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Intranasal Immunization of Pneumococcal pep27 Mutant Attenuates Allergic and Inflammatory Diseases by Upregulating Skin and Mucosal Tregs

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Conventional immunization methods such as intramuscular injections lack effective mucosal protection against pathogens that enter through the mucosal surfaces. Moreover, conventional therapy often leads to adverse events and compromised immunity, followed by complicated outcomes, leading to the need to switch to other options. Thus, a need to develop safe and effective treatment with long-term beneficial outcomes to reduce the risk of relapse is mandatory. Mucosal vaccines administered across mucosal surfaces, such as the respiratory or intestinal mucosa, to prompt robust localized and systemic immune responses to prevent the public from acquiring pathogenic diseases. Mucosal immunity contains a unique immune cell milieu that selectively identify pathogens and limits the transmission and progression of mucosal diseases, such as allergic dermatitis and inflammatory bowel disease (IBD). It also offers protection from localized infection at the site of entry, enables the clearance of pathogens on mucosal surfaces, and leads to the induction of long-term immunity with the ability to shape regulatory responses. Regulatory T (Treg) cells have been a promising strategy to suppress mucosal diseases. To find advances in mucosal treatment, we investigated the therapeutic effects of intranasal pep27 mutant immunization. Nasal immunization protects mucosal surfaces, but nasal antigen presentation appears to entail the need for an adjuvant to stimulate immunogenicity. Here, a novel method is developed to induce Tregs via intranasal immunization without an adjuvant to potentially overcome allergic diseases and gut and lung inflammation using lung–gut axis communication in animal models. The implementation of the pep27 mutant for these therapies should be preceded by studies on Treg resilience through clinical translational studies on dietary changes.
This content is subject to copyright.
Citation: Iqbal, H.; Rhee, D.-K.
Intranasal Immunization of
Pneumococcal pep27 Mutant
Attenuates Allergic and Inflammatory
Diseases by Upregulating Skin and
Mucosal Tregs. Vaccines 2024,12, 737.
https://doi.org/10.3390/vaccines
12070737
Academic Editor: Jingyou Yu
Received: 13 June 2024
Revised: 29 June 2024
Accepted: 1 July 2024
Published: 3 July 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Review
Intranasal Immunization of Pneumococcal pep27 Mutant
Attenuates Allergic and Inflammatory Diseases by Upregulating
Skin and Mucosal Tregs
Hamid Iqbal 1,2 and Dong-Kwon Rhee 2, *
1Department of Pharmacy, CECOS University, Peshawar 25000, Pakistan; hamid.bukhari11@gmail.com
2School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea
*Correspondence: dkrhee@skku.edu
Abstract: Conventional immunization methods such as intramuscular injections lack effective mu-
cosal protection against pathogens that enter through the mucosal surfaces. Moreover, conventional
therapy often leads to adverse events and compromised immunity, followed by complicated out-
comes, leading to the need to switch to other options. Thus, a need to develop safe and effective
treatment with long-term beneficial outcomes to reduce the risk of relapse is mandatory. Mucosal
vaccines administered across mucosal surfaces, such as the respiratory or intestinal mucosa, to prompt
robust localized and systemic immune responses to prevent the public from acquiring pathogenic
diseases. Mucosal immunity contains a unique immune cell milieu that selectively identify pathogens
and limits the transmission and progression of mucosal diseases, such as allergic dermatitis and
inflammatory bowel disease (IBD). It also offers protection from localized infection at the site of entry,
enables the clearance of pathogens on mucosal surfaces, and leads to the induction of long-term
immunity with the ability to shape regulatory responses. Regulatory T (Treg) cells have been a
promising strategy to suppress mucosal diseases. To find advances in mucosal treatment, we investi-
gated the therapeutic effects of intranasal pep27 mutant immunization. Nasal immunization protects
mucosal surfaces, but nasal antigen presentation appears to entail the need for an adjuvant to stimu-
late immunogenicity. Here, a novel method is developed to induce Tregs via intranasal immunization
without an adjuvant to potentially overcome allergic diseases and gut and lung inflammation using
lung–gut axis communication in animal models. The implementation of the pep27 mutant for these
therapies should be preceded by studies on Treg resilience through clinical translational studies on
dietary changes.
Keywords: Treg cells; nasal vaccine; mucosal tolerance; allergy; inflammatory diseases
1. Introduction
Mucosal vaccines administer elicit mucosal immunity at the site of administration
and in certain other mucosal compartments depending on the routes of administration.
Mucosal vaccines can target specific mucosal surfaces, such as respiratory, genital, or
intestinal mucosa. Both nasal and sublingual vaccines can elicit mucosal immunity in the
upper and lower respiratory tract, stomach, and small intestine. Oral, rectal, and vaginal
vaccines induce mucosal immunity in the GI tract, colon, and rectum [
1
4
]. Furthermore,
mucosal immunization vaccines offer several other advantages over traditional systemic
vaccination by inducing higher levels of antibodies and protecting mucosal surfaces from
pathogen infection [
3
,
4
]. With the advent of COVID-19, it has become even more important
to block pathogens at the nasal mucosa entrance. Therefore, to prevent the colonization
of respiratory pathogens, the need for nasal vaccines is a prerequisite to overcoming
conventional injectable vaccines [
5
]. Nasal immunization is considered to be the most
effective method of inducing mucosal immunity in the nasopharynx, lungs, and vagina [
6
],
but whether or not it can stimulate skin and intestinal mucosal immunity remains unknown.
