Peritoneal B Cells Play a Role in the Production of Anti-polyethylene Glycol (PEG) IgM against Intravenously Injected siRNA-PEGylated Liposome Complexes

Abstract

Polyethylene glycol (PEG)-modified (PEGylated) cationic liposomes are frequently used as delivery vehicles for small interfering RNA (siRNA)-based drugs because of their ability to encapsulate/complex with siRNA and prolong the circulation half-life in vivo. Nevertheless, we have reported that subsequent intravenous (IV) injections of siRNA complexed with PEGylated cationic liposomes (PLpx) induces the production of anti-PEG immunoglobulin M (IgM), which accelerates the blood clearance of subsequent doses of PLpx and other PEGylated products. In this study, it is interesting that splenectomy (removal of spleen) did not prevent anti-PEG IgM induction by IV injection of PLpx. This indicates that B cells other than the splenic version are involved in anti-PEG IgM production under these conditions. In vitro and in vivo studies have shown that peritoneal cells also secrete anti-PEG IgM in response to the administration of PLpx. Interleukin-6 (IL-6) is a glycoprotein that is secreted by peritoneal immune cells and has been detected in response to the in vivo administration of PLpx. These observations indicate that IV injection of PLpx stimulates the proliferation/differentiation of peritoneal PEG-specific B cells into plasma cells via IL-6 induction, which results in the production of anti-PEG IgM from the peritoneal cavity of mice. Our results suggest the mutual contribution of peritoneal B cells as a potent anti-PEG immune response against PLpx.

INTRODUCTION

Small interfering RNA (siRNA) has gained considerable attention as a powerful tool for silencing specific genes.^1,2) Onpattro^® is siRNA drug encapsulated in a lipid nanoparticle for the treatment of polyneuropathies caused by hereditary transthyretin amyloidosis. This drug has been approved for clinical use,³⁾ and its further development continues at the preclinical and clinical levels.⁴⁾ Poor circulation stability has given siRNA therapeutics an unfavorable pharmacokinetics profile.⁵⁾ It is of utmost importance that suitable delivery systems be developed. These systems include lipid nanoparticles (LNPs),⁶⁾ stable nucleic acid-lipid particles (SNALP),⁷⁾ and cationic liposomes⁸⁾ that could protect siRNA from degradation and improve targeting to specific tissues.⁹⁾ Modification with polyethylene glycol (PEG), so called PEGylation, has become the standard in vivo delivery system to improve stability and extend circulation following administration compared with non-PEGylated delivery system.¹⁰⁾

Despite the fact that PEGylation efficiently improve the circulation half-life of delivery system, many findings have demonstrated a loss of long-circulation properties of PEGylated delivery systems, like liposomes,^11–13) proteins,¹⁴⁾ micelles,¹⁵⁾ and LNP,¹⁶⁾ upon repeated intravenous (IV) injections via a phenomenon referred to as accelerated blood clearance (ABC) phenomenon.” In the case of PEGylated liposomes, the initiator of ABC was confirmed to be anti-PEG immunoglobulin M (IgM) produced in response to the first dose. The production level of anti-PEG IgM, however, is now known to be substantially influenced by not only the dose, length and density of PEG,¹³⁾ but also the species of the encapsulated drugs.^17–19) Encapsulation of chemotherapeutic agents such as doxorubicin¹⁷⁾ and oxaliplatin¹⁸⁾ within PEGylated liposomes has attenuated/abrogated anti-PEG IgM production. On the other hand, compared with empty PEGylated liposomes, siRNA complexed with PEGylated cationic liposomes (PLpx) significantly enhances anti-PEG IgM production in an siRNA-sequence-dependent manner.^19–21) It has been reported that poly U and GU-rich sequences in siRNA stimulate toll-like receptor 7, which results in induction of inflammatory cytokines.²²⁾ For example, siRNA for β-galactosidase (siβ-gal), which has an immunostimulatory sequence,²²⁾ strongly induces anti-PEG IgM production, while siRNA for green fluorescent protein (GFP) (siGFP), which lacks such an immunostimulatory sequence, weakly induces anti-PEG IgM production. Immune activation by PEG as well as siRNA might attenuate the efficacy of PLpx-based drugs. However, the exact mechanism by which PLpx elicits anti-PEG IgM production is not yet fully understood.

