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. Author manuscript; available in PMC: 2024 May 15.
Published in final edited form as: Arthritis Rheumatol. 2021 Mar 8;73(5):769–778. doi: 10.1002/art.41585

Suppression of Rheumatoid Arthritis by Enhanced Lymph Node Trafficking of Engineered Interleukin-10 in Murine Models

Eiji Yuba 1,2, Erica Budina 1, Kiyomitsu Katsumata 1, Ako Ishihara 1, Aslan Mansurov 1, Aaron T Alpar 1, Elyse A Watkins 1, Peyman Hosseinchi 1, Joseph W Reda 1, Abigail L Lauterbach 1, Mindy Nguyen 1, Ani Solanki 3, Takahiro Kageyama 4, Melody A Swartz 1,5,6, Jun Ishihara 1,7,*, Jeffrey A Hubbell 1,6,*
PMCID: PMC11095083  NIHMSID: NIHMS1962091  PMID: 33169522

Abstract

Objective.

Rheumatoid arthritis (RA) is a major autoimmune disease that causes synovitis and joint damage. Although clinical trials using interleukin-10 (IL-10), an anti-inflammatory cytokine, have been performed as a potential treatment of RA, its therapeutic effects have been limited, potentially due to insufficient residence in lymphoid organs, where antigen recognition primarily occurs. Here, we engineered IL-10 as a fusion with serum albumin (SA).

Methods.

SA-fused IL-10 was recombinantly expressed. After intravenous injection to mice, retention of SA-IL-10 at lymph node (LN), immune cell compositions at paws, and therapeutic effect on arthritis model mice were assessed.

Results.

SA fusion to IL-10 led to enhanced LN accumulation compared with unmodified IL-10. Intravenous SA-IL-10 treatment restored immune cell composition in the paws to a normal status, elevated the frequency of suppressive M2 macrophages, reduced IL-17A amount in the paw-draining LN, and protected joint morphology. Intravenous SA-IL-10 treatment showed similar efficacy as treatment with an anti-TNF-α antibody. SA-IL-10 was equally effective when administered intravenously, locally or subcutaneously, which benefits clinical translation of this molecule.

Conclusion.

SA fusion to IL-10 is a simple but effective engineering strategy for RA therapy and holds clinical translational potential.

Keywords: interleukin-10, albumin fusion, lymph node targeting, tolerance

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease that is currently controlled through treatment with inhibitors of inflammatory pathways. Pathological features of RA are synovitis and joint destruction, which cause severe pain and joint dysfunction (1, 2). Although the causal antigen for RA has not been fully elucidated, collagen recognition by immune cells plays a key role. During progression of RA, autoantigen-specific T cells, especially Th17 cells, are activated and produce inflammatory cytokines including IL-17. Inflammatory cytokines, such as TNF-α and IL-6, in the joint induce activation of macrophages and neutrophils as mediators of the inflammatory response. These inflammatory cells infiltrate the joints and cause various inflammatory responses including activation of osteoclasts that destroy the bones in the joint (3). The current strategy for RA treatment is symptomatic, and, considering that many inflammatory cytokines are involved RA progression, various biological therapeutics such as antibodies or soluble receptors for TNF-α have been developed and approved for clinical use (4).

As another type of biological therapeutic, administration of anti-inflammatory cytokines has been studied for treatment of RA to induce systemic suppression of inflammation or tolerance. IL-10 is one such anti-inflammatory cytokine (5–7), and various attempts have been performed to explore IL-10-based autoimmune disease therapeutics (6–8). However, the therapeutic effect of IL-10 in autoimmune disease is still controversial, possibly because of its short circulating half-life and its uncontrolled biodistribution after systemic administration (8). To improve these drawbacks for IL-10, molecular engineering of IL-10 has been employed. Poly(ethylene glycol) (PEG) has been grafted to IL-10 to prolong circulation (9); however, PEGylation generally induces significant decreases of protein bioactivity, since controlling the extent of modification and the modification site is challenging. An IL-10 variant that targets an extracellular matrix protein splice variant that is present in sites of chronic inflammation has been produced by fusion of an antibody fragment to promote accumulation of IL-10 within the inflamed site, showing enhanced function in the active collagen-induced arthritis (CIA) model (10–12). IL-10 accumulation in the inflamed site directly suppresses inflammation at the disease site, whereas IL-10 also binds to IL-10 receptor-expressing cells such as macrophages, dendritic cells and Th17 (13, 14) present in secondary lymphoid organs, which induces various biological responses including differentiation of T cells, polarization of antigen presenting cells, decrease of CD86 expression on antigen presenting cells, and suppression of inflammatory responses (15). Therefore, IL-10 delivery to the secondary lymphoid organs would be a promising approach to induce systemic suppression of inflammation or tolerance, whereas successful candidates have not yet been developed.