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Vaccines 2024,12, 737 2 of 16
Most antigens are not sufficient to induce mucosal immunity and require immune-
boosting adjuvants such as cholera toxin and heat-labile enterotoxin. However, a clinical
study found that an inactivated nasal influenza vaccine containing adjuvants caused facial
paralysis (Bell’s palsy) in some people [
7
]. Moreover, mucosal adjuvants can impair the
olfactory system of mice [
8
]. Therefore, nasal immunization is preferred to be administered
without adjuvants to avoid pathological conditions.
Current treatments for allergic dermatitis, rhinitis, asthma, and inflammatory bowel
disease (IBD) do not offer a cure, but provide temporary relief;
β
-2 agonists and inhaled cor-
ticosteroids can be used for mild asthma symptoms, while antibodies to type 2-dependent
cytokines (IL-4, IL-5, and IL-13) can be used for severe allergies [
9
]. However, these
treatments often lead to drug resistance or side effects, resulting in a switch to other
therapies [10,11]. Therefore, safe and highly effective treatments need to be developed.
Regulatory T (Treg) cells have been known to suppress inflammatory responses
[12,13]
.
Treg cells can be utilized to maintain immune homeostasis by relieving excessive inflam-
mation or preventing autoimmunity after a pathogenic event. In murine models, Tregs
can regulate both low- and high-level inflammation caused by type 2-hypocytokine and
type 2-hypercytokine secretion, respectively, and in human cells, they can alleviate allergic
airway inflammation [
9
]. Non-toxic Treg cells on the oral and nasal mucosal surfaces are
induced similarly to cells in the gut. Tolerance of intestinal Treg is induced only when
low amounts of antigen are delivered, and at high antigen doses, anergy is induced [
14
].
Respiratory tolerance has mechanisms similar to intestinal oral tolerance mechanisms [
15
],
but it is not known whether or not nasal and mucosal tolerance can be regulated within the
nasal cavity.
2. Pneumococcal Pep27 Induction during Invasion and Lack of Sepsis Induction by
pep27 Mutant
Streptococcus pneumoniae (pneumococcus) is carried asymptomatically in the nasophar-
ynx of healthy individuals, and this serves as a major reservoir for pneumococcal infec-
tions [
16
]. Pneumococcus causes various potentially life-threatening infections such as
pneumonia, bacteremia (sepsis), and meningitis [17]. A prerequisite for invasive pneumo-
nia is that pneumococci must colonize the nasopharynx before they can progress to invasive
pneumonia and disseminate to the lung, bloodstream, and central nervous system [
18
]. In
pneumococci, bacterial lysis releases cell wall components and pneumolysin toxin, and
subsequently triggers pro-inflammatory responses. Moreover, mutations in the major
autolysin (LytA) reduce pneumococcal virulence [19].
Pep27 is an effector molecule of the vncRS operon that mediates vancomycin resistance
and autolysis [
20
,
21
]. However, via microarray analysis, we discovered that a number
of pneumococcal genes were induced upon invasion of the human lung cell line A549.
We confirmed that these target genes were indeed induced upon invasion of A549 cells
via real-time PCR, which demonstrated that not only pep27, but also vncR and vncS were
activated via pneumococcal infection. Furthermore, when we constructed 15 mutants of
the genes induced during A549 invasion and tested them for attenuation of cytotoxicity
in vitro
after infection with A549 cells, we confirmed reduced toxicity in mice via intranasal
infection (pneumonia model) or intraperitoneal injection (sepsis model). Of those genes,
the pep27 gene of the vncRS operon was more prominently induced than normal controls
(Table 1). However, the most significantly induced gene was always pep27, and the pep27
mutant was found to have the least toxicity and vaccine efficacy. The lysis-resistant pep27
mutant (
pep27) gives rise to reduced cytotoxicity to host cells, resulting in decreased
inflammation and death [
22
,
23
]. The
pep27 mutant could be a potentially useful mutation
for use in an inexpensive live-attenuated vaccine that could be used to elicit mucosal
immunity and attenuate virulence. Based on these features, we immunized the mice three
times, once/week, and measured the relevant parameters for experimental purposes, as
shown in Figure 1. Thus,
pep27 makes the pneumococci incapable of infiltrating into the
lungs, blood, and brain [
18
], resulting in a virtually non-cytotoxic and highly safe agent
Vaccines 2024,12, 737 3 of 16
that does not cause death after injection into the brains of immunocompromised mice [
24
].
Furthermore, intranasal immunization with
pep27, without any adjuvant, demonstrated
long-term protective efficacy [18].
Table 1. Effects of
pep27 on pneumococcal virulence and
pep27 immunization efficacy in multiple
disease models.
Disease Model pep27 Mechanism Ref.