We previously demonstrated the essential role of the spleen in the production of anti-PEG IgM against empty PEGylated liposomes.²³⁾ Splenectomy (removal of spleen) significantly attenuates the production of anti-PEG IgM following IV administration of PEGylated liposomes.¹⁴⁾ In the same context, we have reported that anti-PEG IgM was produced from spleen cells following IV injection of PLpx.²⁰⁾ We also found, however, that splenectomy fails to completely prevent the production of anti-PEG IgM following the administration of either empty PEGylated liposomes or PLpx; in experimentation, some anti-PEG IgM production has been observed in splenectomized mice.^14,19) Particularly, PLpx induced full level of anti-PEG IgM even in splenectomized mice compared with sham-operated mice. Both empty PEGylated liposomes and PLpx induce anti-PEG IgM in T cell-independent manner, but immunostimulatory nucleic acids on PLpx further modulate immune system. This prompts the assumption that other lymphoid cells/tissues other than the spleen contribute to anti-PEG IgM production following the IV administration of immunostimulatory PLpx.

In the present study, therefore, we demonstrated the contribution of immune cells in the peritoneal cavity to secretion of anti-PEG IgM following IV injection of PLpx in both normal and splenectomized mice. We demonstrated that peritoneal cells as well as spleen cells participate in anti-PEG IgM production following the injection of PLpx complexed with immunostimulatory siRNA. In addition, interleukin-6 (IL-6) secreted by peritoneal immune cells, in response to IV injections of PLpx, stimulated the proliferation/differentiation of peritoneal PEG-specific B cells, particularly B1a cells, in plasma cells and, thereby, triggered the production of anti-PEG IgM. Our results suggest that IV injections of PLpx could influence immune cells not only in the spleen, but also in the peritoneal cavity.

MATERIALS AND METHODS

Materials and Animals

Hydrogenated egg phosphatidylcholine (HEPC), palmitoyloleoyl phosphatidylcholine (POPC), and dioleoyl phosphatidylethanolamine (DOPE) were purchased from NOF (Tokyo, Japan). Samples of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG₂₀₀₀-DSPE) were kindly donated by Nippon Fine Chemical (Osaka Japan). Cholesterol (Chol) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The cationic lipid, O,O′-ditetradecanoyl-N-(alpha-trimethylammonio acetyl) diethanolamine chloride (DC-6-14) was purchased from Sogo Pharmaceutical (Tokyo, Japan). DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) was purchased from Thermo Fisher Scientific (MA, U.S.A.). All other reagents were of analytical grade.

Animals

Male BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). C.B-17/lcr-scid/scid (SCID) mice were purchased from Japan CLEA (Tokyo, Japan). The mice were 4-8 weeks old at the beginning of each experiment. All animal experiments were conducted following evaluation and approval by the Animal and Ethics Review Committee of Tokushima University (Approved Number: T2019-47).

siRNA

siRNA was chemically synthesized by Hokkaido System Science (Hokkaido, Japan). The sequences of the siRNAs for GFP (siGFP) are 5′-GGCUACGUCCAGGAGCGCATTdTdT-3′ (sense) and 5′-UGCGCUCCUGGACGUAGCCTTdTdT-3′ (antisense). The sequences of siRNA for β-galactosidase (siβ-gal) have an immunostimulatory motif²²⁾: 5′-CUACACAAAUCAGCGAUUUUUdTdT-3′ (sense) and 5′-AAAUCGCUGAUUUGUGUAGUUdTdT-3′ (antisense). Annealing was performed by heating mixtures at 90 °C for 5 min, which was followed by cooling at room temperature.

Preparation of siRNA-PEGylated Cationic Liposome Complex

siRNA complexed with PEGylated cationic liposomes (PLpx) was prepared as described previously.²⁰⁾ Cationic liposomes, composed of DOPE:POPC:Chol:DC-6-14 (3 : 2 : 3 : 2, molar ratio), were prepared via a thin-film hydration and extrusion with a polycarbonate membrane with successive pore sizes of 400, 200, 100, and 80 nm. In the biodistribution study, fluorescent lipid dye, DiI, was mixed with cationic liposome components to achieve 1% of DiI against total lipids (molar ratio). For PEGylation, mPEG₂₀₀₀-DSPE (10 mg/mL) was mixed with cationic liposomes to achieve 5% of PEG-DSPE against total lipids (molar ratio) and incubated at 37 °C for 60 min, which is so-called a post-insertion technique to incorporate PEG in outer lipid layer of the preformed liposomes.²⁴⁾ The size and zeta potential of the PEGylated cationic liposomes were 98 nm and 12 mV, which was determined via a Zetasizer Nano (Malvern Instruments, U.K.). Finally, PEGylated cationic liposomes were mixed with either siGFP or siβ-gal at a ratio of 3.81 (nitrogen/phosphate ratio, N/P ratio), and the mixture then was vigorously vortexed for 15 min to form PEGylated cationic liposome-siRNA complex (GFP PLpx or β-gal PLpx) through electrostatic interaction. The size and zeta potential of GFP PLpx were 190 nm and 9 mV, and those of β-gal PLpx were 160 nm and 3 mV, respectively. Agarose electrophoresis analysis showed that almost all siRNAs were loaded onto PLpx without free siRNA. To detect PEG-specific cells, fluorescence-labeled PEGylated liposomes, composed of HEPC:Chol:mPEG₂₀₀₀-DSPE:DiI (1.85 : 1 : 0.15 : 0.02, molar ratio), were prepared as described previously.²⁵⁾ The size and zeta potential of prepared PEGylated liposomes were 106 nm and −8.8 mV. Concentration of phospholipids in the liposomes were determined via colorimetric assay.²⁶⁾

