pathogens
ArticleSuppressive Effect of Lactococcus lactis subsp. cremoris
YRC3780 on a Murine Model of Japanese Cedar Pollinosis
Kenji Uchida 1, *, Kenichi Iida 1 , Ikumi Fujioka 1 , Satoshi Hachimura 2 and Osamu Kaminuma 3, *
1
2

3

*

R&D Center, Yotsuba Milk Products Co., Ltd., Sapporo 061-1264, Japan
Research Center for Food Safety, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Tokyo 113-8657, Japan
Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University,
Hiroshima 734-8553, Japan
Correspondence: uchida_k@yotsuba.co.jp (K.U.); okami@hiroshima-u.ac.jp (O.K.);
Tel.: +81-11-377-5561 (K.U.); +81-82-257-5819 (O.K.)

Abstract: Accumulating evidence suggests that Lactococcus lactis subsp. cremoris YRC3780 isolated
from kefir has the potential to alleviate allergic responses. Herein, we investigated the effect ofYRC3780 on a murine model of Japanese cedar pollinosis (JCP). BALB/c mice immunized with cedarpollen extract (CPE) exhibited an increase in serum immunoglobulin E and developed nasal inflammatory responses including sneezing, nasal hyperresponsiveness, and nasal eosinophil accumulation
upon intranasal allergen challenge. These responses were suppressed by the oral administration of
YRC3780, although the effects on CPE-induced sneezing response and eosinophil infiltration were notstatistically significant. Total fecal microbiota diversity was not affected by allergen immunization andchallenge or by YRC3780 administration. However, the abundances of Bifidobacteriales, Veillonellaceae,
Lactococcus, and Lactococcus lactis were larger and that of Bacteroides was smaller in YRC3780-treatedmice compared with those in CPE-challenged and YRC3780-untreated mice. Our findings suggest
the usefulness of YRC3780 for alleviating JCP.
Citation: Uchida, K.; Iida, K.; Fujioka,
I.; Hachimura, S.; Kaminuma, O.
Suppressive Effect of Lactococcus lactis

Keywords: allergic rhinitis; eosinophil; gastrointestinal microbiota; immunoglobulin E; Japanesecedar pollinosis; Lactococcus lactis subsp. cremoris YRC3780; nasal hyperresponsivenesssubsp. cremoris YRC3780 on a Murine
Model of Japanese Cedar Pollinosis.
Pathogens 2022, 11, 1347. https://doi.org/10.3390/pathogens11111347

1. Introduction

Academic Editor: Tingfan Leung

In recent years, the number of people with allergic diseases has been increasing annually worldwide. Specifically in Japan, the prevalence of allergic rhinitis (AR) as of 2019 was
49.2%, representing an increase of about 10% in 10 years [1]. Approximately 80% of AR patients in Japan (38.8% of the total population) suffer from Japanese cedar pollinosis (JCP) [1].
The current treatment of AR including JCP consists mainly of medications that relieve
symptoms such as sneezing, rhinorrhea, and nasal congestion, and downregulate nasal
inflammation. Subcutaneous and recently approved sublingual immunotherapies have thepotential to modulate disease activity. However, prescribing medicines for the multitudeof patients with AR raises concerns about substantial costs and morbidity from serious
side effects [2]. Inspired by the successful oral application of bioproducts, particularly insublingual immunotherapy, we became interested in the potential of food products thatexhibit immunomodulatory effects to prevent the development and alleviate the symptomsof AR. Recent reports suggest that in addition to Benifuuki tea [3] and fermented fruit juice
beverages [4], several foods containing lactic and acetic acid bacteria and bifidobacteria
display efficacy in AR including JCP [5–15]. For example, based on double-blind placebocontrolled trials for JCP, Lactobacillus paracasei KW3110, Lactobacillus acidophilus L-55 andL-92, Lactobacillus casei Shirota, and Bifidobacterium longum BB536 have been reported to
improve the patients’ symptoms to some degree [5–10].