Lethal pneumococcal D39 strain Decreases virulence and allows rapid clearance due to lower levels of
capsular polysaccharide [18]
Lethal pneumococcal pneumonia pep27-attenuated lactoferrin induced the vncRS operon to prevent lysis,
in vivo cytokine production, and subsequent lung inflammation [24]
Lethal pneumococcal pneumonia Shows resistance to lysis and reduced cytotoxicity, resulting in decreased
inflammation and death, enabling effective mucosal protection [25]
Lethal pneumococcal pneumonia Reduces morbidity and mortality against pneumococcal and
influenza infections [26]
Pneumococcal pneumonia pep27 immunization impairs serotype-independent colonization by
increasing IgA-, Th1-, and Th17-type cytokine responses [27]
Pneumococcal colonization
Non-transformable
pep27
comD immunization significantly diminished
colonization levels regardless of serotype [28]
Nasal infection with S. pneumoniae,S. aureus,
K. pneumoniae
pep27 inhibited colony formation of pathogens and induced
noncanonical Wnt and subsequent IL-17 secretion [29]
Korean Red Ginseng + pep27 before lethal
pneumococcal challenge
Korean Red Ginseng enhanced
pep27 vaccine efficacy by inhibiting ROS
production, apoptotic signaling, and inflammation [30]
DSS-induced Colitis
pep27 significantly attenuated the expression levels of pro-inflammatory
cytokine caspase-14 to attenuate experimental colitis through the
restoration of functional Tregs and healthy gut microbiota composition
[31]
DSS-induced Colitis Tregs elicited by pep27 were able to suppress Wnt5a expression to help
restore immunological tolerance and provide a robust antioxidant milieu [32]
Ovalbumin-induced asthma
pep27 immunization suppresses TH2 cytokine and pulmonary eosinophil
accumulation, and goblet cell proliferation, maintaining a balance between
Th1, Th2, and Treg cells
[33]
Ovalbumin-induced Allergic rhinitis
pep27 reduced the activation of the NLRP3 inflammasome in the nasal
mucosa by suppressing NF-κB activation through downregulating TLR2
and TLR4 expression
[34]
Oxazolone-induced Atopic dermatitis pep27 upregulated Treg and epithelial barrier function and inhibited
TSLP and Th2 expression [35]
Intranasal immunization with attenuated erythromycin-resistant
pep27 and inacti-
vated markerless
pep27 could protect a host from lethal pneumococcal challenge serotype
independently; it also lowers bacterial colonization in the nasopharynx [
18
,
25
], suggesting
that
pep27 may be able to provide mucosal immunity against pneumococcal diseases
and could represent an efficient mucosal vaccine. Additionally,
pep27 immunization
protected against heterologous strains in bronchoalveolar lavage fluid at the nasophar-
ynx, suggesting that
pep27 immunization provides a wide range of cross-protection,
demonstrating long-lasting immunity [
27
]. Furthermore,
pep27
comD immunization
significantly increased the survival time after heterologous challenges, and diminished
colonization levels independent of serotype [28], as shown in Figure 2and Table 1.
Vaccines 2024,12, 737 4 of 16
Vaccines 2024, 12, x FOR PEER REVIEW 3 of 16
be used to elicit mucosal immunity and aenuate virulence. Based on these features, we
immunized the mice three times, once/week, and measured the relevant parameters for
experimental purposes, as shown in Figure 1. Thus, Δpep27 makes the pneumococci
incapable of inltrating into the lungs, blood, and brain [18], resulting in a virtually non-
cytotoxic and highly safe agent that does not cause death after injection into the brains of
immunocompromised mice [24]. Furthermore, intranasal immunization with Δpep27,
without any adjuvant, demonstrated long-term protective ecacy [18].
Figure 1. pep27 immunization in various inammatory diseases. Mice were intranasally
immunized with pep27, 3 times, once a week, with 1 × 108 CFU. Experimental diseases were
induced via the appropriate methods for specied days until the mice were sacriced. Collectively,
several parameters such as IgG level, M2 macrophages, and Treg cells were induced via pep27
immunization. However, inammatory mediators using cytokine and chemokine transcription
factors in pneumococcal and virus models were suppressed. Image created using the BioRender
application.
Table 1. Eects of Δpep27 on pneumococcal virulence and Δpep27 immunization ecacy in
multiple disease models.
Disease Model
Δpep27 Mechanism
Ref.
Lethal pneumococcal D39 strain
Decreases virulence and allows rapid clearance due to lower levels of
capsular polysaccharide
[18]
Lethal pneumococcal
pneumonia
Δpep27-attenuated lactoferrin induced the vncRS operon to prevent lysis, in
vivo cytokine production, and subsequent lung inflammation
[24]
Lethal pneumococcal
pneumonia
Shows resistance to lysis and reduced cytotoxicity, resulting in decreased
inflammation and death, enabling effective mucosal protection
[25]
Lethal pneumococcal
pneumonia
Reduces morbidity and mortality against pneumococcal and influenza
infections
[73]
Pneumococcal pneumonia
Δpep27 immunization impairs serotype-independent colonization by
increasing IgA-, Th1-, and Th17-type cytokine responses
[26]
Figure 1.
pep27 immunization in various inflammatory diseases. Mice were intranasally immunized
with
pep27, 3 times, once a week, with 1
×
10
8
CFU. Experimental diseases were induced via the
appropriate methods for specified days until the mice were sacrificed. Collectively, several parameters
such as IgG level, M2 macrophages, and Treg cells were induced via
pep27 immunization. However,
inflammatory mediators using cytokine and chemokine transcription factors in pneumococcal and
virus models were suppressed. Image created using the BioRender application (biorender.com).
Mechanistically, vncRS is activated by lactoferrin in serum and is required for the
development of pneumonia and sepsis. When the VncS sensor is exposed to lactoferrin, it
is phosphorylated, and the phosphate group is transferred to the VncR response regulator,
allowing the VncRS operon to be induced, which in turn secretes the effector Pep27, which
is thought to cause bacterial lysis and release, leading to host lung inflammation. Deletion
of the effector Pep27 does not induce lysis, and it is incompetent for invasion into the lungs
and blood, confirming that pep27 is essential for the inflammatory response and sepsis [
24
].
