The evaluation of lymph node cell proliferation response by liposomes loaded with major histocompatibility complex class II binding aquaporin 4 antigen peptide

ABSTRACT

Autoimmune responses to aquaporin 4 (AQP4) cause neuromyelitis optica (NMO); thus, specific immunotolerance to this self-antigen could represent a new NMO treatment. We generated the liposome-encapsulated AQP4 peptide 201-220 (p201-220) to induce immunotolerance. Liposomes were generated using phosphatidylserine and the polyglycidol species PG8MG. The in vivo tissue distribution of the liposomes was tested using an ex vivo imaging system. To confirm the antigen presentation capacity of PG8MG liposomes, dendritic cells were treated with PG8MG liposome-encapsulated AQP4 p201-220 (AQP4-PG8MG liposomes). Immunotolerance induction by AQP4-PG8MG liposomes was evaluated using the ex vivo cell proliferation of lymph node cells isolated from AQP4 p201-220-immunized AQP4-deficient mice. Fluorescent dye-labeled PG8MG liposomes were distributed to the lymph nodes. AQP4 p201-220 was presented on dendritic cells. AQP4-PG8MG liposomes were tended to suppress immune responses to AQP4 p201-220. Thus, the encapsulation of AQP4 peptides in PG8MG liposomes represents a new strategy for suppressing autoimmune responses to AQP4.

Graphical Abstract

Aquaporin 4 antigen peptide-encapsulated liposome has the potential to induce immunotolerance through antigen presentation on dendritic cells.

antigen-specific immunotolerance, aquaporin 4 (AQP4), neuromyelitis optica (NMO)

Abbreviations

Abbreviations
 
• AQP4:

  aquaporin 4

 
• AQP4 PG8MG liposome:

  AQP4 p201-220-encapsulated PEG(+)20%PG8MG liposome

 
• DC:

  dendritic cell

 
• DiR:

  1,1′-dioctadecyl tetramethyl indotricarbocyanine iodide

 
• DSPE:

  1,2-dioctadecanoyl-sn-glycero-3- phosphoethanolamine

 
• DSPS:

  1,2-distearoyl-sn-glycero-3-phospho-l-serine

 
• MHCII:

  major histocompatibility complex class II

 
• NMO:

  neuromyelitis optica

 
• p201-220:

  peptide 201-220

 
• PEG:

  polyethylene glycol

Autoimmune diseases are caused by the induction of immune responses to self-antigens, including islet autoantigen in type 1 diabetes (Warshauer et al.2020) and myelin basic protein in multiple sclerosis (Weissert 2017). Neuromyelitis optica (NMO) is an autoimmune disease leading to visual loss and limb weakness arising from demyelination and axonal injury (Graber et al.2008). The pathogenesis of NMO is related to autoimmune response and production of an autoantibody against aquaporin 4 (AQP4). Antibodies against AQP4, an ion channel expressed in astrocytes in the central nervous system (Graber et al.2008; Steinman et al.2016), are observed in 75% of patients with NMO (Pilli et al.2017), and immunization of mice with AQP4 can cause paralysis, a symptom of NMO (Sagan et al.2016). Moreover, lymphocyte infiltration is observed in tissues affected by NMO, such as the optic nerve and spinal cord (Lucchinetti et al.2002; Pilli et al.2017). The presence of anti-AQP4 antibody in patient serum is used as a diagnostic marker for NMO (Crout, Parks and Majithia 2016). In addition, T cells with autoreactivity against AQP4 peptide are present in patients with NMO (Vaknin-Dembinsky et al.2016).

Immunosuppressants are widely used to treat NMO. For example, azathioprine and cyclophosphamide improve the clinical symptoms of NMO (Xu et al.2016). Meanwhile, side effects such as leukopenia or recurrent infections are serious risks of such systemic immunosuppression (Torres et al.2015). To avoid these side effects, a treatment specific for the pathogenic mechanism is highly anticipated. This notion has been discussed and evidenced in preclinical mouse models of NMO (Steinman et al.2016). Lymph node cells from mice immunized with full-length AQP4 protein display immune responses to AQP4 peptides, and AQP4 peptide 201-220 (p201-220) was experimentally identified as a dominant immunogenic epitope (Vogel et al.2017). Immunization with AQP4 p201-220 was also reported to induce immune responses in the spinal cord and optic nerve (Sagan et al.2016).