Anti-PEG IgM Production in Splenectomized Mice

Splenectomized mice were prepared 1 d before liposome injection by removing the spleen through a flank incision as described previously.²³⁾ As a control, sham-operated mice were prepared by the same procedure without removal of the spleen. Some splenectomized mice were intravenously (IV) injected with GFP PLpx while some received injections of β-gal PLpx, and the same procedure was applied to sham-operated mice (0.75 µmol phospholipids and 12.5 µg siRNA/mouse). At 2–5 d after injection, blood was collected and centrifuged to obtain sera. Anti-PEG IgM in the sera was measured via enzyme-linked immunosorbent assay (ELISA) (Anti-PEG IgM ELISA kit, Nano T-Sailing, Tokushima, Japan) according to manufacturer’s protocol without modification.

In Vitro Secretion of Anti-PEG IgM Production from Splenic Cells, Peritoneal Cells, Lymph Node Cells, or Blood Cells

In order to confirm which cells contribute to secretion of anti-PEG IgM, immune cells in spleen, peritoneal cavity, lymph nodes and blood were cultured following the injection of PLpx. Some splenectomized mice were IV injected with GFP PLpx while some received injections of β-gal PLpx, and the same procedure was applied to sham-operated mice. Three days later, when immune cells are activated but do not secrete anti-PEG IgM, the spleen and lymph nodes were collected and suspended in RPMI-1640 medium. Then, cell suspensions were prepared by pressing tissue through a cell strainer (100 µm, Greiner Bio-One, Kremsmünster, Austria) followed by lysis of the red blood cells as described previously.²⁵⁾ Peritoneal cells were obtained by washing the peritoneal cavity with 2.5 mL of Dulbecco’s phosphate buffered saline (PBS) (pH 7.4) followed by lysis of the red blood cells. The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin and 100 µg/mL streptomycin and 50 µM 2-mercaptoethanol at a cell density of 2.0 × 10⁶ cells/mL for 2 d in vitro. Anti-PEG IgM in the supernatant was measured via ELISA, as described above.

Contribution of Spleen Cells and Peritoneal Cells to Anti-PEG IgM Production by siRNA-PLpx in the Cells of Adoptively Transferred Mice

In order to confirm the secretion of anti-PEG IgM in vivo, either the spleen cells or peritoneal cells suspended in Dulbecco’s PBS (1 × 10⁷ live cells confirmed by trypan blue assay) from naïve mice were adoptively transferred into immune-deficient SCID mice via either IV injection or intraperitoneal (i.p.) injection, respectively. One day later, either GFP PLpx or β-gal PLpx was IV injected into the mice. At day 5 following the PLpx injection, the anti-PEG IgM in the serum was measured via ELISA, as described above.

Detection of PEG-Specific Cells via Flow Cytometry

Some splenectomized mice were IV injected with GFP PLpx while some received injections of β-gal PLpx, and the same procedure was applied to sham-operated mice. Three days later, spleen cells and peritoneal cells were obtained from the mice as described above. In order to detect PEG-specific cells, a PEG-specific assay was performed according to a procedure established in our previous study.²⁵⁾ Briefly, the cells were incubated with DiI-labeled PEGylated liposomes in combination with either anti-mouse IgM antibody-APC conjugate (Biolegend) or anti-mouse CD138 antibody-APC conjugate (Biolegend) for 30 min at 4 °C. After washing twice with cold Dulbecco’s PBS, the cells were analyzed by flow cytometry (Gallios, Beckman Coulter). After gating on lymphocytes identified by forward scatter and side scatter, PEG-specific B cells and PEG-specific plasma cells were considered as DiI⁺ IgM⁺ cells and DiI⁺ CD138⁺ cells, respectively (Supplementary Fig. 1). The percentage of PEG-specific B cells and PEG-specific plasma cells was shown as DiI⁺ IgM⁺ cells/total IgM⁺ cells*100 and DiI⁺ CD138⁺ cells/total CD138⁺ cells*100. To distinguish between B cell subsets, anti-mouse CD5-FITC conjugate, anti-mouse CD23-PE/Cy7 conjugate, and anti-mouse/human B220-APC conjugate (Biolegend) were used instead of anti-mouse IgM and anti-mouse CD138. Each B cell subset was defined as DiI⁺ B220⁺ CD23⁻ CD5⁺ (PEG-specific B1a cells), DiI⁺ B220⁺ CD23⁻ CD5⁻ (PEG-specific B1b cells), or DiI⁺ B220⁺ CD23⁺ CD5⁻ (PEG-specific B2 cells).

