4. Discussion
To investigate effects of MQL on allergic immune responses, we used an
OVA-induced allergic mouse model, and showed that MQL suppresses
Th2-related immune responses such as serum IgE production and Th2
cytokine production by splenocytes. In our previous studies, we revealed
that MQL was one of active compounds in Rosae multiflorae fructus
extract, which can ameliorate asthma, rhinitis, and food
allergies21,40. Furthermore, Nguyen et al .
reported that Rosae multiflorae fructus has an anti-allergic
effect by suppressing antigen-specific T cell activation and
proliferation in vivo 41. Our results in
vivo study also suggest that MQL suppresses splenocyte proliferation
and IL-2 production, thereby inhibiting Th2-related immune responses.
Therefore, we focused on CD4+ T cells and investigated
the anti-allergic mechanisms of MQL.
Th2-dominant responses, showing increased Th2 (IL-4, IL-5, and IL-13)
cytokines and IgE production, are characteristic immune responses in
patients with allergic diseases such as food allergy, asthma, and
AD42–44. We previously reported some strategies to
treat allergic diseases45–47. The first involves
regulating the Th1/Th2 immune balance by suppressing excessively induced
Th2-mediated immune responses or increasing Th1-related immune
responses48–50. However, here we confirmed that MQL
treatment does not increase the Th1-associated cytokine IFN-γ. Thus, we
focused on T cell proliferation as reducing T cell clonal expansion and
differentiation represents an alternative way to manage allergic
diseases2. Mechanisms for suppressing T cell
proliferation are typically inducing regulatory T cells (Treg) or
up-regulating HO-1 expression. When we investigated the effects of MQL
on Treg induction, MQL treatment did not increase
CD4+Foxp3+ T cell populations (data
not shown). Therefore, in the present study, we investigated the effects
of MQL on Th2-dominant allergic immune responses and mechanisms involved
in HO-1 expression.
HO-1 is known to have various physiological functions including a
protective effect against oxidative stress12,13. In
particular, it was reported that up-regulation of HO-1 in
CD4+ T cells suppresses their proliferation by
inhibiting IL-2 production7. Similarly, our results
showed that MQL suppresses CD4+ T cell proliferation
and IL-2 production induced by anti-CD3/CD28 antibody stimulation, while
HO-1 expression was increased. Based on these results, we hypothesize
that MQL may be able to suppress the CD4+ T
cell-mediated immune responses by inhibiting proliferation of
CD4+ T cells via up-regulation of HO-1. Furthermore,
we speculate that HO-1 is a key regulator of CD4+ T
cell proliferation.
Up-stream signals of HO-1 have been reported by many previous studies.
Notably, Nrf2, which is normally repressed by Keap1, translocates to the
nucleus upon activation and binds to ARE sequences of the HO-1 gene.
Especially, about the regulation of HO-1 on cell proliferation,
activation of MAPKs (ERK, JNK, and p38) as up-stream signals of Nfr2
plays as factors, which are able to regulate cell proliferation.
However, the specific MAPKs involved depend on the administered
compounds. For example, it was reported that quercetin enhances
Nrf2/HO-1 activity via both ERK and p38 signaling
pathways51,52 and suppresses T cell proliferation by
enhancing HO-1 expression20.
Quercetin-3-O-β- D-glucuronopyranoside, also known as
isoquercitrin, can also up-regulate HO-1 expression via ERK
pathways53. Although in a different cell type, in a
recent study, Lee et al. revealed that MQL up-regulates HO-1
expression via the Nrf2 pathway in human hepatoma cells (HepG2) but does
not activate nuclear factor-kappa B (NF-κB)54. In this
study, we found that MQL activated Nrf2-HO-1 signals through
phosphorylation of ERK in CD4+ T cells. In experiments
using MAPK inhibitors, we confirmed that U0126 (ERK inhibitor) most
significantly suppressed HO-1 expression, and MQL strongly increased ERK
phosphorylation in CD4+ T cells. Although treatment
with SB20350 (p38 inhibitor) suppressed HO-1 expression, it was
relatively weak compared with the effect of U0126. Moreover, p38
phosphorylation was not detected following MQL treatment. Therefore, our
results demonstrate that ERK activation is upstream of MQL-induced HO-1
expression in CD4+ T cells.
Ras is a small GTP-binding protein with three isoforms (H-Ras, K-Ras,
and N-Ras), and controls growth via ERK activation and other
intracellular signaling pathways55. Membrane-bound Ras
(GTP-bound active form) can activate Raf by recruiting Raf and promoting
the formation of B-Raf /C-Raf complexes or homodimers. Activated Raf
protein can then induce phosphorylation of
ERK1/234,56. Previous studies have reported that HO-1
expression can be up-regulated by activating the Ras-Raf-ERK signaling
pathway57,58. Based on these findings, we investigated
whether MQL-induced HO-1 up-regulation was associated with C-Raf
phosphorylation. Our results showed that MQL could induce
phosphorylation of C-Raf in CD4+ T cells, indicating
that C-Raf could be a potential up-stream signal of the ERK-Nrf2
signaling pathway.