Concerning the induction of immunotolerance to specific antigens, dendritic cells (DCs), a major antigen-presenting cell type, have important roles. DCs capture and process exogenous antigens in peripheral tissues and present them to T cells. Antigen presentation can cause T cell activation or inactivation depending on the maturation stage of DCs (Kishimoto and Maldonado 2018). In the absence of danger signals or under inflammatory conditions, DCs remain in an immature state, in which antigen presentation causes T cell inactivation, leading to immunotolerance (Kishimoto and Maldonado 2018). The delivery of self/harmless antigens to DCs induces antigen-specific immunotolerance. A recent review identified antigen-encapsulated liposomes as an effective modality for inducing antigen-specific immunotolerance (Kishimoto and Maldonado 2018). Immunotolerance induced by antigen-encapsulated liposomes is suspected to occur through several mechanisms. First, antigen-encapsulated liposomes can be incorporated into DCs via endocytosis. Second, self-antigen peptides in these liposomes are released in the endosomes of DCs (Pujol-Autonell et al.2017). Subsequently, the released antigen binds to major histocompatibility complex class II (MHCII) in early endosomes, permitting its presentation on the DC surface (Kishimoto and Maldonado 2018). The resulting MHCII–peptide complex suppresses T cells that have an ability to specifically interact with the presented peptide (Mahnke, Ring and Enk 2016; Pearson et al.2017).

Previous research found that carboxylated polyglycidol liposomes such as PG8MG enhanced the delivery of contents to DCs via accelerated endocytosis (Kuwahara 2014; Yuba et al.2013). In addition, the modification of liposomes using polyethylene glycol (PEG) can change tissue distribution by residence in the blood (Dadashzadeh et al.2010). In this study, we generated liposomes containing PG8MG and PEG and analyzed their effects on immunotolerance to AQP4.

Materials and methods

Liposome preparation

A lipisome mixture containing 1,2-distearoyl-sn-glycero-3-phospho-l-serine, 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine, (DSPE)-PG8MG, DSPE-PEG 2000 (NOF America), cholesterol (FUJIFILM Wako Pure Chemical), and dioctadecyl tetramethyl indotricarbocyanine iodide (DiR) was produced (Table 1). The AQP4 p201-220 peptide (TAG Copenhagen) was then dissolved in water and then encapsulated by the liposome. The mixture was heated at 60 °C, resuspended by extensive swirling, and put through 5 freeze-thaw cycles at −80 and 60 °C, respectively. Then, the suspension was extruded through 0.1-µm Whatman Nuclepore Track-Etched Membranes (GE Healthcare, IL, USA) at 70 °C using LIPEX extruders (TRANSFERRA Nanosciences). The resulting suspension was dialyzed overnight using Slide-A-Lyzer™ G2 Dialysis Cassettes 20K MWCO (Thermo Fisher Scientific). The produced liposomes were concentrated via ultrafiltration in an Amicon Ultra-15 Centrifugal Filter Unit (100K molecular weight cutoff) at 1700 g for 30 min (Millipore Sigma). The resulting suspension was diluted with 1 m m HEPES solution, and the particle diameter and zeta potential were measured using Zetasizer Nano series Nano-ZS (Malvern Instruments). The compositions and properties of the liposomes generated in this study are summarized in Table 1.

Table 1.

Composition and properties of liposomes

Name	Composition of lipid DSPS:chol:DSPE-PG8MG:PEG-2000:DiR (molar ratio)	Antigen peptide	Diameter (nm)	Zeta potential (mV)
PEG(+)10%PG8MG	78:7:10:5:0.5	None	93	−47.6
PEG(+)20%PG8MG	68:7:20:5:0.5	None	100.3	−48.1
PEG(+)30%PG8MG	58:7:30:5:0.5	None	111.6	−56.1
PEG(−)10%PG8MG	83:7:10:0:0.5	None	104.4	−46.1
PEG(−)20%PG8MG	73:7:20:0:0.5	None	94.7	−46.4
PEG(−)30%PG8MG	63:7:30:0:0.5	None	86.7	−48.0
Control-PG8MG	68:7:20:5:0	None	155.8	−16.0
AQP4-PG8MG	68:7:20:5:0	AQP4	114.5a	−61.0a
		p201-220	164.4b	−45.7b

PEG, polyethylene glycol; AQP4, aquaporin 4; DSPS, 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine; chol, cholesterol; DiR, 1,1′-dioctadecyl tetramethyl indotricarbocyanine iodide; 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine; p201-220; peptide 201-220.

a

Used in Figure 2.

b

Used in Figure 4.

