, was moderately to highly correlated with the degree of
infiltration of CD4 + T cells in the peripheral blood of patients with colorectal cancer. Additionally, GNG7
expression positively correlated with the degree of infiltration of B cells, macrophages, neutrophils and
dendritic cells(37). Gnb3 encodes the G protein β3 subunit and is associated with obesity, diabetes,
Page 9/19
depression and tumors(38–40). REEP5 and REEP6 play important roles in IL-8-stimulated activation of
CXCR1(41). CXCR1 can be used as a marker of AR(42). Liliane et al. found that C3 induces mice to form
a peptide-mediated skin tolerance model by regulating DC function, and that C3-deficient mice fail to
produce Treg cells required for the induction of tolerance(43). From the above reports, it appears that C3
and UBA52 may be involved in the regulation of local immune tolerance. However, the role of other genes
with a large degree of activation or response in the regulation of local immune tolerance and
inflammation needs to be explored in future studies.

Declarations
Statements

The experimental protocols and procedures were conducted in accordance with the guidelines issued by
the Experimental Animal Ethics Committee.

Funding

Anonymised for funding.

Declaration of Conflicting Interests

The authors reported no potential conflict of interest.

Supplemental Material

Supplemental material for this article is available online.

Acknowledgment

We thank anonymised for group (Wuhan, China) for providing technical assistance for this experiment.

Author Contributions

Anonymized for review and editing (lead); writing the original draft (lead); formal analysis (lead).
Anonymized for conceptualization (lead); writing, review, and editing (equal). Anonymized for software
(equal); anonymized for writing, review, and editing (equal). Anonymized for Methodology (lead);
Anonymized for writing – review and editing (equal); anonymized for conceptualization (supporting);
writing – original draft (supporting); anonymized for writing – review and editing (equal).

References
1. Brozek JL, Bousquet J, Agache I, Agarwal A, Bachert C, Bosnic-Anticevich S, et al. Allergic Rhinitis and
its Impact on Asthma (ARIA) guidelines-2016 revision. J Allergy Clin Immunol. 2017;140(4):950-8.