Uptake of IV Injected siRNA-PLpx by Peritoneal Cells

Either DiI-labeled GFP PLpx or DiI-labeled β-gal PLpx was IV injected into splenectomized mice. One day later, peritoneal cells were collected and stained with APC-conjugated anti-mouse/human B220 as described above. The uptake of PLpx by B cells (B220⁺) and non-B cells (B220⁻) was determined via flow cytometry (Gallios).

Cytokine Secretion by Peritoneal Cells

Either GFP PLpx or β-gal PLpx was IV injected into splenectomized mice. At 4 and 9 h post-injection, peritoneal lavage was obtained by washing the peritoneal cavity with 1.5 mL of Dulbecco’s PBS. Cytokines (IL-6, tumor necrosis factor (TNF)-α, interferon-γ (IFN-γ)) in the peritoneal lavage were determined via a Quantikine ELISA kit (R&D system, Minneapolis, MN, U.S.A.) according to manufacturer’s protocol without modification.

Statistical Analysis

All data are expressed as the mean ± standard deviation (S.D.) (n = 3 animals). Statistical analysis was performed with a two-tailed unpaired t test and one-way ANOVA followed by a Tukey post-hoc test using GraphPad InStat software (GraphPad Software, CA, U.S.A.). p < 0.05 was considered statistically significant.

RESULTS

Injection of siRNA for β-Galactosidase-PEGylated Cationic Liposome Complex (β-Gal PLpx) Induced Anti-PEG IgM in the Absence of a Spleen

The effect of splenectomy on anti-PEG IgM production was investigated following the injection of PLpx. The PLpx was prepared by simple mixing of siRNA with PEGylated cationic liposome and the size and zeta potential of each liposome formulation were shown in Table 1. The PLpx were IV injected into either splenectomized mice or sham-operated control mice. At indicated time points, sera were collected and the level of anti-PEG IgM production in the sera was evaluated (Figs. 1A, B). In the sham-operated control mice, in both PLpxs, the induction of anti-PEG IgM was observed and the induction level reached a peak on days 4-5 following injection (Fig. 1A), which is consistent with our previous study in which anti-PEG IgM level was maximized on day 5 and gradually decreased by day 10.¹⁹⁾ The PLpx containing strong immunostimulatory siRNA (siβ-gal) (β-gal PLpx) caused robust anti-PEG IgM production, by comparison with the GFP-PLpx that contained a less-stimulatory version of siRNA (siGFP), which is consistent with our previous report.²⁰⁾ In the splenectomized mice, it is interesting that the anti-PEG IgM induced by GFP-PLpx was completely diminished, while the anti-PEG IgM induced by β-gal-PLpx was maintained (Fig. 1B). In addition, similar anti-PEG IgM responses were observed when splenectomy was performed before and after injection of PLpx (Supplementary Fig. 2). These results indicate the contribution that cells/tissues other than the spleen made to anti-PEG IgM production following the injection of the β-gal-PLpx, which contains a powerful immunostimulatory siRNA (siβ-gal).

Table 1. Physicochemical Properties of the Prepared Liposomes

Formula	Particle size (nm)	PDI	Zeta potential (mV)
PEGylated liposome	106 ± 5	0.089	−9
PEGylated cationic liposome	98 ± 7	0.076	12
GFP PLpx	190 ± 3	0.124	9
β-Gal PLpx	160 ± 2	0.115	3

Fig. 1. Anti-PEG IgM Was Produced Following the Injection of a siRNA-PEGylated Cationic Liposome Complex (PLpx) in Splenectomized Mice

Either siGFP complexed with PEGylated cationic liposomes (GFP PLpx) or siβ-gal complexed with liposomes (β-gal PLpx) was IV injected into (A) sham-operated control or (B) splenectomized mice (0.75 µmol phospholipids and 12.5 µg siRNA/mouse). Then, serum was collected at indicated time points after the injection. Anti-PEG IgM in the serum was determined via ELISA. Each value represents the mean ± S.D. (n = 3 animals). * p < 0.05, ** p < 0.01 vs. no treatment.