Vaccines 2024,12, 737 5 of 16
Vaccines 2024, 12, x FOR PEER REVIEW 5 of 16
Figure 2. Therapeutic eect of pep27 mutant vaccine in various inammatory diseases. The response
to Δpep27 immunization via the intranasal route was analyzed using gene signatures predominant
in dierent organs of mice. Δpep27 immunization aenuated disease development in the mouse
model by suppressing aberrant gene expression and dysregulated immune responses. These
disturbances deplete Treg cells and subsequently induce inammatory mediators, including
cytokines, chemokines, and inammatory pathways. In contrast, ∆pep27 had a remedial eect that
preserved mucosal integrity through Treg upregulation, suggesting a promising candidate therapy
for clinical application with a potent anti-inammatory mucosal immune mechanism. Image
created using the BioRender application.
Figure 2. Therapeutic effect of pep27 mutant vaccine in various inflammatory diseases. The response
to
pep27 immunization via the intranasal route was analyzed using gene signatures predominant
in different organs of mice.
pep27 immunization attenuated disease development in the mouse
model by suppressing aberrant gene expression and dysregulated immune responses. These dis-
turbances deplete Treg cells and subsequently induce inflammatory mediators, including cytokines,
chemokines, and inflammatory pathways. In contrast,
pep27 had a remedial effect that preserved
mucosal integrity through Treg upregulation, suggesting a promising candidate therapy for clinical
application with a potent anti-inflammatory mucosal immune mechanism. Image created using the
BioRender application.
Vaccines 2024,12, 737 6 of 16
3. Gut–Brain, Gut–Lung, and Gut–Liver Axis
The gut contains a variety of substances, including food, medications, if any, and
secretions from the body, such as stomach acid and bile salts. As such, our gut microbiome
can be altered by these substances. While there are many types of microorganisms in the
gut, what they are fed can select for certain microorganisms, which in turn produces specific
microbial products, and/or metabolites. In the gut, various hydrolyzed and transformed
products and metabolites, such as short-chain fatty acids (SCFAs), dopamine, tyrosine,
tryptophan, trimethylamine N-oxide, and urolithin A, are present and involved in gut–brain
axis (GBA) communication. These metabolites are absorbed through the intestinal wall
and enter the bloodstream, where they are eventually distributed to all organs. However,
some of the metabolites enter through the blood–brain barrier and affect or modulate the
central nervous system [
36
]. However, the GBA network appears to be interconnected
by the vagus nerve, with signals from the gut reaching the afferent vagus nerve in the
brain; in return, the CNS sends signals to the gut via efferent vagal neurons. Thus, the
gut microenvironment can be monitored via gut neurons and transmitted to the brain via
the vagus nerve, and vice versa [
37
]. The GBA plays an important role in homeostasis
to regulate various functions of the gut and brain, including immune function, barrier
permeability, and the gut reflex [3740].
A variety of microbial metabolites can exert different effects; for example, butyric
acid mitigates cognitive impairment and taurine enhances memory [
36
]. Thus, the gut
microbiome can actually act as a multifactorial cause of gut and brain diseases [
38
,
39
,
41
].
Increased populations of harmful bacteria and abnormal gut microbiota composition can
lead to increased gut dysbiosis and immune dysfunction through GBA interactions, which
can lead to brain disorders such as depression, high sensitivity to stress, and neurode-
generative disorders [
37
39
,
41
]. The gut microbiome can modulate brain structure and
function, and in turn, the brain can modulate the microbial environment and microbiome
composition within the gut microbiome. Collectively, these pathways are referred to as the
microbiota–gut–brain axis (microbiota–GBA), which represents a comprehensive concept of
biochemical signaling and interactions between the brain, gut bacteria, and gastrointestinal
tract [38].
Accumulating evidence suggests that SCFAs consist of acetate, lactate, butyrate, propi-
onate, and succinate, which are produced through fermentation of fiber-rich diet by bacteria
(e.g., Prevotella,Bacteroides, and Ruminococcus) [
42
]. High-fructose diets reduce SCFAs, pro-
mote gut dysbiosis, and induce neuroinflammation [
43
]. SCFAs can be considered a leading
link in the immune axis between the gut and lungs. Increased luminal butyrate production
promotes mucosal healing and encourages the production of protective mucus along the
intestinal epithelium [
44
]. Indeed, SCFAs are known to modulate immune homeosis and
mucosal defense, thus contributing to barrier functions. Several Lactobacillus species are
known to secrete lactate-producing bacteria, a precursor for SCFA-producing bacteria.
Interestingly, alterations in the nasal microbial community including airways also
affect the composition of intestinal microbiota. In addition, constant exposure of mucosal
surfaces, particularly the respiratory and gastrointestinal tracts, to microbes and antigens
from the environment makes these surfaces valuable for shaping tolerogenic responses in
autoimmune and allergic disease. Numerous studies have shown that 2
·
5
µ
L of inoculum
consisting of fluids, particles, or even microorganisms deposited into the nasal cavity
of mice can later be detected in the gastrointestinal tract (GIT) [
45
]. This indicates that
the mucosal immune system of the GIT may serve as a primary sensor of any foreign
antigens that are introduced into the nasal cavity [
46
]. For example, manifestations of
pneumonia due to Pseudomonas aeruginosa or multi-drug resistant Staphylococcus aureus
in lungs are believed to trigger gut injury [
47
]. Furthermore, several gastrointestinal
disorders have manifestations in the respiratory tract, for example, about half of IBD
patients with known alterations in their intestinal microbiota composition have abnormal
lung function. COPD (chronic obstructive pulmonary disease) patients show intestinal
hyper-permeability with a high prevalence of IBD [
48
], thus suggesting that the “gut–lung
Vaccines 2024,12, 737 7 of 16
axis” is a bi-directional communication network where many respiratory infections are
often accompanied by gastrointestinal symptoms [
49
]. Communication in the gut–lung axis
comprises many direct and indirect pathways. Moreover, disturbing the lung microbiome
with the antibiotic neomycin can significantly attenuate the severity of the experimental
autoimmune encephalomyelitis (EAE) by sensitizing the autoreactivity of brain T cells
via microglia. Thus, lung microbiome dysbiosis can regulate brain autoimmunity [
50
].