In the present study, we engineered IL-10 by fusion of serum albumin (SA), seeking to explore if IL-10’s therapeutic effects on RA would be improved. We made the observation that SA fusion to IL-10 not only enhanced circulation time but also accumulation in the lymph nodes (LNs). Here, suppression effects of arthritis by engineered IL-10 were evaluated using a passive collagen antibody-induced arthritis (CAIA) model and an active CIA model in the mouse. We found that SA fusion to IL-10 enhanced accumulation of IL-10 into LNs after intravenous injection. SA-fused IL-10 significantly improved the anti-inflammatory effects of IL-10 in the two murine RA models and functioned similarly to TNF-α blockade.

Materials and Methods

Production and purification of recombinant proteins

The sequences encoding for the mouse serum albumin without pro-peptide (25 to 608 amino acids of whole serum albumin), mouse IL-10, and a (GGGS)2 linker were synthesized and subcloned into the mammalian expression vector pcDNA3.1(+) by Genscript. A sequence encoding for 6 His was added at the C-terminus for further purification of the recombinant protein. Suspension-adapted HEK-293F cells were routinely maintained in serum-free FreeStyle 293 Expression Medium (Gibco). On the day of transfection, cells were inoculated into fresh medium at a density of 1 × 106 cells/mL. 2 μg/mL plasmid DNA, 2 μg/mL linear 25 kDa polyethylenimine (Polysciences), and OptiPRO SFM media (4% final concentration, Thermo Fisher) were sequentially added. The culture flask was agitated by orbital shaking at 135 rpm at 37°C in the presence of 5% CO2. Seven days after transfection, the cell culture medium was collected by centrifugation and filtered through a 0.22 μm filter. Culture media was loaded into a HisTrap HP 5 mL column (GE Healthcare), using an ÄKTA pure 25 (GE Healthcare). After washing the column with wash buffer (20 mM NaH2PO4, 0.5 M NaCl, pH 8.0), protein was eluted with a gradient of 500 mM imidazole (in 20 mM NaH2PO4, 0.5 M NaCl, pH 8.0). The protein was further purified with size exclusion chromatography using a HiLoad Superdex 200PG column (GE Healthcare) using PBS as an eluent. All purification steps were carried out at 4°C. The expression of the proteins was verified as >90% pure by SDS-PAGE. Purified proteins were tested for endotoxin via HEK-Blue TLR4 reporter cell line and endotoxin levels were confirmed to be less than 0.01 EU/mL. Protein concentration was determined through absorbance at 280 nm using NanoDrop (Thermo Scientific).

Detection of SA-IL-10 binding to FcRn

SPR measurements were carried out with Biacore X100 instrument. Recombinant mouse FcRn (Acro Biosystems) was immobilized via amine coupling on a C1 chip (GE Healthcare) for ~200 RU according to the manufacturer’s instructions. SA-IL-10 was flowed at decreasing concentrations in the running buffer (0.01 M monobasic anhydrous sodium phosphate, pH 5.8, 0.15 M NaCl) at 30 μL/min at room temperature. The sensor chip was regenerated with PBS, pH 7.4 for every cycle. Specific binding of SA-fused proteins to FcRn was calculated by comparison to a non-functionalized channel used as a reference. The Kd value of the SA-IL-10 was determined by fitting 1:1 Langmuir binding model to the data using BIAevaluation software (GE Healthcare).

Mice

BALB/c female mice at 7 wk of age and DBA/1J male mice at 8 wk of age were obtained from the Jackson Laboratory. Experiments were performed with approval from the Institutional Animal Care and Use Committee of the University of Chicago.