Peritoneal Cells Produced Anti-PEG IgM Following an Injection of β-Gal PLpx

To address the possibility that cells other than splenic cells could contribute to anti-PEG IgM production, peritoneal cells, lymph node cells, blood cells, and spleen cells, which are abundant for immune cells, were collected from the mice following the IV administration of PLpx, and these cells were then cultured in vitro. In the culture supernatant, anti-PEG IgM was determined. The spleen cells, from the mice received either β-gal PLpx or GFP-PLpx, induced anti-PEG IgM production (Fig. 2A). It is noteworthy that peritoneal cells and lymph node cells from the mice received β-gal PLpx, which then induced a slight production of anti-PEG IgM (Fig. 2A). Neither lymph node cells nor blood cells from the mice that received GFP-PLpx produced anti-PEG IgM (Fig. 2A)

Fig. 2. Anti-PEG IgM Was Produced from Not Only Spleen Cells but Also from Peritoneal Cells Following the Injection of β-Gal PLpx

(A) Either GFP PLpx or β-gal PLpx was IV injected into sham-operated mice (0.75 µmol phospholipids and 12.5 µg siRNA/mouse). Spleen cells, peritoneal cells, lymph node cells and blood cells were collected at Day 3 after the injection and cultured for a further 2 d. Anti-PEG IgM in cell supernatant was determined via ELISA. (B) Peritoneal cells of BALB/c mice were adoptively transferred (1.0 × 10⁷ cells/mouse) into SCID mice by i.p. injection. As a control, spleen cells from BALB/c mice were transferred (1.0 × 10⁷ cells/mouse) into SCID mice by IV injection. β-gal PLpx (0.75 µmol phospholipids and 12.5 µg siRNA/mouse) were IV injected into the mice on Day 1 after adoptive transfer. Then, serum was collected on Day 5 after the PLpx injection. Anti-PEG IgM in the serum was determined via ELISA. Each value represents the mean ± S.D. (n = 3 animals) * p < 0.05, *** p < 0.001.

In order to verify the predominant contribution that peritoneal cells make to anti-PEG IgM production under in vivo conditions, we performed an adoptive transfer of peritoneal cells from immunocompetent mice into immunodeficient SCID mice. After i.p. adoptive transfer of peritoneal cells that included B cells, β-gal PLpx was IV injected into SCID mice and anti-PEG IgM production was evaluated 5 d later (Fig. 2B). As a positive control, spleen cells were adoptively transferred via IV injection into SCID mice. The SCID mice reconstituted with immunocompetent spleen cells efficiently produced anti-PEG IgM following injection of β-gal PLpx, while SCID mice without reconstitution did not (Fig. 2B). Interestingly, the SCID mice reconstituted with immunocompetent peritoneal cells produced noticeable amount of anti-PEG IgM, which indicates that, in addition to spleen cells, peritoneal cells efficiently contribute to anti-PEG IgM production following injections of β-gal PLpx.

The Occurrence of PEG-Specific B Cells Is Increased in the Peritoneal Cavity Following an Injection of β-Gal PLpx

As a reaction to PEG in mice, we have described the presence of PEG-specific B cells, which produce anti-PEG IgM.²⁵⁾ Our previous study showed that the number of PEG-specific B cells increased on day 3 after injection of PEGylated liposomes. Therefore, in the present study, we evaluated the number of PEG-specific cells in the spleen and peritoneal cavity on day 3 after IV injection of PLpx. In the present study, we examined the effect that the injection of PLpx exerts on the number of PEG-specific cells in the spleen and peritoneal cavity. Both β-gal PLpx and GFP PLpx tended to increase the number of PEG-specific B cells in the spleen (Fig. 3A, left). Only β-gal PLpx significantly increased PEG-specific plasma cells that are known to mount a massive antibody response (Fig. 3A, right). These results suggest that β-gal PLpx induces differentiation from B cells in the spleen to plasma cells, which leads to anti-PEG IgM production. In the peritoneal cavity cells, β-gal PLpx significantly increased PEG-specific B cells (Fig. 3B, left) and tended to increase plasma cells (Fig. 3B, right), but GFP PLpx did not. The increase of PEG-specific B cells was confirmed in splenectomized mice which received with β-gal PLpx (Supplementary Fig. 3). It is worth noting that the total numbers of B cells and T cells in the peritoneal cavity were not affected by the injections of either of the forms of PLpxs (Supplementary Fig. 4).

Fig. 3. PEG-Specific B Cells and Plasma Cells Were Increased in the Spleen and in the Peritoneal Cavity Following the Injection of PLpx

Either GFP PLpx or β-gal PLpx was IV injected into sham-operated mice (0.75 µmol phospholipids and 12.5 µg siRNA/mouse). (A) Spleen cells and (B) peritoneal cells were collected at Day 3 after the injection. (Left panel) PEG-specific B cells and (right panel) PEG-specific plasma cells were stained and determined via flow cytometry. Each value represents the mean ± S.D. (n = 3 animals) * p < 0.05, ** p < 0.01 vs. no treatment.