Page 10/19
 2. Zhang Q, Zhu W, Zou Z, Yu W, Gao P, Wang Y, et al. A Preliminary Study in Immune Response of
BALB/c and C57BL/6 Mice with a Locally Allergic Rhinitis Model. Am J Rhinol Allergy.
2023:19458924231157619.
3. Bernal AM, Fernandez-Brando RJ, Bruballa AC, Fiorentino GA, Pineda GE, Zotta E, et al. Differential
Outcome between BALB/c and C57BL/6 Mice after Escherichia coli O157:H7 Infection Is Associated
with a Dissimilar Tolerance Mechanism. Infect Immun. 2021;89(5).
4. Shin JU, Kim SH, Noh JY, Kim JH, Kim HR, Jeong KY, et al. Allergen-specific immunotherapy induces
regulatory T cells in an atopic dermatitis mouse model. Allergy. 2018;73(9):1801-11.
5. Smaldini PL, Trejo F, Cohen JL, Piaggio E, Docena GH. Systemic IL-2/anti-IL-2Ab complex combined
with sublingual immunotherapy suppresses experimental food allergy in mice through induction of
mucosal regulatory T cells. Allergy. 2018;73(4):885-95.
6. Bachmann MF, Kundig TM. Allergen-specific immunotherapy: is it vaccination against toxins after
all? Allergy. 2017;72(1):13-23.
7. van Wijk F, Knippels L. Initiating mechanisms of food allergy: Oral tolerance versus allergic
sensitization. Biomed Pharmacother. 2007;61(1):8-20.
8. Liu EG, Yin X, Swaminathan A, Eisenbarth SC. Antigen-Presenting Cells in Food Tolerance and
Allergy. Front Immunol. 2020;11:616020.
9. Kucuksezer UC, Ozdemir C, Akdis M, Akdis CA. Precision/Personalized Medicine in Allergic Diseases
and Asthma. Arch Immunol Ther Exp (Warsz). 2018;66(6):431-42.
10. Lou W, Wang C, Wang Y, Han D, Zhang L. Responses of CD4(+) CD25(+) Foxp3(+) and IL-10-secreting
type I T regulatory cells to cluster-specific immunotherapy for allergic rhinitis in children. Pediatr
Allergy Immunol. 2012;23(2):140-9.
11. Boonpiyathad T, Sokolowska M, Morita H, Ruckert B, Kast JI, Wawrzyniak M, et al. Der p 1-specific
regulatory T-cell response during house dust mite allergen immunotherapy. Allergy. 2019;74(5):976-
85.
12. Zaleska A, Eiwegger T, Soyer O, van de Veen W, Rhyner C, Soyka MB, et al. Immune regulation by
intralymphatic immunotherapy with modular allergen translocation MAT vaccine. Allergy.
2014;69(9):1162-70.
13. Soyer OU, Akdis M, Ring J, Behrendt H, Crameri R, Lauener R, et al. Mechanisms of peripheral
tolerance to allergens. Allergy. 2013;68(2):161-70.
14. Dunston D, Ashby S, Krosnowski K, Ogura T, Lin W. An effective manual deboning method to prepare
intact mouse nasal tissue with preserved anatomical organization. J Vis Exp. 2013(78).
15. Voynow JA, Fischer BM, Malarkey DE, Burch LH, Wong T, Longphre M, et al. Neutrophil elastase
induces mucus cell metaplasia in mouse lung. American Journal of Physiology-Lung Cellular and
Molecular Physiology. 2004;287(6):L1293-L302.
16. Karupiah G. Type 1 and type 2 cytokines in antiviral defense. Veterinary immunology and
immunopathology. 1998;63(1-2):105-9.