Binding of the proteins to splenocytes or LN-derived cells

Single-cell suspensions were obtained by gently disrupting the spleen and popliteal LN through a 70-μm cell strainer. Red blood cells were lysed with ACK lysing buffer (Quality Biological) for splenocytes. Cells were counted and re-suspended in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin (all from Life Technologies). 1 × 105 cells/well were seeded in a 96 well microplate and were incubated with 2 μg/100 μL of SA, or SA-IL-10 for 30 min on ice. After 4-times washing by PBS, cells were further incubated with anti-mouse albumin antibodies (abcam) for 20 min on ice. After 3-times washing by PBS, cells were incubated with 1 μg/mL AlexaFluor 647-labeled anti-Rabbit IgG (Jackson ImmunoResearch), anti-B220 (RA3–6B2, BioLegend), anti-CD3e (145–2C11, BD Biosciences), anti-CD4 (RM4–5, BD Biosciences), anti-CD8 (53–6.7, BD Biosciences), anti-CD11c (HL3, BD Biosciences), anti-CD45 (30-F11, BD Biosciences) and anti-F4/80 (T45–2342, BD Biosciences) antibodies for 20 min on ice. Cells were analyzed by flow cytometry as described below.

Plasma pharmacokinetics of the proteins

IL-10 or SA-IL-10 (equivalent to 35 μg of IL-10) was injected intravenously into female BALB/c mice. Blood samples were collected in protein-low binding tubes at 1, 5, 10, and 30 min, and 1, 4, 8 and 24 hr after injection, followed by overnight incubation at 4°C. IL-10 concentrations in serum were measured by IL-10 Mouse Uncoated ELISA kit (Invitrogen) according to the manufacturer’s protocol. Exponential two-phase decay (Y = Ae−αt + Be−βt) fitting was used to calculate the half-life. Fast clearance half-life, t1/2,α; slow clearance half-life, t1/2,β. Data were analyzed using Prism software (v8, GraphPad).

CAIA model

Arthritis was induced in female BALB/c mice by intraperitoneal injection of anti-collagen antibody cocktail (1.0 mg/mouse, Chondrex) on day 0, followed by intraperitoneal injection of LPS (25 μg/mouse, Chondrex) on day 3. On the day 3, mice were intravenously, subcutaneously (mid-back), or via footpad injected with PBS, wt IL-10, SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of Rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell) before LPS injection. Joint swelling was scored every day according to the manufacture’s protocol (Chondrex). On the last day of scoring, the hind paws were fixed in 10% neutral formalin (Sigma-Aldrich), decalcified in Decalcifer II (Leica), and then provided for histological analysis. Paraffin-embedded paws were sliced at 5 μm thickness and stained with H&E. The images were scanned with a Pannoramic digital slide scanner and analyzed using a Pannoramic Viewer software. The severity of synovial hyperplasia and bone resorption for the arthritis model was scored by three-grade evaluation (0–2) according to the previously reported criteria with slight modifications as follows: 0, normal to minimal infiltration of pannus in cartilage and subchondral bone of marginal zone; 1, mild to moderate infiltration of marginal zone with minor cortical and medullary bone destruction; 2, severe infiltration associated with total or near total destruction of joint architecture. The scores in both hind paws were summed for each mouse (score per mouse total, 0–4). The histopathological analyses were performed in a blinded fashion.

CIA model

Male DBA/1J mice (8 wk old) were immunized by subcutaneous injection at the base of the tail with bovine collagen/complete Freund’s adjuvant (CFA) emulsion (Hooke Kit, Hooke Laboratories). Three weeks later, a booster injection of bovine collagen/incomplete Freund’s adjuvant (IFA) emulsion (Hooke Kit, Hooke Laboratories) was performed. After the booster injection, mice were inspected every day, and joint swelling was scored according to the manufacture’s protocol (Hooke Laboratories). When showing total score of 2–4 (defined as Day 0), mice were intravenously injected with PBS, SA-IL-10 (each equivalent to 43.5 μg of IL-10), or 200 μg of Rat anti-mouse TNF-α antibody (clone XT3.11, Bio X Cell). On the last day of scoring, hind paws were collected and histological analyses were employed as described above.

In vivo bio-distribution study

To make fluorescently labeled protein, wt IL-10, and SA-IL-10 were incubated with 8-fold molar excess of using DyLight 800 NHS ester (Thermo Fisher) for 1 hr at room temperature, and unreacted dye was removed by a Zebaspin spin column (Thermo Fisher) according to the manufacturer’s instruction. BALB/c mice were intraperitoneal injected by anti-collagen antibody cocktail (1.0 mg/mouse) on day 0, subsequently 10 μg of LPS was injected to right hind paw on day 3. The following day, 20 μg of DyLight 800-labeled proteins were intravenously injected. After 4 hr, organs harvested from the disease model were imaged with the Xenogen IVIS Imaging System 100 (Xenogen) under the following conditions: f/stop: 2; optical filter excitation 745 nm; excitation 800 nm; exposure time: 5 sec; small binning. Each organ was weighed to normalize the fluorescence signal from each organ.