Received: 22 September 2022
Accepted: 12 November 2022
Published: 14 November 2022
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https://www.mdpi.com/journal/pathogens

Pathogens 2022, 11, 1347

2 of 10Lactococcus lactis subsp. cremoris YRC3780 (hereafter YRC3780) is isolated from kefir, a
traditional fermented milk product habitually consumed in the Caucasus region. YRC3780
has exhibited immunomodulatory effects in several animal studies, even in its heat-killed
form. For example, in a bowel cancer-implanted mouse model, the serum IL-2 leveland natural killer cell activity increased after oral live YRC3780 administration [16]. The
allergen-induced production of IL-4 was suppressed, and IL-12 production was enhanced
by co-culture of immunized mouse splenocytes with YRC3780, suggesting a beneficial effecton the Th1/Th2 balance. In an atopic dermatitis-like murine model of skin inflammation,oral heat-killed YRC3780 administration alleviated allergen-induced dermal responses
and decreased the expression of IL-4 and IL-33 in CD4+ T cells in the Peyer’s patches
and draining lymph nodes, respectively [17]. The effectiveness of live YRC3780 in humanallergy as well has been suggested in several clinical studies [18,19]. In those rodent andhuman studies, no harmful effect of YRC3780 was reported.
In this study, we used an experimental model of JCP [20] in which mice are immunized
and challenged with cedar pollen extract (CPE) to evaluate the efficacy of YRC3780. Thismurine JCP model (Figure 1) exhibits allergic responses similar to those in patients with
JCP, i.e., elevation of the serum immunoglobulin E (IgE) level, CPE-induced sneezing
response, eosinophil accumulation in the nasal mucosa, and nasal hyperresponsiveness(NHR), a pathophysiological feature by which AR symptoms are augmented [21]. We
previously demonstrated the efficacy of dexamethasone and sublingual immunotherapy
in the similar allergen-immunized and -challenged mouse models [22,23]. The effect ofheat-killed YRC3780 on these allergen-induced responses was investigated by monitoring
fluctuations in fecal microbiota.

Figure 1. Experimental schedule for the mouse model of Japanese cedar pollinosis. BALB/c mice
immunized by intraperitoneal (i.p.) injection of cedar pollen extract (CPE) plus alum were challenged
by intranasal (i.n.) allergen injection. Mice were fed with standard rodent diet, CE-2 (Crea-Japan Inc.,Tokyo, Japan), supplemented with or without Lactococcus lactis subsp. cremoris YRC3780 (YRC3780)
throughout the experimental period. Blood and feces sampling and nasal response assessment were
performed on the indicated days. The detailed experimental procedure is described in the Section 2.

2. Materials and Methods
2.1. Preparation of YRC3780
YRC3780 was isolated by Yotsuba Milk Products Co., Ltd. (Hokkaido, Japan), inoculated into M17 broth (Merck KGaA, Darmstadt, Germany), and cultivated for 16 h at 30 ◦ C.
We confirmed in a separate experiment that the resulting YRC3780 showed approximately
3–4 × 1011 colony-forming units/g. Then the cells were harvested by 8000 rpm centrifugation at 4 ◦ C for 10 min, washed twice, and resuspended in sterilized distilled water.