Although the number of bacteria in the lungs is much lower than that in the gut, it has been
shown that changes in the lung microbiome could affect the brain, perhaps through the
lung–brain axis, which is functionally similar to the gut–brain axis.
Another organ involved in the regulation of the gut microbiome is the liver, which
also affects gut function through the gut–liver axis. Bile salts, which are necessary for
lipid digestion in vertebrates, are synthesized in the liver, conjugated with amino acids
such as glycine and taurine by primary host enzymes, and secreted into the small intestine.
Once secreted, bile salts are deconjugated by secondary gut bacteria that possess bile salt
hydrolase (BSH) to separate amino acids from bile salts [
51
]. A variety of gut bacteria can
conjugate bile acids, including the genera Actinobacterium,Bacillus,Bacteroides,Bifidobac-
terium,Clostridium,Fusobacterium, and Lactobacillus. Furthermore, bile amidates have also
been produced by the genera Clostridium,Bifidobacterium, and Enterococcus [
52
]. In addition,
deconjugation of glycine-conjugated bile acids is associated with the genus Gemmiger, and
deconjugation of taurocholic acid is linked to the genera Eubacterium and Ruminococcus [
39
].
This suggests that different microbes may have the same substrate specificity and may
share functions with other microbes. Moreover, gut microbiota imbalances and changes
in bile acids are involved in the regulation of inflammatory responses through bile acids
receptors, but conversely, bile acid receptor changes can also affect the abundance of gut
microbes [53].
The bile salt hydrolase (bsh) gene is characterized as an acyltransferase that forms bile
acid amidates [
53
55
]. Furthermore, human gut metagenomic analysis has shown that
BSH activity and abundance in the human gut is associated with IBD [
56
]. Bacterial bile
acid amidates act as ligands and activate host receptors responsible for the transcription of
the aryl hydrocarbon receptor (AHR) and the Pregnane X receptor (PXR) [
55
]. PXR levels
correlate well with intracellular bile acid levels in the gut and liver, and inhibiting PXR
increases IL-8 and TNF-
α
and decreases IL-10 and TGF-
β
, suggesting a worsening of IBD
symptoms. Thus, PXR agonist exhibited anti-inflammatory effects by inhibiting NF-
κ
B
and suppressing cytokine secretion in mice with dextran sulfate sodium (DSS)-induced
colitis [53].
Elevated levels of gut bacteria-derived secondary bile acids (i.e., lithocholic acid (LCA)
and deoxycholic acid (DCA)) harm the intestinal barrier, leading to dysbiosis and increased
gut inflammatory responses. In addition, a lack of BSH-positive bacteria can lead to
intestinal inflammation and bile acid metabolic dysfunction [
57
]. Increasing intestinal
BSH activity by administering BSH-competent probiotics or introducing them via fecal
transplantation can provide various health benefits to the host. Therefore, to overcome gut
inflammation, research exploring the regulation of the farnesoid X receptor (FXR, including
PXR), the master regulator of bile acid homeostasis, and its impact on the gut microbiome
is of considerable importance [57].
Since bile acids can be converted into different metabolites by various gut bacteria, it
is necessary to determine which bacterial enzymes degrade/transform bile acids and how
they affect the gut. Also, humans and rodents differ significantly in terms of the regulatory
activity of certain bile acids on FXR receptors, so rodent results should be considered with
caution when comparing them to human results. To fully understand bile acid-dependent
FXR function, especially in mucosal immune regulation, we need to understand how
different parameters in rodents and humans change in response to inflammation, diet, and
microbial imbalance [53].
Vaccines 2024,12, 737 8 of 16
4. Treg Cells for Brain Diseases
Impaired Treg function can be modulated by either functional deficiency including
impaired stability of Treg cells or numerical deficiency of Treg cells. Impaired stability of
Treg cells seems to be the cause of autoimmune diseases such as multiple sclerosis (MS). To
overcome this conundrum, several methods have been developed [
58
]. To reverse low Treg
stability prior to the implementation of Treg therapy, rapamycin (mTORC1 inhibitor) could
restore Treg cells in autoimmune disease [59].
Immunologically, the brain acts as a part of the systemic immune response system,
connecting not only the gut–brain axis but also the neural network, and Tregs play a
pivotal role in inflammation. In particular, an emerging view is that brain Treg cells directly
support tissue regeneration and repair processes by suppressing glial reactivity to neuronal
damage. Brain Treg cells are attractive therapeutic targets in all neurological diseases,
from neuroinflammatory disorders to neurodegenerative diseases and even psychiatric
disorders [60].
The GBA has a bi-directional association. Thus, brain function is affected by intestinal
inflammation, and conversely, brain disorders can cause IBD. Similarly, Treg function is
controlled by GBA in a bi-directional manner via the neuroimmune response. SCFAs are
known to limit mucosal inflammation via the induction of Tregs [
61
]. Consistent with
this, intranasal immunization of
pep27 showed that the microbiome composition of
pep27-immunizaed colitis mice was positively correlated with gut Treg induction and
negatively associated with proinflammatory cytokines [
31
], presumably via the GBA or the
lung–gut axis.