LN microscopy

BALB/c mice were intravenously injected with DyLight594-labeled wt IL-10 (43.5 μg) or SA-IL-10 labeled with equimolar amounts of dye. 24 hr after injection, popliteal LNs were harvested and frozen in dry ice with optimal cutting temperature (OCT) compound. Tissue slices (10 μm) were obtained by cryo-sectioning. The tissues were fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. After washing with PBS-T, the tissues were blocked with 2% BSA in PBS-T for 1 hr at room temperature. The tissues were stained with anti-mouse CD3 antibody (1:100, 145–2C11, BioLegend) or anti-mouse peripheral node addressin (PNAd) antibody (1:200, MECA79, BioLegend) and Alexa Fluor 488 donkey anti-rat (1:400, Jackson ImmunoResearch). The tissues were washed three times and then covered with ProLong gold antifade mountant with 4′, 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). An IX83 microscope (Olympus) was used for imaging with 10x magnification for CD3 staining, and a Leica SP8 3D Laser Scanning Confocal microscope with 20x magnification for PNAd staining. Images were processed using ImageJ software (NIH).

LN pharmacokinetics

wt IL-10, or SA-IL-10 (each equivalent to 35 μg of IL-10) was injected intravenously into CAIA mice. Popliteal, mesenteric, cervical LNs were collected at 30 min, and 1, 4, 8, 24, 32, 48 and 72 hr after injection, and were subsequently homogenized using Lysing Matrix D and FastPrep-24 5G (MP Biomedical) for 40 s at 5,000 beats/min in T-PER tissue protein extraction reagent (Thermo Scientific) with cOmplete™ proteinase inhibitor cocktail (Roche). After homogenization, samples were incubated overnight at 4°C. Samples were centrifuged (5,000 g, 5 min), and the total protein concentration and IL-10 concentration were analyzed using a BCA assay kit (Thermo Fisher) and IL-10 Mouse Uncoated ELISA kit (Invitrogen), respectively. Simultaneously, cytokine levels in the LN extract were measured using Mouse Uncoated ELISA kit (Invitrogen) or Ready-SET-Go! ELISA kits (eBioscience) according to the manufacturer’s protocol. For detection of GM-CSF, wt IL-10 or SA-IL-10 (each equivalent to 35 μg of IL-10) was injected intravenously twice with a 3 day interval into CAIA mice. The day following the last injection, popliteal LNs were collected for detection of GM-CSF.

Flow cytometry

CAIA mice were intravenously injected with PBS, wt IL-10, or SA-IL-10 (each equivalent to 43.5 μg of IL-10). Eight days after, blood and hind paws were harvested. Red blood cells in blood were lysed with ACK lysing buffer (Quality Biological), followed by antibody staining for flow cytometry. Paws were digested in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% FBS, 2 mg/mL collagenase D and 40 μg/mL DNase I (Roche) for 60 min at 37°C. Single-cell suspensions were obtained by gently disrupting through a 70-μm cell strainer. Antibodies against the following molecules were used: anti-mouse CD3 (145–2C11, BD Biosciences), CD4 (RM4–5, BD Biosciences), anti-mouse CD8α (53–6.7, BD Biosciences), anti-mouse CD25 (PC61, BD Biosciences), anti-mouse CD45 (30-F11, BD Biosciences), CD44 (IM7, BD Biosciences), CD62L (MEL-14, BD Biosciences), PD-1 (29F.1A12, BD Biosciences), NK1.1 (PK136, BD Biosciences), Foxp3 (MF23, BD Biosciences), F4/80 (T45–2342, BD Biosciences), MHC II (M5/114.15.2, BioLegend), CD206 (C068C2, BioLegend), Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), CD11b (M1/70, BioLegend), CD11c (HL3, BD Biosciences), B220 (RA3–6B2, BioLegend). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Staining was carried out on ice for 20 min if not indicated otherwise, and intracellular staining was performed using the Foxp3 staining kit according to manufacturer’s instructions (BioLegend). Following a washing step, cells were stained with specific antibodies for 20 min on ice prior to fixation. All flow cytometric analyses were done using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).