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Following the heat treatment at 100 ◦ C for 10 min, the cells were lyophilized and mixed
with the standard rodent diet (CE-2, Crea-Japan Inc., Tokyo, Japan) at the concentration of
0.2% (w/w).
2.2. Administration of YRC3780 to MiceSeven-week-old specific-pathogen-free female BALB/c mice were purchased from
CLEA-Japan Inc. The mice received Milli-Q water and CE-2 under controlled conditionsof temperature (25 ± 2 ◦ C), humidity (50 ± 10%), and light (12 h light–dark cycle). Afterthe habituation period of at least 7 days, the YRC3780 group received CE-2 containing
0.2% YRC3780 throughout the experimental period (Figure 1). Due to the requirementof the stable quality YRC3780 in CE-2 for 45-day consecutive administration, we used its
heat-killed form in this study.
2.3. Mouse JCP Model
The experimental murine model of JCP was developed as previously described [20]with minor modifications (Figure 1). In brief, BALB/c mice were immunized with an
intraperitoneal injection of 10 µg CPE (Cedar Pollen Extract-cj, Cosmo bio, Tokyo, Japan)
emulsified with 0.5 mg alum (Imject Alum; Thermo Fisher Scientific Inc., Chicago, IL, USA)on days 0, 7, 14, and 21 (Figure 1). On days 35–38 and 42–45, the mice were challenged
once daily with the two-time intranasal administration of 5.75 µL per nostril of 0.1 mg/mL
CPE solution with a 5 min interval. The Normal group was prepared for negative control
with the intraperitoneal and intranasal injection of saline in the same manner as CPE
immunization and challenge (Figure 1).
Immediately after the last allergen challenge, nasal symptoms were evaluated bycounting the number of sneezes for 5 min. After 6 h, nasal hyperresponsiveness (NHR)
was evaluated by counting the number of sneezes for 5 min following the intranasal
administration of 5 µL per nostril of 100 mM histamine (Nacalai Tesque Inc., Kyoto, Japan)
solution. Immediately after the NHR assessment, nasal lavage was performed as described
previously [23]. The number of leukocytes in the nasal lavage fluid (NALF) was counted
using a hemocytometer. Through the microscopic examination of the cytocentrifugedpreparation stained with Diff-Quik (Sysmex, Kobe, Japan), differential cell classification
based on morphologic criteria was performed for at least 200 cells.
2.4. Measurement of Serum IgE Level
The allergen-induced IgE response was evaluated by measuring the total serum
IgE. In order to reduce the invasive influence on the mice, peripheral blood collection
was performed on day 44, which was close to, but different from, the days for nasal
response assessment and feces sampling. The serum IgE level was assessed using an
LBIS Mouse IgE ELISA Kit (Fujifilm Wako Pure Chemical, Osaka, Japan) according to the
manufacturer’s instructions.
2.5. Microbiota Analysis
Metagenomic next-generation sequencing analysis was performed for investigating
fecal microbiota. Feces collected from each mouse on days 0 and 42 was applied for
DNA extraction using a ZymoBIOMICS DNA Miniprep Kit (ZYMO Research, Irvine, CA,
USA) according to the manufacturer’s instructions. The V4 region of 16S rDNA wasamplified with KAPA Hifi HS ReadyMix (NIPPON Genetics, Tokyo, Japan), F515 primer(50 -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-30 ),and R806 primer (50 -TCGTCGGCAGCGTCAGATGTGTATAAGACAGGTGYCAGCMGCC
GCGGTAA-30 ), and then purified using AMPure XP (Beckman Coulter, Brea, CA, USA).
The DNA library was prepared using a Nextera XT DNA Library Preparation Kit (Illumina
Inc., San Diego, CA, USA), and then analyzed on iSeq100 (Illumina Inc.). The resulting
FastQ sequence was applied for the OTU and quantitative insights into microbial ecology
analyses in FASMAC (Kanagawa, Japan). The taxonomic classification was performed

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4 of 10based on the Greengenes database provided by Lawrence Berkeley National Laboratory(Berkeley, CA, USA) and the SILVA database (SILVA131) provided by the Max Planck
Institute for Marine Microbiology and Jacobs University (Bremen, Germany). Changes
in the diversity of the microbiota at day 42 from the start of the experiment were evaluated by assessing the OTU number, Chao1 index, and Shannon diversity index, and thecomposition of several bacterial species.
2.6. Statistical Analysis
The data are presented as the mean ± standard error of the mean. The difference
among three groups was analyzed by using the Kruskal–Wallis test with Dunnet’s multiple
comparison tests in BellCurve for Excel (Social Survey Research Information Co., Tokyo,
Japan). A p-value less than 0.05 was considered to indicate statistical significance.
3. Results
3.1. Effect of YRC3780 on Serum IgE Response
The murine JCP model (Figure 1) was established by immunization and challengewith CPE as described previously [20]. The administration of YRC3780 was achieved by
the free oral intake of diet containing 0.2% YRC3780. The allergen-induced IgE response
was evaluated by assessing the serum IgE level in the peripheral blood. The serum IgElevel was significantly elevated in allergen-immunized and -challenged mice (Control
group) in comparison with non-immunized and -challenged mice (Normal group). The
allergen-induced serum IgE response was significantly suppressed in YRC3780-treatedmice (YRC3780 group) (Figure 2).