Animal ethics

All protocols for animal experiments were approved by the Takeda Institutional Animal Care and Use Committee (Protocol No.: AU-00 020791).

Tissue distribution analysis of liposome

Liposomes labeled with DiR were subcutaneously administered at the dorsal surface in the middle of the left and right inguinal lymph nodes of 8-week-old female wild-type C57BL/6N mice (Charles River Laboratories Japan). After 24 h, when liposomes have been effectively incorporated into tissues (Allen et al.1991; Liu, Mori and Huang 1991; Drummond et al.1999), the mice were anesthetized and sacrificed to collect blood, inguinal lymph nodes, livers, and kidneys. Tissues were placed in the light tight chamber of the IVIS Spectrum imaging system (PerkinElmer) and imaged ex vivo. The fluorescence of DiR-labeled liposomes was measured at excitation and emission wavelengths of 710 and 760 nm, respectively. Data acquisition and analysis were performed using Living Image Software (version 4.4, PerkinElmer). Fluorescence from the region of interest was quantified as the average radiant efficiency.

In vitro antigen presentation assay

DCs were differentiated from mouse bone marrow as described previously (Inaba et al.2009). Tibias were isolated from C57BL/6N mice and cut at both ends with scissors. Bone marrow was obtained by flushing out each of the shafts with RPMI-1640 (FUJIFILM Wako Pure Chemical) using a syringe. Then, red blood cells were lysed with ammonium chloride solution by letting the mixture stand for 3 min. The bone marrow cells were washed twice with RPMI-1640. Viable cells were suspended in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin (FUJIFILM Wako Pure Chemical), 100 µg/mL streptomycin (FUJIFILM Wako Pure Chemical), 50 µm beta-mercaptoethanol (FUJIFILM Wako Pure Chemical), and 20 ng/mL GM-CSF (FUJIFILM Wako Pure Chemical) and seeded at 1 × 10⁶ cells/well in a 24-well plate. The cells were cultured for 6 days and incubated with AQP4-PG8MG liposomes for another 5 days. The cells were collected and frozen at −80 °C until use. Antigen presentation was analyzed using the ProPresent assay (ProImmune) as described previously (Xue et al.2016). Briefly, after MHCII (I-A^b) was immunoprecipitated, the amino acid sequences of peptides in the MHCII immunoprecipitates were analyzed via liquid chromatography–mass spectrometry.

Evaluation of the neurological symptoms of mice immunized with AQP4 p201-220

Wild-type C57BL/6N mice (Charles River Laboratories Japan) were immunized with Complete Freund's adjuvant containing 100-400 µg of AQP4 p201-220. The mice received 200 ng of pertussis toxin on days 0 and 2. The clinical score was measured for 24 days and defined as follows: 0, no disease; 1, tail tone loss; 2, impaired lighting, 3, severe paralysis or paraplegia; 4, quadriplegia; and 5, moribund or death. Among all the animals included in this study, incidence rate was calculated as the percentage of animals with a clinical score of 1-5. Cumulative score was calculated as the sum of the clinical score for each day from day 0 to day 24.

Ex vivo immunotolerance assay

Immunotolerance to AQP4 p201-220 was evaluated in AQP4-deficient mice (Kitaura et al.2009) (Riken BioResource Research Center). AQP4 p201-220 was dissolved in phosphate-buffered saline (FUJIFILM Wako Pure Chemical), and 10 mg of AQP4 p201-220 were orally administered to each AQP4-deficient mouse 1, 4, and 7 days before immunization. On the same days, AQP4-PG8MG liposomes were subcutaneously administered into the back of the mice at doses of 154, 154, and 77 µg/mouse, respectively. Then, mice were immunized with Complete Freund's adjuvant containing 100 µg of AQP4 p201-220 on day 0. Eleven days after immunization, mice were sacrificed, and the left and right inguinal lymph nodes were collected. Lymph node cells were seeded on a 96-well plate and cultured for 3 days in the presence of AQP4 p201-220 (0.1-10 µg/mL). ATP content was measured using Cell-titer Glo (Promega) to evaluate cell proliferation in response to the peptide.

Statistical analysis

William's test was used to evaluate the clinical score following AQP4 p201-220 immunization. Dunnett's test was used to evaluate differences between the control and test groups in the in vivo immunotolerance assay. Tukey's test was used to evaluate differences among the experimental groups concerning the tissue distribution of liposomes.