Safety assessments

BALB/c mice were intravenously injected with PBS, wt IL-10, or SA-IL-10 (each equivalent to 43.5 μg of IL-10). Two days after injection, blood samples collected from mice were analyzed using a COULTER Ac•T 5diff CP hematology analyzer (Beckman Coulter) according to the manufacturer’s instructions. Spleen weight was also measured. Serum samples collected from protein-injected mice were analyzed using Biochemistry Analyzer (Alfa Wassermann Diagnostic Technologies) according to the manufacturer’s instructions. For evaluation of general immunosuppression, PBS and 100 μg anti-TNF-α were injected intraperitoneally every two days beginning on day 0 for 14 days. FTY720 (1 mg/kg body weight) was administered orally every day. SA-IL-10 (equivalent to 43.5 μg of IL-10) was injected subcutaneously on days 0 and 8. C57BL/6 mice were challenged subcutaneously in the front hocks on day 5 with 10 μg endotoxinfree ovalbumin, 50 μg alum, and 5 μg monophosphoryl lipid A (MPLA). Mice were bled on days 13 (a) and 19 (b), and plasma was analyzed for anti-ovalbumin total IgG titers.

Statistical analysis

Statistically significant differences between experimental groups were determined using Prism software (v8, GraphPad). Where one-way ANOVA followed by Tukey’s HSD post hoc test was used, variance between groups was found to be similar by Brown-Forsythe test. For non-parametric data, Kruskal-Wallis test followed by Dunn’s multiple comparison test was used. For single comparisons, a two-tailed Student’s t-test was used. The symbols *, **, *** and **** indicate P values less than 0.05, 0.01, 0.001 and 0.0001, respectively; ns, not significant.

Results

Albumin-fused IL-10 binds to neonatal Fc receptor (FcRn) and APCs and accumulates within the LNs

Wild type (wt) mouse IL-10, and SA-fused mouse IL-10 were recombinantly expressed, and the molecular weight of the fusion protein was correspondingly higher than for wt IL-10 as determined by SDS-PAGE; in addition, most of the SA-IL-10 existed as a monomer under non-reducing conditions (Fig. 1a). Surface plasmon resonance (SPR) analysis revealed that SA-IL-10 binds to FcRn with micromolar order of Kd (Fig. 1b). The binding ability of these proteins to splenocytes and single cells isolated from the popliteal LN was further evaluated by flow cytometry (Fig. 1c). SA-fused IL-10 exhibited high binding to macrophages and dendritic cells in both splenocytes and LN-derived cells. After intravenous administration of fluorescently-labeled SA-IL-10, significantly higher fluorescence signals were observed within the popliteal LN compared with wt IL-10 (Fig. 1d). Interestingly, higher fluorescence signals were located surrounding high endothelial venules (HEVs), where antigen presenting cells (APCs) reside (16).

Fig. 1. Albumin fusion to IL-10 provided FcRn binding and resulted in LN accumulation.

Fig. 1.

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(a) SDS-PAGE analysis for wt IL-10 and SA-IL-10. (b) Binding analysis of SA-IL-10 to FcRn. (c) Splenocytes (i) or single cells from the popliteal LN (ii) were incubated with SA or SA-IL-10 for 30 min on ice. Binding of each protein to immune cells was detected by co-staining with an anti-SA antibody and antibodies for specific markers of each immune cell population. (d) Immunofluorescence images of the popliteal LN after intravenous injection of DyLight594-labeled wt IL-10 or SA-IL-10. T cells and high endothelial venules (HEVs) were respectively stained with anti-CD3 or anti-PNAd antibodies.

Albumin-fused IL-10 shows prolonged blood circulation

SA is known to demonstrate long circulation via FcRn-mediated recycling on vascular endothelial cells (17, 18). As expected, SA-IL-10 showed significantly prolonged blood circulation compared with wt IL-10 (Fig. S1a). Fig. S1b represents the fluorescence signals from major organs of mice intravenously injected with DyLight800-labled proteins. Reflecting its long circulation properties, SA-IL-10 showed higher signals in the heart, lungs and spleen than that of wt IL-10.