Figure 2. Effect of YRC3780 on the serum immunoglobulin E (IgE) response. Cedar pollen extractimmunized mice were challenged by intranasal allergen injection with (YRC3780) or without (Control)
oral administration of YRC3780. Non-immunized and -challenged mice (Normal) were the negative
control. The serum IgE level was evaluated on day 44, as shown in Figure 1. Data are expressed asmeans ± standard error of the mean of 12 mice in each group. * p < 0.05, ** p < 0.01.

3.2. Effect on Nasal Responses
The effect of YRC3780 on allergen-induced nasal responses was investigated. Intranasal CPE administration evoked a significant increase in the frequency of sneezes in
the Control group. The allergen-induced sneezing response was slightly, but not significantly, alleviated by YRC3780 administration (Figure 3a, p = 0.08). Our previous study
demonstrated that the allergen-induced sneezing response in this model represents T celldependent nasal NHR rather than IgE-dependent mast cell degranulation [24]. Therefore,
the effect of YRC3780 on allergen-induced NHR was examined. The histamine-evokedsneezing response was significantly augmented in the Control group, indicating the development of NHR. The NHR response was significantly suppressed by YRC3780 administration (Figure 3b).

Pathogens 2022, 11, 1347

5 of 10Figure 3. Effect of YRC3780 on allergen-induced sneezing and nasal hyperresponsiveness (NHR).
Cedar pollen extract-immunized mice were challenged by intranasal allergen injection with (YRC3780)
or without (Control) oral administration of YRC3780. Non-immunized and -challenged mice (Normal)
were the negative control. The allergen-induced sneezing response was evaluated by counting thefrequency of sneezes immediately after the last allergen challenge (a). Allergen-induced NHR was
evaluated by counting the frequency of histamine-induced sneezes 6 h after the last allergen challenge(b). Data are expressed as means ± standard error of the mean of 12 mice in each group. * p < 0.05,** p < 0.01.

3.3. Effect on Eosinophil Accumulation in the Nasal Mucosa
The allergen-induced accumulation of eosinophils in the nasal mucosa was evaluatedby the ratio of eosinophils to total inflammatory cells and the number of eosinophils
recovered in nasal lavage fluid (NALF). CPE challenge to immunized mice evoked asignificant increase in the ratio of eosinophils in NALF. The allergen-induced increasein the NALF eosinophil ratio was suppressed by YRC3780 administration (Figure 4a).
Essentially the same, but not significant, tendency was also observed in the number of
NALF eosinophils.

Figure 4. Effect of YRC3780 on allergen-induced nasal eosinophil accumulation. Cedar pollen
extract-immunized mice were challenged by intranasal allergen injection with (YRC3780) or without(Control) oral administration of YRC3780. Non-immunized and -challenged mice (Normal) were thenegative control. The ratio (a) and number (b) of eosinophils in the nasal lavage fluid (NALF) was
evaluated 6 h after the last allergen challenge. Data are expressed as means ± standard error of themean of 11 (Control) or 12 (YRC3780, Normal) mice. * p < 0.05, ** p < 0.01.

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6 of 103.4. Influence on Fecal Microbiota
3.4.1. Diversity of Microbiota
The change in the diversity of fecal microbiota during the experimental period was
examined, as shown in Figure 1. The basal levels of operational taxonomic unit (OTU)
number (3914 ± 608) and indicators of α-diversity such as Chao1 index (15678 ± 1615) and
Shannon diversity index (4.21 ± 0.10) were comparable to those demonstrated in previous
studies [25]. A slight increase in the OTU number was observed in the Normal group at
day 42 from the start of the experiment. CPE immunization and challenge tended to
upregulate the OTU number, but not significantly, regardless of YRC3780 administration(Figure 5a). Probably due to the spontaneous fluctuation of the OTU number, a slight increment in the Chao1 index, and conversely, a reduction in the Shannon diversity index wereobserved in the Normal group, although these parameters were not affected by allergen or
YRC3780 treatment (Figure 5b,c). Since these differences were not statistically significant,the negligible contribution of YRC3780, if any, to the total diversity of gastrointestinal
microbiota was suggested.