Page 11/19
17. Busch RA, Jonker MA, Pierre JF, Heneghan AF, Kudsk KA. Innate Mucosal Immune System Response
of BALB/c vs C57BL/6 Mice to Injury in the Setting of Enteral and Parenteral Feeding. JPEN J
Parenter Enteral Nutr. 2016;40(2):256-63.
18. Petrovic R, Bufan B, Arsenovic-Ranin N, Zivkovic I, Minic R, Radojevic K, et al. Mouse strain and sex
as determinants of immune response to trivalent influenza vaccine. Life Sci. 2018;207:117-26.
19. Goverse G, Olivier BJ, Molenaar R, Knippenberg M, Greuter M, Konijn T, et al. Vitamin A metabolism
and mucosal immune function are distinct between BALB/c and C57BL/6 mice. European journal of
immunology. 2015;45(1):89-100.
20. Camuesco D, Rodriguez-Cabezas ME, Garrido-Mesa N, Cueto-Sola M, Bailon E, Comalada M, et al.
The intestinal anti-inflammatory effect of dersalazine sodium is related to a down-regulation in IL-17
production in experimental models of rodent colitis. Br J Pharmacol. 2012;165(3):729-40.
21. Melgar S, Karlsson A, Michaelsson E. Acute colitis induced by dextran sulfate sodium progresses to
chronicity in C57BL/6 but not in BALB/c mice: correlation between symptoms and inflammation. Am
J Physiol Gastrointest Liver Physiol. 2005;288(6):G1328-38.
22. Melgar S, Drmotova M, Rehnstrom E, Jansson L, Michaelsson E. Local production of chemokines
and prostaglandin E2 in the acute, chronic and recovery phase of murine experimental colitis.
Cytokine. 2006;35(5-6):275-83.
23. Sasaki S, Ishida Y, Nishio N, Ito S, Isobe K. Thymic involution correlates with severe ulcerative colitis
induced by oral administration of dextran sulphate sodium in C57BL/6 mice but not in BALB/c mice.
Inflammation. 2008;31(5):319-28.
24. Tsuchiya T, Fukuda S, Hamada H, Nakamura A, Kohama Y, Ishikawa H, et al. Role of gamma delta T
cells in the inflammatory response of experimental colitis mice. J Immunol. 2003;171(10):5507-13.
25. Chen X, Oppenheim JJ, Howard OM. BALB/c mice have more CD4+CD25+ T regulatory cells and
show greater susceptibility to suppression of their CD4+CD25- responder T cells than C57BL/6 mice.
J Leukoc Biol. 2005;78(1):114-21.
26. Lee K, Byun J, Kim B, Yeon J, Tai J, Lee SH, et al. TRPV4-Mediated Epithelial Junction Disruption in
Allergic Rhinitis Triggered by House Dust Mites. Am J Rhinol Allergy. 2021;35(4):432-40.
27. Palomares O, Yaman G, Azkur AK, Akkoc T, Akdis M, Akdis CA. Role of Treg in immune regulation of
allergic diseases. European journal of immunology. 2010;40(5):1232-40.
28. van Loosdregt J, Fleskens V, Tiemessen MM, Mokry M, van Boxtel R, Meerding J, et al. Canonical Wnt
signaling negatively modulates regulatory T cell function. Immunity. 2013;39(2):298-310.
29. Warner JR, McIntosh KB. How common are extraribosomal functions of ribosomal proteins? Mol
Cell. 2009;34(1):3-11.
30. Sun XX, DeVine T, Challagundla KB, Dai MS. Interplay between ribosomal protein S27a and MDM2
protein in p53 activation in response to ribosomal stress. J Biol Chem. 2011;286(26):22730-41.
31. Pirkkala L, Nykänen P, Sistonen L. Roles of the heat shock transcription factors in regulation of the
heat shock response and beyond. FASEB journal : official publication of the Federation of American
Societies for Experimental Biology. 2001;15(7):1118-31.
Page 12/19
32. Somasundaram T, Bhat SP. Developmentally dictated expression of heat shock factors: exclusive
expression of HSF4 in the postnatal lens and its specific interaction with alphaB-crystallin heat
shock promoter. J Biol Chem. 2004;279(43):44497-503.
33. Bianchi M, Crinelli R, Giacomini E, Carloni E, Radici L, Scarpa ES, et al. A negative feedback
mechanism links UBC gene expression to ubiquitin levels by affecting RNA splicing rather than
transcription. Sci Rep. 2019;9(1):18556.
34. Peyrou M, Cereijo R, Quesada-Lopez T, Campderros L, Gavalda-Navarro A, Linares-Pose L, et al. The
kallikrein-kinin pathway as a mechanism for auto-control of brown adipose tissue activity. Nat
Commun. 2020;11(1):2132.
35. Pelosi A, Menardy F, Popa D, Girault JA, Herve D. Heterozygous Gnal Mice Are a Novel Animal Model
with Which to Study Dystonia Pathophysiology. J Neurosci. 2017;37(26):6253-67.
36. Laurin N, Ickowicz A, Pathare T, Malone M, Tannock R, Schachar R, et al. Investigation of the G
protein subunit Galphaolf gene (GNAL) in attention deficit/hyperactivity disorder. J Psychiatr Res.
2008;42(2):117-24.
37. Fang C, Zhong R, Qiu C, Zou BB. The Prognostic Value of GNG7 in Colorectal Cancer and Its
Relationship With Immune Infiltration. Front Genet. 2022;13:833013.
38. Ye Y, Sun Z, Guo A, Song LS, Grobe JL, Chen S. Ablation of the GNB3 gene in mice does not affect
body weight, metabolism or blood pressure, but causes bradycardia. Cell Signal. 2014;26(11):2514-
20.
39. Sheu SY, Handke S, Brocker-Preuss M, Gorges R, Frey UH, Ensinger C, et al. The C allele of the GNB3
C825T polymorphism of the G protein beta3-subunit is associated with an increased risk for the
development of oncocytic thyroid tumours. J Pathol. 2007;211(1):60-6.
40. Lopez-Leon S, Janssens AC, Gonzalez-Zuloeta Ladd AM, Del-Favero J, Claes SJ, Oostra BA, et al.
Meta-analyses of genetic studies on major depressive disorder. Mol Psychiatry. 2008;13(8):772-85.
41. Park CR, You DJ, Park S, Mander S, Jang DE, Yeom SC, et al. The accessory proteins REEP5 and
REEP6 refine CXCR1-mediated cellular responses and lung cancer progression. Sci Rep.
2016;6:39041.
42. Francis JN, Jacobson MR, Lloyd CM, Sabroe I, Durham SR, Till SJ. CXCR1+CD4+ T cells in human
allergic disease. J Immunol. 2004;172(1):268-73.
43. Fossati-Jimack L, Ling GS, Baudino L, Szajna M, Manivannan K, Zhao JC, et al. Intranasal peptide-
induced tolerance and linked suppression: consequences of complement deficiency. Immunology.
2015;144(1):149-57.