Peritoneal B cells are known to generally divide into one of three subsets: B1a cells, B1b cells, and B2 cells.²⁷⁾ Flow cytometry analysis was performed to identify the population of peritoneal PEG-specific B cells in splenectomized mice that made the largest contribution to anti-PEG IgM production (Fig. 4). Injection of GFP PLpx into the splenectomized mice increased none of the B-cell subsets in the peritoneal cavity cells, which is agreement with the results shown in Fig. 3B. Injection of β-gal PLpx tended to increase the number of PEG-specific B1a cells compared with other B cell subsets. This indicates that B1a cells could contribute to anti-PEG IgM production in the peritoneal cavity.

Fig. 4. PEG-Specific B1a Cells in Peritoneal B Cells Were Increased Following Injection of PLpx

Either GFP PLpx or β-gal PLpx was IV injected into splenectomized mice (0.75 µmol phospholipids and 12.5 µg siRNA/mouse). Peritoneal cells were collected at Day 3 after the injection. PEG-specific B1a cells, B1b cells, or B2 cells were stained and determined via flow cytometry. Each value represents the mean ± S.D. (n = 3 animals)

Both β-Gal PLpx and GFP PLpx Interact with Peritoneal Cells

B cells differentiate and secrete antibody following direct interaction with antigens through B cell receptor on the surface of B cells. Therefore, the levels of interaction of each PLpx with peritoneal cells affect the anti-PEG IgM production. To gain insight into the factors that could contribute to the anti-PEG IgM response following PLpx administration into splenectomized mice, the interactions of the PLpx with the peritoneal B cells was evaluated (Fig. 5). Both β-gal PLpx and GFP PLpx efficiently interacted with peritoneal B cells to a similar extent. In addition, both β-gal PLpx and GFP PLpx efficiently interacted with non-B cells in the peritoneal cavity. It appears that the higher anti-PEG IgM production in the β-gal PLpx compared with that in the GFP PLpx (Fig. 1) could be independent of the interaction of the β-gal PLpx with the peritoneal cavity B cells.

Fig. 5. Both GFP PLpx and β-Gal PLpx Were Interacted with Peritoneal B Cells and Non-B Cells

Either DiI-labeled GFP PLpx or DiI-labeled β-gal PLpx was IV injected into splenectomized mice (0.75 µmol phospholipids and 12.5 µg siRNA/mouse). Then, peritoneal cells were collected at Day 1 after the injection. These cells were stained with anti-B220 antibody. The interactions of PLpx with B cells (DiI⁺B220⁺) and non-B cells (DiI⁺B220⁻) with peritoneal B cells were determined via flow cytometry. Each value represents the mean ± S.D. (n = 3 animals) ** p < 0.01 vs. no treatment.

siRNA for β-Gal PLpx Induced IL-6 Production in the Peritoneal Cavity, but GFP PLpx Did Not

Inflammatory cytokines are known to contribute to B-cell activation and proliferation, which results in antibody production.²⁸⁾ We evaluated the production of inflammatory cytokines, TNF-α, IFN-γ and IL-6, in the peritoneal cavity in response to IV injection of β-gal PLpx into the splenectomized mice (Fig. 6). IV injection of β-gal PLpx induced production of IL-6 in the peritoneal cavity at 4 h post treatment, which returned to normal levels at 9 h. This injection, however, induced the production of neither TNF-α nor IFN-γ. On the other hand, injection of GFP PLpx barely increased the production of any cytokines. Given that IL-6 is an essential cytokine for B cell proliferation and differentiation,²⁹⁾ these results suggest that induction of IL-6 is likely to contribute to anti-PEG IgM production from peritoneal B cells following injection of the β-gal PLpx.

Fig. 6. β-Gal PLpx Induced IL-6 Secretion in the Peritoneal Cavity

Either GFP PLpx or β-gal PLpx was IV injected into the splenectomized mice (0.75 µmol phospholipids and 12.5 µg siRNA/mouse). At 4 or 9 h after the injection, 1.5 mL of PBS was injected into the peritoneal cavity and the peritoneal lavage was collected. IL-6, IFN-γ, and TNF-α in the lavage was determined via ELISA. Each value represents the mean ± S.D. (n = 3 animals) * p < 0.05 vs. no treatment.