ROS such as superoxide anion radicals (·O2-), hydroxyl
radicals (·OH), and hydrogen peroxide
(H2O2) are constantly produced as a
byproduct of mitochondrial oxidative metabolism. Excessively generated
ROS, however, can induce oxidative stress, which underlies various
diseases, while normal ROS levels can regulate cell growth by modulating
proliferation and differentiation. Specifically, ROS activate cellular
pathways such as MAPK, JAK/STAT, Nrf2, and NF-κB
signals59–61. Furthermore, intracellular ROS are
known to induce HO-1 expression62,63. Consistent with
our results, it was reported that intracellular ROS activates the
Raf-MEK-ERK signaling pathways64 and could induce
ERK1/2 phosphorylation by activating Ras39,65.
Importantly, flavonoids are antioxidant secondary phenolic metabolites
naturally produced in fruits and vegetables, and are able to against
oxidative stress66. However, the flavonoids can also
increase intracellular ROS levels as prooxidants. For example, McNally
et al , revealed that curcumin increases HO-1 expression via ROS
generation67. In addition, it has been reported that
quercetin can also act as a prooxidant capable of producing
intracellular ROS37,68. In this study, we found that
MQL induces ROS production in CD4+ T cells, which
enhances HO-1 expression via Raf-ERK activation. Consequently, this
leads to suppression of IL-2 production and CD4+ T
cell proliferation.
TMA as a hapten has been known to induce allergic AD by evoking
Th2-dominant immune responses in mice26,69–71.
Haptens act as antigens by generating hapten-protein complexes through
conjugation with self-proteins then activate adaptive immune responses
when recognized by APCs such as DCs71. Langerhans
cells, which are DCs that reside in the epidermis, induce naïve
CD4+ T cell differentiation by presenting antigens
after migrating into T cell-rich areas, such as the paracortex of the
DLNs72. To characterize the effects of MQL on a
TMA-induced AD-like mouse model, we administered MQL either orally or
topically, since both oral and topical therapies are effective for
treating AD. Considering that this was a disease model, we used a higher
dosage of MQL (4 and 10 mg/kg) than in the OVA-induced Th2-dominant
mouse model (MQL, 2 and 4 mg/kg). Our results revealed that AD symptoms
such as ear swelling, tissue infiltration of inflammatory cells, and IgE
production were ameliorated by MQL in both topical and oral treatments.
Interestingly, serum IgE levels were strongly reduced by oral
administration of MQL compared with topical administration, possibly
because of IgE production involves a systemic immune response. However,
in DLNs, which represent peripheral immune responses, IL-13 levels
induced by TMA were dramatically reduced by topical treatment of MQL
compared with oral administration. These results indicate that MQL may
be effective as an oral or topical drug depending on the internal or
external causes of atopic dermatitis. Further anti-proliferative effects
of MQL were seen in allergic mouse models as Th2 (IL-13) and Th1 (IFN-γ)
cytokines decreased, but not IL-12 (cytokine derived from APC). Fig. 6
shows that IFN-γ is suppressed by MQL topical treatment, but not IL-12,
while oral administration of MQL did not affect IFN-γ production but
increased IL-12 production. More evidently, IFN-γ/IL-12 ratios showed
that MQL treatment decreases IFN-γ production compared with the TMA
alone group as follows: Ratio of IFN-γ/IL-12 in the naïve group (oral,
1.3; topical, 1.7), TMA-induced AD group (oral, 2.4; topical, 8.9), MQL
4 group (oral, 2.3; topical, 6.5), and MQL 10 group (oral, 2.2; topical,
5.3). Therefore, we believe that MQL has the potential to be developed
as a therapeutic agent for AD.
In conclusion, we demonstrated that
MQL suppressed Th2-related immune
responses by reducing CD4+ T cell proliferation via
up-regulation of HO-1 expression. Mechanistically, MQL increased HO-1
expression via activation of the Raf-ERK-Nrf2 pathway and by generating
ROS in CD4+ T cells. Furthermore, we verified that
these effects of MQL on CD4+ T cells leads to
alleviated allergic diseases such as an atopic dermatitis in
vivo . It is possible that MQL may provide clinical benefits on other
allergic and CD4+ T cell-mediated diseases. The
application MQL on various diseases remains the focus of further
studies.