Figures

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Figure 1

General flow diagram of experiment

Figure 2

Trends in the number of sneezes at 0th week, 4th week to 15th week for each group.

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Figure 3

Changes in serum OVA-specific antibodies (IgE, IgG1, IgG2a) in each group. n=12 for each group in the
first eight weeks and n=6 for each group from 9th week to 15th week.

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Figure 4

Changes in spleen culture supernatant cytokines (IL-4, IFN-γ, IL-10) in each subgroup. *, P<0.05. **,
P<0.01.

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Figure 5

Histopathological staining of mouse nasal mucosa. A. HE staining of the nasal mucosa, eosinophils are
indicated by arrows. B. PAS staining of the nasal mucosa and goblet cells are indicated by arrows. (1) 8th
BALB/c PBS group (2) 8th BALB/c OVA group, (3) 8th C57BL/6 PBS group, (4) 8th C57BL/6 OVA group,
(5) 15th BALB/c PBS group (6) 15th BALB/c OVA group, (7) 15th C57BL/6 PBS group (8) 15th C57BL/6
PBS group. C. Average number of eosinophils per visual field D. Mean grading of goblet cells per visual
field. *, P<0.05. **, P<0.01.

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Figure 6

A. Immunohistochemical results of eosinophil chemotactic factor (eotaxin) in nasal mucosa (40×); (1)
8th BALB/c PBS group (2) 8th BALB/c OVA group, (3) 8th C57BL/6 PBS group, (4) 8th C57BL/6 OVA
group, (5) 15th BALB/c PBS group (6) 15th BALB/c OVA group, (7) 15th C57BL/6 PBS group (8) 15th
C57BL/6 PBS group. same below. B. Immunohistochemical results of TGF-β in nasal mucosa (40×). C.
Immunohistochemical results of IL-10 in nasal mucosa (40×). D. Eotaxin expression in each group. E.
TGF-β expression in each group. F. IL-10 expression in each group. **, p<0.01. *, p<0.05.

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Figure 7

A. Differential gene volcano plot. (a) 15th BALB/c OVA group vs 15th BALB/c PBS group.(b) 15th
C57BL/6 OVA group vs 15th C57BL/6 PBS group.(c) A. 15th BALB/c OVA group vs 15th C57BL/6 OVA
group. B. Results of GO-KEGG analysis. (a) 15th BALB/c OVA group vs 15th BALB/c PBS group.(b) 15th
C57BL/6 OVA group vs 15th C57BL/6 PBS group.(c) 15th BALB/c OVA group vs 15th C57BL/6 OVA
group.(d) Results of mice at 15th week compared to 8th week. C. Results of GSEA analysis. D. The top ten
core genes were analyzed.

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