DISCUSSION

Immune activation by siRNA-based drugs represents a significant drawback to a full deployment of RNA interference (RNAi) in clinical settings.³⁰⁾ Accordingly, understanding the mechanism of any immune responses to siRNA-based drugs is a crucial for the development of safe and effective siRNA-based or nucleic acids-based therapeutics. Accordingly, in the present study, we employed immunostimulatory siRNA siβ-gal,²²⁾ and purposefully also used the less immunostimulatory version of siRNA, siGFP. Here, we were able to demonstrate how IV injection of the siRNA-PEGylated cationic liposome complex (PLpx) induced the production of anti-PEG IgM (Fig. 1). It appears that not only spleen cells but also peritoneal cells contributed to PLpx-induced anti-PEG IgM production (Fig. 2). Immunostimulatory siβ-gal complexed with the liposomes (β-gal PLpx) induced abundant anti-PEG IgM production. Those significantly increased the number of PEG-specific B cells in spleen cells and in the B1 cells in the peritoneal cavity cells, as well as promoting the differentiation from B1 cells to plasma cells (Figs. 3, 4), which in turn led to abundant anti-PEG IgM production. To the best of our knowledge, this is the first report that has described how PEG-specific B cells in the peritoneal cavity contribute to the secretion of anti-PEG IgM that is induced by the IV injection of siRNA-containing PEGylated nanoparticles such as PLpx. Further studies are needed to confirm the contribution of lymph nodes, rather than spleen and peritoneal cavity, to anti-PEG IgM production in future.

In a previous study, we reported that the anti-PEG IgM production level induced by PLpx predominantly depends on the immunostimulatory activity of siRNA.²⁰⁾ We also reported that the spleen plays an essential part in an anti-PEG IgM response against PEGylated liposomes.¹⁴⁾ In the present study, we used splenectomized (no spleen) mice to show how the IV injection of a β-gal PLpx containing the immunostimulatory potential of siβ-gal significantly overrides the compromising effect of splenectomy and succeeds in inducing anti-PEG IgM (Fig. 1), which presumably is secreted from peritoneal cells (Fig. 2). The production of anti-PEG IgM was almost the same between sham-operated group and splenetomized mice, suggesting major origin of anti-PEG IgM is peritoneal cavity. On the other hand, as shown in Fig. 2, splenic cells secrete more anti-PEG IgM than peritoneal cells after injection of β-gal PLpx. This discrepancy might be attribute to the alternative role of peritoneal cavity in the absence of spleen. As shown in Fig. 5, it was interesting that we could detect the interaction of PLpx with the cells in the peritoneal cavity following the IV injection of PLpx. IL-6 secretion was also detected from the cells of the peritoneal cavity following IV injection (Fig. 6). Bally et al.³¹⁾ reported that liposomal doxorubicin can reach the peritoneal cavity following IV injection. These results suggest that IV-injected PLpx reaches the peritoneal cavity where it affects the local immunity and in turn induces anti-PEG IgM production in mice. A more detailed study is required, however, to account for the mechanism of how PLpx moves into the peritoneal cavity from blood circulation following IV injection. On the other hand, our recent study demonstrated that PEGylated liposomes induced anti-PEG IgM following all tested route of injection (intravenous, intraperitoneal, intramuscular, subcutaneous), but the level of anti-PEG IgM induction varied with routes.³²⁾ Accumulation amounts of PEGylated liposomes in each lymphoid tissue might affect the production of anti-PEG IgM.

It is noteworthy that splenectomy subverted/abrogated anti-PEG IgM production following the IV injection of a less immunostimulatory form of siRNA (GFP PLpx) (Figs. 1, 2). That observation excludes the contribution of peritoneal cells to the immunogenic response against the less-immunostimulatory GFP PLpx. The literature contains reports that poly U and GU-rich siRNA stimulates innate immunity to produce inflammatory cytokines via signaling by toll-like receptor 7 (TLR7).^22,33) We have demonstrated that IV injection of β-gal PLpx stimulates the secretion of IL-6 into serum in a TLR7-dependent manner, while GFP PLpx did not.²⁰⁾ Similarly, in the current study, IV injection of β-gal PLpx induced a production of IL-6 in the peritoneal cavity (Fig. 6) with a simultaneous increase in PEG-specific B cells and plasma cells in the peritoneal cavity (Fig. 3). IL-6 is known to regulate B cell proliferation and differentiation into plasma cells.²⁹⁾ Accordingly, the induction of IL-6 by β-gal PLpx could contribute to the proliferation/differentiation of PEG-specific B cells and plasma cells in the peritoneal cavity, which would result in a robust anti-PEG IgM response. It is worth noting that despite the fact that IV injection of GFP PLpx did not trigger a significant production of IL-6 in the peritoneal cavity (Fig. 6), the higher sensitivity of spleen cells against PEG along with higher PLpx accumulation in the spleen, compared with that in the peritoneal cavity, could account for the pronounced anti-PEG IgM immune response observed against GFP PLpx in normal “non splenectomized” mice.