Figure 5. Effects of YRC3780 on fecal microbiota diversity. Cedar pollen extract-immunized mice werechallenged by intranasal allergen injection with (YRC3780) or without (Control) oral administration of
YRC3780. Non-immunized and -challenged mice (Normal) were the negative control. The microbiota
composition in feces was analyzed on days 0 and 42, as indicated in Figure 1. Changes in theoperational taxonomic unit (OTU) number (a), Chao1 index (b), and Shannon diversity index (c) fromthe start of the experiment were calculated. Data are expressed as mean ± standard error of the meanof 12 mice in each group.

3.4.2. Bacterial Species
The influence of oral YRC3780 administration on several fecal bacterial species wasevaluated by analyzing the change in microbiota composition during the experimental
period. Spontaneous decreases in Bifidobacteriales and Bacteroides and an increase in Veillonellaceae were observed even in the Normal group at day 42 from the start of the experiment(Figure 6). Immunization and challenge with CPE significantly decreased the compositionof Veillonellaceae, Lactococcus, and Lactococcus lactis. In the Control group, the compositionof Bifidobacteriales, Veillonellaceae, Lactococcus, and Lactococcus lactis was significantly higher
and that of Bacteroides was lower in the YRC3780 group. Even when compared with theNormal group, the YRC3780 group showed significantly higher abundances of Bifidobacteriales, Lactococcus, and Lactococcus lactis and lower levels of Bacteroides (Figure 6), suggesting
the existence of the allergen-independent effect of YRC3780. In contrast, the CPE-mediateddownmodulation of Veillonellaceae composition recovered with YRC3780 administration;
this outcome was in parallel with the effect of YRC3780 on allergen-induced serum IgE,
NHR, and nasal eosinophil responses.

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7 of 10Figure 6. Effect of YRC3780 on changes in fecal bacterial species. Cedar pollen extract-immunized
mice were challenged by intranasal allergen injection with (YRC3780) or without (Control) oral administration of YRC3780. Non-immunized and -challenged mice (Normal) were the negative control.
The microbiota composition in feces was analyzed on days 0 and 42, as indicated in Figure 1. Thechange ratios in the abundance of Bifidobacteriales (a), Bacteroides (b), Veillonellaceae (c), Lactococcus (d),and Lactococcus lactis (e) from the start of experiment were calculated. Data are expressed asmean ± standard error of the mean of 12 mice in each group. * p < 0.05, ** p < 0.01, *** p < 0.001.

4. Discussion
The present study demonstrated that YRC3780 has the potential to alter the pathogenesis of AR by downregulating IgE production, NHR development, and nasal inflammation
with decreasing eosinophil ratio. Our findings suggest a possible relationship between
these beneficial effects of YRC3780 and its influence on several gastrointestinal bacterial
species, despite a lack of effect on total microbiota diversity.
The downmodulating effect of YRC3780 on IgE synthesis is supported by our previous
study demonstrating decreased IL-4 expression in splenocytes in vitro and in Peyer’s patch
CD4+ T cells in vivo with YRC3780 treatment [17]. Since the mice were immunized with
CPE, the total IgE response was expected to reflect a CPE-specific IgE response. Therefore,
this effect is probably related to the tendency toward suppression of the allergen-induced
sneezing response principally caused by IgE-mediated mast cell degranulation. As further
proof of concept, we recently demonstrated the suppression of IgE- and mast cell-dependent
itchy eyes in adult patients with AR in a randomized, double-blind, placebo-controlled,
8-week trial of drinkable yogurt containing YRC3780 [19]. A decreased allergen-induced
serum IgE response in CPE-immunized mice that received a Lactobacillus helveticus strain
has also been reported [26].
The allergen-induced sneezing response is related to NHR. In our murine AR model,
the development of NHR was largely dependent on allergen-specific T cells. The augmentation of the histamine-induced sneezing response in allergen-immunized and -challenged
mice, as shown in this study, was suppressed by depleting CD4+ T cells. Furthermore, micePathogens 2022, 11, 1347