Results

Selection of liposomes for in vivo analysis

Antigen responses in rodent NMO are represented by the proliferative responses of lymph node cells (Sagan et al.2017). Thus, we first sought to effectively deliver antigen to lymph nodes using liposomes. We prepared liposomes with various amounts of PG8MG and PEG and confirmed that the composition did not affect the general properties of liposomes such as diameter and zeta potential (Table 1). We tested the tissue distribution of the prepared liposomes using fluorescent dye to identify an optimal liposome composition (Figure 1a). In this study, the DiR solution, which is not accumulated in tissues, showed low fluorescence, reflecting the accumulation of liposome in the tissue (Figure 1a). All tested PG8MG liposomes were detected in the lymph nodes (Figure 1b and c). To estimate the tissue incorporation efficiency, blood fluorescence, which reflects low liposome uptake in tissues (Allen et al.1991), was measured. The blood residence of PEG(+)10%PG8MG liposomes was significantly higher than PEG(+)30%PG8MG liposomes (Figure 1d), indicating that higher PG8MG content may reduce blood residence. To estimate liposome clearance via the liver, liver fluorescence was measured. The liver accumulation of PEG(−)30%PG8MG liposomes was significantly higher than that of PEG(+)10%PG8MG liposomes (Figure 1e). Despite the distribution difference of some PG8MG liposomes in the blood and liver, their accumulation in the lymph node was not markedly affected by the percentage of PG8MG and surface PEG modification. These results suggested that PG8MG liposome can act as a carrier for peptide delivery to the lymph node. To prevent the low tissue uptake and rapid clearance of PG8MG liposome from the body, we selected PEG(+)20%PG8MG liposome for peptide delivery to the lymph node.

Figure 1.

Tissue distribution of PG8MG liposomes. (a) Schema of the experiment. Fluorescent dye-labeled liposomes were administered to C57BL/6N mice. Fluorescence in tissue was measured 1 day after administration. DiR solution was used as a negative control. (b) The tissue distribution of fluorescent dye-labeled PG8MG liposomes. (c) Fluorescence intensity of the lymph nodes. (d) Fluorescence intensity of blood. (e) Fluorescence intensity of the liver. Data are presented as the mean + SD, n = 4. #: P < .05 by Tukey's test.

Induction of AQP4 p201-220 presentation by DCs

We then examined whether PG8MG liposomes can induce antigen presentation. To answer this question, we encapsulated AQP4 p201-220 in PEG(+)20%PG8MG liposomes (hereafter referred to as AQP4-PG8MG liposomes), which can accumulate in the lymph node without undergoing widespread distribution in the blood and liver, and incubated them with DCs differentiated in vitro. Then, the amino acid sequences presented on MHCII in DC were analyzed as depicted in Figure 2a. Liquid chromatography–mass spectrometry detected 16 peptide fragments derived from AQP4 p201-220 sequences. All identified peptide fragments included amino acid residues 205-215 of AQP4 (Figure 2b).

Figure 2.

Aquaporin 4 peptide 201-220 (p201-220) presentation via major histocompatibility complex class II (MHCII) on dendritic cells (DCs). (a) Schema of the experiment. DCs were differentiated from bone marrow cells and treated with AQP4-PG8MG liposomes for 5 days. The peptide–MHCII complexes were collected via immunoprecipitation, and peptide sequences on MHCII were analyzed using liquid chromatography–mass spectrometry. (b) Amino acid sequences presented on MHCII.

In vivo induction of immune responses by AQP4p201-220

Prior to evaluating the effects of AQP4-PG8MG liposomes on immunotolerance, we optimized the dose of AQP4 p201-220 antigen and sampling time of lymph node cells based on the clinical score following AQP4 p201-220 immunization (Figure 3a). The neurological symptom was measured from day 0 to day 24 and recorded as a 5-point scale clinical score. The incidence rates of the clinical symptoms, which were calculated as the percentage of animals with a score of 1-5 among the total number of animals included, in mice treated with 100, 200, and 400 µg of AQP4 p201-220 were 62.5%, 75.0%, and 75.0%, respectively (Figure 3b). Clinical symptoms first appeared in the 100, 200, and 400 µg groups on days 13, 13, and 10, respectively (Figure 3b). The mean maximum clinical scores in these 3 groups were 0.88, 1.63, and 1.50, respectively (Figure 3c). The maximal clinical score in the 100, 200, and 400 µg groups was recorded on days 16, 16-17, and 16, respectively (Figure 3c). The mean cumulative clinical scores, which were calculated as the sum of the clinical score for each day from day 0 to day 24, in the 100-, 200-, and 400-µg groups were 2.81, 6.13, and 5.13, respectively. The cumulative clinical score was significantly higher in the 200 and 400 µg groups than in the control group (Figure 3d).