One of the triggering mechanisms of Treg that act in this bi-directional way is medi-
ated by liver vagal afferent nerves, which indirectly sense the gut environment and relay
the sensing inputs to the brain parasympathetic nerves as well as enteric neurons. This
therefore shows that brain Treg upregulation also upregulates gut Treg function via the
vagus nerve [
40
]. Thus, intestinal homeostasis may be regulated by the hepatic vago-vagal
GBA reflex to maintain intestinal Treg cell numbers [
40
], suggesting that treating intestinal
inflammation using brain Treg upregulation may be a more feasible therapeutic approach
than modulating intestinal Treg function by administering other chemicals or neuromodu-
lators in the future. Studies are underway to enhance brain Treg function to treat multiple
sclerosis [
62
,
63
], Parkinson’s disease [
64
], glioblastoma [
65
], and other neurological diseases
such as epilepsy, neurotrophic pain, and stroke [
58
,
66
,
67
]. Tregs can be classified as either
natural Tregs (nTregs) or induced Tregs (iTregs). Enhancement of nTregs can be achieved by
(1) stabilizing nTreg function and survival, (2) manipulating antigen-specific Treg reactions,
(3) using immunomodulators peripherally to induce Treg populations, or directing the
adoptive transfer of Treg cells. iTreg populations are generated via direct administration of
low doses of Treg inducers such as IL-2, CD3 monoclonal antibody, or GM-CSF. In addition,
the use of these immunologic agents to proliferate dysfunctional Tregs ex vivo and then
perform autologous adoptive transplantation is being investigated [
58
]. Several approaches
that combine these therapies with brain delivery methods to enhance efficacy are also
currently under intensive development [60].
5. Treg Cells for Inflammatory or Allergic Diseases
Currently, novel microbiome approaches to overcome IBD include either fecal mi-
crobiome transplantation (FMT) [
68
70
] or ingestion of microbial strains that induce Treg
function that can suppress intestinal inflammation [
71
,
72
]. However, these methods re-
quire antibiotic treatment to eliminate microbial imbalances prior to bacterial treatments.
Vancomycin is administered to purge vegetative C. difficile, which produces toxins and
causes inflammation and diarrhea, but does not kill the spore forms that cause germination
when treatment is discontinued. This is because antibiotic treatment causes a lack of bene-
ficial Firmicutes and results in an increase in bile acid, which in turn allows germination
of C. difficile spores. Therefore, discontinuation of antibiotic therapy and/or incomplete
sterilization may cause a recurrence of C. difficile disease. Recently, oral administration
Vaccines 2024,12, 737 9 of 16
of SER-109 (from the fecal microbiota of healthy donors) has been shown to significantly
reduce the recurrence rate of C. difficile (12%) compared with that of the placebo group
(40%) [
69
]. Moreover, SER-109 resulted in a significant improvement in disease-specific
quality of life scores from as early as week 1 compared with those of patients treated
with the placebo, with steady and sustained improvement continued through to week 8
post-dose [
70
]. Moreover, the gut microbiome is subject to dietary modifications even after
these treatments [
73
,
74
]. Therefore, an alternative approach that is not affected by diet
would be preferable, and more efficient methods of Treg induction and maintenance are
required. To date, it is unknown whether nasal immunization can upregulate Tregs in the
skin and gut due to the lack of characterization or vaccination of the nasal mucosa.
Although intestinal Treg enrichment has been studied, the intestinal Treg population
is continuously modulated by diet and other medications and interacts reciprocally with
the gut–brain, gut–lung and gut–liver networks to maintain homeostasis [
75
77
]. Diets
and drugs targeting specific microbiomes are available to improve host disease, but most
do not seem to be universally applicable [
78
]. Therefore, even if Treg cells are enriched
with a specific diet, medication, or appropriate therapy, the Treg population remains intact
while under the control of diet and medication. Therefore, one of the key factors in actually
achieving a Treg-enhancing effect is Treg cell stability and/or Treg resilience.
6. Intranasal Immunization of pep27 Protects against Pathogens and Influenza
Virus Infection
In our approach to intranasal immunization using
pep27 for the prevention of
pneumococcal diseases, microarray and system biology analyses of human lung cells
after
pep27 infection unraveled unexpected features predicting the preventive effect of
influenza virus infection and intestinal abnormality. Thereafter, a series of experiments
on this prediction were performed and showed that the prediction was true [
26
,
31
,
32
].
pep27 provides transient non-specific protection from heterologous bacteria through non-
canonical Wnt upregulation. Nasal immunization with
pep27 can inhibit colonization
by Staphylococcus aureus and Klebsiella pneumoniae, indicating non-specific resistance to
respiratory pathogens [29].
Injectable pneumococcal vaccines, including the 23-valent polysaccharide vaccine
and the 13-valent conjugate vaccine, do not provide mucosal immunity and do not pro-
vide complete protection against secondary pneumococcal infection following primary
influenza virus infection [79]. To address these challenges, we determined whether or not
pep27 could protect mice against secondary pneumococcal infection following influenza
virus infection. Surprisingly,
pep27 protected mice against secondary pneumococcal
infection after influenza virus infection by lowering the influenza virus burden in the
lungs. In contrast, the unimmunized group of mice had a nearly 60% higher mortality rate
following pneumococcal infection due to higher bacterial loads.
pep27 vaccination alone
can prevent influenza and pneumococcal infections by reducing viral titers in the lungs
after infection. Overall,
pep27 immunization is a novel and safe method to overcome
both invasive pneumococcal disease and serious secondary infections following influenza
infection during influenza epidemics [26], as shown in Figure 2and Table 1.