8 of 10transplanted with in vitro-differentiated T cell subsets developed significant NHR uponallergen challenge independent of IgE- and mast cell-related responses [24]. These observations indicate that CD4+ T cells are likely one of the primary targets of YRC3780-mediated
NHR suppression. In addition to the ability of YRC3780 to suppress IL-4 activity, we
recently demonstrated that YRC3780 augments the production of IFN-γ in the co-culture of
antigen-presenting cells and CD4+ T cells. In this regard, YRC3780 potentially improves theTh1/Th2 balance. A similar effect has been reported in studies of other lactic acid bacteria.
In a murine model of birch pollen allergy, Repa et al. demonstrated the upregulation of
IgG2a and IFN-γ production by Lactococcus lactis and Lactobacillus plantarum administration [27]. The downregulation of allergen-induced peritoneal eosinophilia and increased
serum IgG2a/IgG1 ratio in CPE-immunized mice by administrating a Lactobacillus gasseristrain has also been reported [28].
Allergen-induced local eosinophil accumulation is also mediated by CD4+ T cells. We
demonstrated in an earlier study [29] that the airway eosinophilia in allergen-specific T celltransferred mice was abrogated by anti-IL-5 neutralizing antibody treatment. In addition,
administering a Lactobacillus paracasei strain to perinatal maternal mice decreased birch
pollen-induced bronchial inflammation with eosinophil accumulation and IL-5 production
in offspring [30]. We need to confirm the effect of YRC3780 on IL-5 synthesis. Wang et al.
demonstrated the reduced expression of IL-33, another eosinophil-activating cytokine, inthe draining lymph nodes of atopic dermatitis model mice treated with YRC3780 [17].
In addition to T cell-derived factors responsible for NHR development, the mechanisms
underlying the effect of YRC3780 on CD4+ T cells remain to be explored.
The modification of gastrointestinal microbiota by several lactic acid bacteria, even in
their heat-killed form, as in this study, has been reported [31,32]. In addition to live microbebased probiotics, the concept of postbiotics has recently attracted attention for maintaining
gut conditions. Postbiotics based on non-viable microorganisms and bacterial-free extracts,
as we used in this study, provide a benefit to the host by enhancing the bioactivities
of probiotics [33]. Although we did not observe an effect on total microbiota diversity,
the composition of several bacterial strains was affected by YRC3780 administration. In
particular, the recovery of Veillonellaceae from allergen-induced downregulation is linked
to YRC37800 s effect on AR responses. A direct effect on allergic responses has not beenreported; however, a strong association of gastrointestinal Veillonellaceae content with foodsensitization conditions [34] and atopy [35] has been reported. Lynch et al. demonstrated
the correlation of bronchial asthma development with the presence of Veillonellaceae inenvironment house dust [36]. The direct effect of Veillonellaceae on allergic responses
deserves further examination.
In conclusion, based on the suppressive effect on allergen-induced serum IgE production, NHR, and nasal eosinophilia, the oral intake of YRC3780, even in its heat-killed
form, is a promising addition to the currently available medication for AR including CPEmediated JCP. Additional research is needed to further delineate the usefulness of YRC3780,
and its underlying mechanisms, including gastrointestinal microbiota modification.
Author Contributions: Conceptualization, K.U. and O.K.; methodology, K.U. and O.K.; software,
K.U., K.I. and I.F.; validation, O.K.; formal analysis, K.U., K.I. and I.F.; investigation, K.U., K.I. andI.F.; resources, K.U., K.I. and I.F.; data curation, K.U., K.I. and I.F.; writing—original draft preparation,
K.U.; writing—review and editing, K.U., K.I., I.F., S.H. and O.K.; visualization, K.U.; supervision,
O.K.; project administration, K.U. and O.K.; funding acquisition, K.U. All authors have read andagreed to the published version of the manuscript.
Funding: This study was funded by Yotsuba Milk Products Co., Ltd.
Institutional Review Board Statement: All animal experiments were performed following theguidelines and approval of the Institutional Animal Care and Use Committee of Hiroshima University(Approval Code: A20-25).
Informed Consent Statement: Not applicable.

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Data Availability Statement: Not applicable.
Conflicts of Interest: K.U., K.I. and I.F. are employees of Yotsuba Milk Products Co., Ltd.

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