Figure 3.

Time and antigen dose dependency of neurological symptoms in mice immunized with AQP4 peptide 201-220 (p201-220). (a) Schema of the experiment. C57BL/6N mice were immunized with AQP4 p201-220 (100, 200, or 400 µg/mouse). (b) Incidence of neurological symptoms. (c) Clinical score for neurological symptom after immunization with AQP4 p201-220. (d) Cumulative clinical score from day 0 to day 24. (c and d) Data are presented as the mean + SD, n = 8. #: P < .05 vs control group by Williams’ test.

We hypothesized that 100 µg dose of AQP4 p201-220 was sufficient for inducing immune cell activation because 62.5% of mice showed clinical symptom. Moreover, the moderate immunization condition might be a suitable factor to assess the response to the liposome treatment. Thus, we selected an AQP4 p201-220 dose of 100 µg of for the immunotolerance assay. As shown in Figure 3b, disease onset occurred 13 days after immunization with 100 µg of AQP4 p201-220. Based on previous findings that immune cell activation in the lymph nodes occurred prior to the infiltration of immune cells into neural tissue and disease onset (Barthelmes et al.2016), we decided to collect lymph nodes 11 days after immunization.

Induction of immunotolerance by AQP4-PG8MG liposomes

Next, we assessed whether AQP4-PG8MG liposomes could induce immunotolerance. In this aim, we evaluated the proliferative response of lymph node cells as an immune response to AQP4 (Figure 4a). In this assay system, the AQP4-deficient mice were pretreated with the liposome. The mice were subsequently immunized with AQP4 p201-220, after which the lymph node cells were collected and restimulated with AQP4 p201-220 to evaluate the proliferative response.

Figure 4.

The effect of AQP4-PG8MG liposomes on the proliferation of lymph node cells in response to immunization with AQP4 peptide 201-220 (p201-220). (a) Schema of the experiment. The proliferation of lymph node cells derived from AQP4-deficient mice was analyzed ex vivo. Mice were divided to the following groups: (i) no treatment, (ii) immunization with AQP4 p201-220, (iii) oral treatment with AQP4 peptides before immunization, (iv) subcutaneous treatment with PG8MG liposomes lacking antigen before immunization, and (v) subcutaneous treatment with AQP4-PG8MG liposomes before immunization. The ex vivo proliferation responses of lymph node cells were evaluated in the presence of AQP4 p201-220 (0.1-10 µg/mL). (b) Ex vivo proliferation responses of groups (i), (ii), and (iii). (c) Ex vivo proliferation responses of groups (i), (ii), (iv), and (v). Data are presented as the mean + SD, n = 4. #: P < .05 vs group (ii) by Dunnett's test.

We observed no ex vivo proliferation response of lymph node cells collected from untreated mice, whereas an autoimmune response was established in mice immunized with AQP4 p201-220. The proliferation response in immunized mice was significantly suppressed by oral treatment with AQP4 p201-220 (Figure 4b). We then tested the effects of liposomes in this assay. Antigen-free control liposomes had no effect on the proliferation of lymphoid cells (Figure 4c). Meanwhile, AQP4-PG8MG liposomes tended to suppress the proliferation response by 21.1%, 27.8%, and 25.8% in the presence of 1, 3, and 10 µg/mL AQP4 p201-220, respectively (Figure 4c).

Discussion

In this study, we developed AQP4 p201-220-encapsulated liposomes using PG8MG that effectively delivered antigen peptides to DCs. We confirmed that the AQP4 p201-220 peptide in the liposome was presented by MHCII on DCs. Pretreatment with AQP4-PG8MG liposomes tended to suppress the immune response to AQP4 p201-220.