During pneumococcal pneumonia, phagocytes produce H
2
O
2
and reactive oxygen
species (ROS) for bacterial removal; nonetheless, the lung is vulnerable to these oxidative
stresses, resulting in extensive cellular and lung damage [
80
]. Thus, we investigated the
therapeutic effect of
pep27 immunization on antioxidant small proline-rich repeat (SPRR)
genes in the lungs and its associated consequences on the gut dysbiosis. We observed
that
pep27 significantly increased the levels of the SPRR genes in the lungs, suggesting a
strengthened alveolar barrier and enhanced resistance to external stressors, resulting in a
robust regenerative and oxidant-stress-relieving mechanism to re-establish immunological
tolerance [
32
,
81
]. Additionally, SPRR genes are involved not only in the establishment of the
physical barrier but also in cell migration and wound healing [
81
,
82
].
pep27, on the other
hand, significantly increased the expression of SPRR genes, resulting in a more strength-
Vaccines 2024,12, 737 10 of 16
ened alveolar barrier and enhanced resistance to external including pneumococci [
32
,
81
].
Similarly, Korean Red Ginseng (KRG), a traditional medicinal herb widely used as an
immune booster, enhanced the efficacy of the
pep27 vaccine by increasing the survival
rate of mice infected with pneumococcus by inhibiting ROS production, suppressing ERK
signaling-mediated cell death and reducing inflammation [
30
]. This suggests that
pep27
immunization blocks ROS and oxidative stress [30,32], as shown in Figure 2and Table 1.
Macrophages are classified into M1 and M2 macrophages, which produce inflam-
matory and anti-inflammatory cytokines, respectively [
83
]. Adoptive transfer of M2
macrophages or the induction of M2 polarization has been shown to suppress experimental
colitis [
84
]. M2 macrophages aid in the resolution of inflammation by downregulating
inflammatory cytokines and secrete copious amounts of IL-10 and TGF-
β
, thereby pro-
tecting against colitis to promote tissue repair and driving epithelial cell regeneration [
83
].
Intranasal
pep27 immunization upregulates colonic M2 macrophages, thereby inhibiting
inflammatory milieu [
32
]. Tregs maintain homeostasis by suppressing excessive immune
activation. Tregs are also very well defined as helpful for resolving and repairing lung
damage caused by infection [85].
Neutrophils play an important role in eliminating pathogens during respiratory in-
fections, and when they are mobilized to the lungs where pathogens have invaded, they
phagocytose the pathogens and then digest and kill them by producing reactive oxygen
species. However, in bacterial pneumonia, this process can lead to excessive lung damage
and respiratory failure. Therefore, neutrophil phagocytosis (efferocytosis), the phagocytic
removal of dead or dying neutrophils, is a key process in resolving lung inflammation.
Tregs interact with alveolar macrophages to promote neutrophil phagocytosis and promote
recovery. Overall, Tregs resolve inflammation and orchestrate tissue protection and airway
system repair in mice and humans [
85
88
]. After three intranasal vaccinations of
pep27,
fluorescence-activated cell sorting (FACS) analysis of splenocytes showed an increase in
Treg expression proportional to the number of immunizations. Additionally, it was con-
firmed that Tregs were induced in serum and bronchoalveolar lavage fluid (BALF) (Kim
GL, manuscript in preparation). Thus, Tregs may be one of the mechanisms by which
pathogen infection is defended against by intranasal pep27 immunization.
7. Intranasal Immunization of pep27 Protects Allergic Diseases
Inactivated serotype 3 S. pneumoniae has been reported to be effective against allergic
diseases, including asthma, via Treg upregulation. These inactivated strains in a mouse
model significantly suppressed the allergic inflammatory responses that are pivotal in the
development and progression of asthma, including Th1 and Th2 cytokine production and
eosinophil recruitment to the airways during or after ovalbumin sensitization [
89
,
90
], but
they are toxic and cannot be used as a vaccine.
Intranasal
pep27 immunization before or after allergen exposure could restore the
necessary balance of Th1/Th2 cells by reducing Th2 activity and maintaining Th1 and Treg
activity disturbed during asthma. Additionally, allergic airway inflammation in the lung
was significantly reduced by
pep27 immunization.
pep27 immunization may provide
long-term protection against asthma without any toxicity [33].
In addition,
pep27 immunization alleviated allergic symptoms such as sneezing and
rubbing frequency and reduced TLR2 and TLR4 expression, Th2 cytokines, and eosinophil
infiltration in the nasal mucosa of an ovalbumin (OVA)-induced allergic rhinitis mouse
model [
34
], shown in Figure 2. Mechanistically,
pep27 reduced the activation of the
NLRP3 inflammasome in the nasal mucosa by downregulating the TLR signaling pathway
and subsequently prevented allergic reactions [34].
In asthma models, IL-27 is an anti-inflammatory cytokine that belongs to the IL-12
family and is primarily expressed on dendritic cells, macrophages, and monocytes [
91
].
Because IL-27 targets Tregs in asthma models [
92
] and reduces respiratory allergy symp-
toms [
93
,
94
], the asthma-relieving effects of
pep27 appear to be due to the suppression of
inflammation by IL-27-induced Tregs.