The marginal zone (MZ) and B1 B lymphocytes are known to participate jointly in the early-phase antibody production against T cell-independent (TI) antigens.^34,35) In our previous studies we noted that PEGylated liposomes and PLpx induce an anti-PEG IgM response in a T cell-independent pathway (acting as a T cell-independent antigen type 2 (TI-2 antigen)).^19,36) No reports, however, have thus far demonstrated the contribution of B1 B lymphocytes to immune responses against PEGylated liposomes and PLpx. B1 cells are a subclass of B lymphocytes that exist mainly in the peritoneal cavity and the spleen. These are known to play an essential role in TI immune responses as well as the splenic MZ B cells. The two types of B1 cells, CD5⁺ B1a cells and CD5⁻ B1b cells, have different repertoires.³⁷⁾ In the present study, we showed that in the absence of splenic B lymphocytes (splenectomized mice), an IV injection of β-gal PLpx tend to increase the peritoneal PEG-specific B1a cells (Fig. 4), and this was correlated with anti-PEG IgM production in splenectomized mice (Figs. 1, 2). In the same context, Kim et al.³⁸⁾ reported a similar phenomenon whereby lactosome, which is a core-shell-type polymeric micelle classified as a TI-2 antigen, triggered lactosome-specific B1 cells in the peritoneal cavity with the subsequent development of a potent anti-lactosome antibody response following IV injection. Characteristics of TI-2 responses is early B cell proliferation and differentiation into plasma cells that start from early phase after immunization,³⁹⁾ which is consistent with our current results (Fig. 1A). In contrast to thymus-dependent (TD) antigen,^40–42) TI-2 responses generally cause neither memory B cell development nor affinity maturation,^39,43–45) and the number of plasma cells decreases significantly after immunization.³⁹⁾ Little is known, however, about how peritoneal B1a cells respond to PEGylated liposomes and PLpx. Further studies are being conducted with PEG-specific peritoneal B1a cells isolated from the peritoneal cavity cells.

To date, RNA-lipoplexes (RNA-LPX) have reached clinical trials as a cancer vaccine for the induction of antitumor immunity.⁴⁶⁾ RNA-LPX are sometimes modified with PEG to prevent aggregation of the particles and to improve the stability in vivo.⁴⁷⁾ In addition, LNPs have been widely utilized for the delivery of siRNA³⁾ and mRNA⁴⁸⁾ and some LNP-based therapeutics have now been approved for clinical uses. LNPs generally include PEG lipids to prevent particle aggregation, although the presence of PEG may reduce the interaction of LNPs with target cells. Therefore, most LNPs employ short acyl-chain PEG lipids to enhance the shedding of PEG lipids from the particles following administration.⁴⁹⁾ Due to the rapid shedding of PEG lipids, LNPs with short acyl-chain PEG lipids attenuate anti-PEG IgM production compared with long acyl-chain PEG lipids.¹⁶⁾ However, a complete attenuation of anti-PEG antibody production has not yet been achieved with the shedding of PEG lipids.^50,51) Other factors that affect anti-PEG IgM production by RNA-LPX or RNA-LNPs include the immunogenicity of RNA and lipids other than PEG lipids.^22,52) As shown in both our previous¹⁹⁾ and the current study, immunostimulatory siRNA in PLpx increased anti-PEG IgM production through activation of innate immune systems such as TLRs. Recent advances in nucleic acid modification, such as ribose 2′-modifications in siRNA⁵³⁾ and pseudouridine in mRNA,⁵⁴⁾ could attenuate the activation of innate immune systems. Empty LNPs without RNA have also shown intrinsic adjuvant activity and induced the secretion of IL-6,⁵⁵⁾ which might enhance the production of anti-PEG IgM. These activations of innate immune systems partly contribute to the vaccine efficacy of mRNA-LNPs, and therefore a novel strategy that could attenuate anti-PEG antibody production without impairing therapeutic efficacy is required for the further development of RNA-LNPs and RNA-LPX. Such studies are required in the future.

CONCLUSION

In this study, we described how IV injection of PLpx induces the production of anti-PEG IgM in mice even in the absence of a spleen. The production of anti-PEG IgM induced by PLpx is attributed to not only spleen B cells but also to peritoneal B cells with the help of the immunostimulatory effect of siRNA. Peritoneal B1a cells could be a contributing factor in the immune responses against PEGylated nanoparticles.

Acknowledgments

We thank Dr. James L. McDonald for his helpful advice in writing the English manuscript. This study was in part supported by the SENSHIN Medical Research Foundation, by the TERUMO LIFE SCIENCE FOUNDATION, by the KOSÉ Cosmetology Research Foundation, by a Grant-in-Aid for Transformative Research Areas (A) (Publicly Offered Research) (21H05526) and a Grant-in-Aid for Fostering Joint International Research (B) (19KK0279) and a Grant-in-Aid for Scientific Research (B) (23H03739) from the Japan Society for the Promotion of Science, and by a research program for the development of an intelligent Tokushima artificial exosome (iTEX) from Tokushima University.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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