Albumin-fused IL-10 reduces immune activity after accumulation within LNs

SA-fused IL-10 showed micromolar affinity to FcRn (Fig. 1b) and accumulation within LNs after intravenous injection (Fig. 1d). Next, the amounts of IL-10 and its pharmacokinetics in the LNs were quantitatively evaluated (Fig. 2a-c). After intravenous injection of wt IL-10, or SA-IL-10 within CAIA mice, IL-10 concentrations in the LNs at various time points were detected using ELISA. SA-IL-10 showed significantly higher IL-10 signals in the joint-draining (popliteal) LN, the mesenteric LN and relatively high signals in a non-draining (cervical) LN compared with wt IL-10 at 4 hr after injection (Fig. 2a). SA-IL-10-injected mice also showed a peak for IL-10 concentration at around 1 hr after injection (Fig. 2b) and 5–18 times higher AUC than wt IL-10 in the LNs (Fig. 2c). Strikingly, SA-IL-10 was detectable even after 3 days in popliteal LN (Fig. 2b). These data indicate that SA-IL-10 immediately accumulated within LNs after intravenous injection and showed higher retention in the LNs compared with wt IL-10.

Fig. 2. Albumin-fused IL-10 accumulated within and suppressed Th17 activation in LNs.

Fig. 2.

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Arthritis (CAIA) was induced by passive immunization of anti-collagen antibodies, followed by intraperitoneal injection of LPS (defined as Day 3). On the day LPS injection, wt IL-10 or SA-IL-10 were intravenously injected into the arthritic mice. IL-10 levels and Th17-relating cytokines in LNs were measured using ELISA. (a) Comparison of IL-10 levels 4 hr after injection of each protein. (b) Pharmacokinetics of wt IL-10 or SA-IL-10 in LNs after intravenous injection. (mean ± SEM; n = 4) (c) AUC of wt IL-10 and SA-IL-10 in various LNs. Th17-relating cytokine levels in joint-draining (popliteal) LN. (d) and a non-draining (cervical) LN (e). (f) GM-CSF levels in the popliteal LN. (mean ± SEM; n = 7) Statistical analyses were done using analysis of a two-tailed Student’s t-test for (d and e) or variance (ANOVA) with Tukey’s test for (a and f). *P < 0.05; **P < 0.01; ****P < 0.0001; ns; not significant.

High concentrations and AUC of SA-IL-10 in the LNs may affect the phenotypes of various immune cells in LNs and other secondary lymphoid organs. Therefore, immune cell populations in the spleen and popliteal LN were analyzed by flow cytometry (Fig. S2). Intravenous injection of SA-IL-10 induced a significant decrease in the frequency of CD3+ T cells and CD45+ lymphocytes in the spleen (Fig. S2a). In addition, the frequencies of CD86+ dendritic cells, granulocytic myeloid-derived suppressor cells (G-MDSCs), and CD86+ M1 macrophages decreased and that of CD206+ M2 macrophages increased after injection of SA-IL-10 compared with PBS or wt IL-10. A similar tendency was observed within the popliteal LNs (Fig. S2b). These data suggest that SA-IL-10 suppressed the activity of APCs and simultaneously activated immunosuppressive M2 macrophages. Deactivation of APCs in the LNs, and high accumulation of IL-10 might suppress the activity of Th17 cells, which play a crucial role in the development of RA (19, 20). We measured Th17-relating cytokines (IL-17, IL-6 and TGF-β) in the LNs in the joint-draining (popliteal) and a non-draining (cervical) LN: compared to treatment with wt IL-10, IL-17 was statistically reduced in the popliteal LN after treatment by SA-IL-10, and levels in the cervical LN were not statistically reduced by either IL-10 variant (Fig. 2d and 2e). Treatment by SA-IL-10 reduced the concentration of GM-CSF in the popliteal LN, whereas wt IL-10 did not (Fig. 2f).

Albumin-fused IL-10 suppresses the development of rheumatoid arthritis

The therapeutic effects of engineered IL-10 in the passive collagen antibody-induced arthritis (CAIA) model were evaluated (Fig. 3). Intravenous injection of SA-IL-10 significantly suppressed the development of arthritis, whereas PBS- or wt IL-10-injected mice exhibited severe inflammation in the paws (Fig. 3a). From histological analysis, intravenous administration of SA-IL-10 significantly suppressed the inflammatory responses in the paws compared with PBS-treated mice and reduced joint pathology (Fig. 3b). The effect of the administration route on therapeutic efficacy was also investigated, comparing intravenous, local (footpad), and subcutaneous (at a distant site, mid-back) administration (Fig. 3c). Strikingly, SA-IL-10 showed quite high suppression effects on CAIA by all of the administration routes tested (Fig. 3c).