Vaccines 2024,12, 737 11 of 16
In bronchial asthma, inflammation is frequently triggered repeatedly, resulting in
increased microvascular permeability and edema, which eventually leads to airway re-
modeling and a thickening of the airway walls [
95
]. Injection of Treg cells after the onset
of chronic asthma disease prevented airway remodeling [
96
], indicating that Tregs can
resolve chronic inflammation in asthma models. Thus, Tregs induced by
pep27 may
also contribute to resolving airway remodeling. In contrast to Tregs, IL-27 appears to act
differently in asthma suppression; when IL 27 was administrated therapeutically, IL 27 did
not ameliorate airway inflammation, airway hyperresponsiveness, and airway remolding.
Prophylactically, administration of IL-27 decreased the concentration of Th2 cytokines and
increased the number of type 1 regulatory T (Tr1) cells in the lungs [
97
,
98
], suggesting
that IL-27 may prevent asthma. Thus, they demonstrated that IL-27 may be useful for
preventive purposes but not for therapeutic purposes. pep27, however, works as both a
therapeutic and preventive method.
Prophylactic and therapeutic analysis showed that
pep27 could elicit anti-inflammatory
Treg-relevant factors and epithelial barrier genes (filaggrin, involucrin, loricrin, and SPRR
proteins). Accordingly, pneumococcal
pep27 immunization upregulated Treg activity,
suppressing epidermal collapse, IgE, and Thymic stromal lymphopoietin (TSLP). On the
other hand, Treg suppression worsened atopic dermatitis through the upregulation of TSLP
and Th2 and the repression of epithelial barrier function compared with the non-suppressed
pneumococcal
pep27 group. In summary, pneumococcal
pep27 immunization alleviated
allergic dermatitis symptoms by upregulating Tregs and epithelial barrier functions and
suppressing TSLP and Th2 to relieve allergic dermatitis symptoms [35].
8. Intranasal Immunization of pep27 Potentially Protects IBD via Anti-Oxidative
SPRR and Anti-Inflammatory M2 Upregulation through Treg Induction
Intranasal
pep27 immunization prevented DSS-induced colitis.
pep27 significantly
mitigated oxidative stress parameters and downregulated pro-inflammatory cytokines and
Wnt5a expressions via Treg induction in the gut. Moreover, pep27 induces upregulation
of the anti-inflammatory genes IL-10 and TGF-
β
1, as well as M2 macrophages, via Treg
induction and tight junction genes.
pep27 also suppresses DSS-induced caspase-14
expression and upregulates Tregs, resulting in healthy microbiota. Inhibition of Treg
function confirmed that
pep27 has therapeutic effects on gut inflammation and caspase-14
via Treg upregulation. Overall, intranasal immunization with
pep27 can attenuate colonic
inflammation via Treg induction and could be a highly pragmatic way to re-establish
immunological tolerance [31,32], as shown in Figure 2and Table 1.
9. Conclusions
To date, treating allergic diseases as well as recurrent inflammatory diseases, including
infections and IBD, remains a challenging task. Despite representing a complex approach
that evaluates immunogenicity, mucosal vaccines have the potential to improve current
applications and extend the utility of vaccines for diseases such as IBD and allergies. Al-
though there have been many attempts to use Tregs, which are effective for hypersensitivity
reactions such as excessive inflammation or allergies, there are many limitations in terms of
functionality. We confirmed that Tregs induced via intranasal immunization were consis-
tently expressed not only in the nasopharynx and lungs, but also in the skin and intestines,
rendering them effective against inflammation/hypersensitivity reactions. Mechanistically,
pep27 suppresses oxidative stress levels, which are closely linked to gut dysbiosis, po-
tentially by increasing the SPRR family in the lungs, suggesting that the lung–gut axis is a
bi-directional communication network. Furthermore, analysis of key genes in the lungs
induced by
pep27 immunization highlighted mucosal protection, particularly in the lungs
and gut, emphasizing the role of Tregs in establishing immune tolerance. Furthermore,
pep27 immunization induced M2 macrophages, an antioxidant milieu, to mitigate the
stress response, and Treg attenuated caspase-14 and Wnt5a expression independent of the
inflammatory environment through the lung–gut axis, suggesting robust anti-inflammatory
Vaccines 2024,12, 737 12 of 16
mucosal tolerance and subsequent restoration of the gut microbiota, ensuring that barrier
integrity is maintained to ensure intestinal immune homeostasis.
pep27 immunization
appears to promote the development and restoration of functional Treg cells in the skin, and
internal and respiratory organs, presumably through mucosal Treg infiltration or induction,
as well as on the gut–liver and gut–brain axis, which in turn may cooperate with the brain
or liver to exert anti-inflammatory effects on the skin and other organs. Moreover, the lung
microbiome can modulate autoimmune diseases in the brain, indicating that the lung–brain
axis may functionally operate similarly to the gut–brain axis (Figure 2, Table 1). To this end,
future studies are warranted to determine whether the pep27 vaccine also induces Tregs in
the brain and liver. Overall,
pep27 may be a promising mucosal vaccine candidate therapy
for clinical applications in allergic and inflammatory diseases (Figures 1and 2, Table 1).
However, due to the complicated nature of IBD and allergic diseases, further clinical trials
optimizing the parameters regulated by
pep27 and the resilience of food-induced changes
in Treg expression are needed to confirm its effectiveness in these diseases.
Author Contributions: H.I. and D.-K.R. collected, analyzed, and reviewed the literature, and wrote
the main manuscript. H.I. and D.-K.R. prepared the figures. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare that they have no conflict of financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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