Our ex vivo imaging analysis of PG8MG liposome illustrated that PG8MG liposomes accumulated in inguinal lymph nodes, which might be where the liposomes drained into after being injected into the middle of the left and right inguinal lymph nodes. Previous reports demonstrated that molecules larger than 16-20 kDa are taken up primarily by the lymphatic system after subcutaneous administration (Porter and Charman 2000), and liposomes containing PG8MG are effectively incorporated in DCs because PG8MG has membrane-fusing ability (Yuba et al.2013). We consider the distribution of PG8MG liposomes to the lymph nodes in this study to be a consequence of a similar mechanism. Multiple studies have analyzed the tissue distribution of liposomes on 24 h administration (Allen et al.1991; Liu, Mori and Huang 1991; Drummond et al.1999). In this study, we evaluated the distribution of liposome in tissues 24 h after administration. However, further kinetics analysis of PG8MG liposome will help understand its distribution.

Our in vitro antigen presentation assay revealed that 16 peptide fragments of AQP4 p201-220 were presented by MHCII on DCs following treatment with AQP4-PG8MG liposomes. We speculated that the structure of MHCII with its peptide-binding groove open at both ends permitted the binding of peptides of different lengths, as suggested in a previous report (Liu et al.2002). It has been reported that 9 amino acids can interact with the binding groove of MHCII (Liu et al.2002). Based on this notion, 9 amino acids among amino acids 205-215 of AQP4 likely bind to MHCII.

In our study, oral treatment with AQP4 p201-220 suppressed the ex vivo proliferation response of lymph node cells. However, oral administration limits the potential of these liposomes as a treatment because of the requirement of large amounts of antigen (Kang, Kim and Kang 1999). There are also issues with orally administered antigen arising from low bioavailability and rapid clearance (Sonaje et al.2009). Previous research reported that the tissue uptake efficiency and plasma residence of a drug were enhanced by encapsulation in liposome (Gabizon, Shmeeda and Barenholz 2003). Thus, our strategy to generate AQP4-PG8MG liposomes could be effective method to overcome limitations in immunotolerance induction via oral antigen administration.

Antigen presentation by DC transmits signal to T cells to induce both immunotolerance and immunization. A previous report showed the maturation status of DC changes the direction of its response (Florez-Grau et al.2018). In the absence of proinflammatory cytokines, DC stays in an immature state; its lack of antigen presentation suppresses the activation of cytotoxic T cells. AQP4-PG8MG liposome, which does not contain proinflammatory factor, is not expected to induce DC maturation. Based on this notion, we consider that AQP4-PG8MG liposome could induce immunotolerance rather than immunization. The anti-AQP4 antibody produced by B cell is thought to be involved in the pathogenesis of NMO (Zeka et al.2016). B cell proliferation occurs in the lymph node, especially in the germinal center, through the interaction with T cells (Allen, Okada and Cyster 2007). As mentioned above, we hypothesize that AQP4 p201-220-encapsulated liposome reduces T cell activation, thereby suppressing B cell proliferation and reducing the amount of anti-AQP4 antibody.

In this study, the ex vivo lymph node proliferation response did not reach statistical significance. In future research, we will further test the suppression of ex vivo proliferation response by AQP4-PG8MG liposomes at a larger scale and in a clinical setting. A previous report has shown the pathological changes in the eye and spinal cord, such as optic nerve inflammation and demyelination of the spinal cord, in the NMO mouse model (Sagan et al.2016). Further analysis of the effect of liposomes on such pathological changes will inform the liposomes’ therapeutic potential.

Previous studies for development of treatment method for NMO focused on using approved drug for other indications, as represented by a clinical study using a combination of immunosuppressant and other anti-inflammatory drugs to prevent clinical symptom of NMO patients (Cree 2015). However, research for generation of new treatment method using knowledge for immunogenicity of AQP4 protein is still slim. The novelty of our study, compared to previous researches, is that we focused on pathogenic mechanism of NMO.

In summary, AQP4 p201-220-encapsulated liposomes may have potential to induce immunotolerance to AQP4 antigen peptides and may be a potential treatment for NMO.

Acknowledgments

The authors thank Dr. Satoru Matsumoto, Mr. Koji Nakano, and Mr. Tomoya Takenaka for helpful discussions about liposome synthesis. The authors also thank Dr. Chihiro Akimoto for reviewing manuscript.

Author contribution

Y.M., Y.N., M.Y., and S.M. designed and conducted the experiments. All authors interpreted the experiment data. Y.M. and S.S. wrote the draft manuscript. All authors reviewed and approved the manuscript.

Funding

The work was funded by Takeda Pharmaceutical Company.

Conflict of interest

There is no conflict of interest.

Disclosure statement

All authors are employee of Takeda Pharmaceutical Company Limited.

Author notes

Y.M. and Y.N. contributed equally to this work.