Fig. 3. Albumin-fused IL-10 suppressed arthritis development more effectively than wt IL-10.

Fig. 3.

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(a) Arthritis (CAIA) was induced by passive immunization with anti-collagen antibodies, followed by intraperitoneal injection of LPS. On the day of LPS injection, PBS, wt IL-10, or SA-IL-10 (equivalent to 43.5 μg of IL-10) was injected intravenously into the arthritic mice. Arthritis scores represent the mean + SEM from 7 mice. (b) Representative H&E images of joints on day 14 in each treatment group. Scale bar, 500 μm. The severity of synovial hyperplasia and bone resorption was scored 0 to 4 as described in Materials and Methods. (mean ± SEM; n = 7). (c) Effect of administration routes on therapeutic effects of SA-IL-10. Arthritis scores represent the mean + SEM from 7 mice. Statistical analyses were done using analysis of variance (ANOVA) with Tukey’s test for (a and c) and a two-tailed Student’s t-test for (b). **P < 0.01; ***P < 0.001; ****P < 0.0001.

As a second arthritis model, the active collagen-induced arthritis (CIA) model was used for evaluation of SA-IL-10 on RA treatment. A single injection of SA-IL-10 to CIA mice induced significant suppression of establishment of arthritis compared with PBS (Fig. 4a). Most mice treated with PBS showed severe inflammation at the paws as indicated by histology and the histological score (Fig. 4b). In contrast, SA-IL-10-treated mice exhibited almost identical status in the paw as naïve mice, and most mice showed a histological score of 1 or less (Fig. 4b). The treatment with anti-TNF-α antibody (αTNF-α), a mouse model of a clinically used antibody drug for treatment of RA, also suppressed the increase of clinical scores compared with PBS-treated CIA mice (Fig. 4c), whereas twice αTNF-α injection did not restore the histology of the joints and histological scores, even though histological score tends to decrease, however not significantly (P = 0.1008) (Fig. 4d). Taken together, these results indicate the highly suppressive effect of inflammation by local or intravenous administration and by even subcutaneous administration of SA-IL-10 and its therapeutic effect was comparable or relatively high with αTNF-α treatment.

Fig. 4. Albumin-fused IL-10 showed improved therapeutic effect on established arthritis.

Fig. 4.

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DBA/1J male mice were subcutaneously injected with bovine collagen/CFA emulsion in the tail base. After three weeks, bovine collagen/IFA emulsion was further injected as a boost. When arthritis scores become 2–4 (defined as Day 0), mice were intravenously injected with PBS, SA-IL-10 (each equivalent to 43.5 μg of IL-10), or with 200 μg of anti-TNF-α antibody. In (c and d), the same treatments were additionally injected to the mice on Day 3. (a and c) Arthritis scores represent the mean + SEM from 9–15 mice. (b and d) Representative H&E histological image of joints on day 16. Scale bars, 500 μm. The severity of synovial hyperplasia and bone resorption was scored 0 to 4 as described in Materials and Methods. Statistical analyses were done using a two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Albumin-fused IL-10 suppresses inflammatory responses in the paws

Next, immune cell populations in the hind paws were analyzed using flow cytometry (Fig. 5a). After intravenous injection of SA-IL-10, frequencies of CD45+ immune cells were significantly decreased compared with PBS- or wt IL-10-treated groups. Within CD45+ cells, the frequencies of B cells and dendritic cells became comparable to levels in healthy mice, and CD11b+ cells were remarkably decreased to the level of healthy mice as well. Among CD11b+ cells, the G-MDSC population was reduced, and the macrophage frequency was recovered to the level of healthy mice. In addition, the frequency of CD206+ M2 macrophages was significantly increased by injection of SA-IL-10 compared with PBS or wt IL-10 treatment, even exceeding that of healthy mice. The analysis of T cell populations in the paws revealed that SA-IL-10 suppressed the change in CD4+ cells and Foxp3+ Treg in CAIA mice (Fig. S3a). Furthermore, SA-IL-10 suppressed a decrease of the Treg frequency in the blood (Fig. S3b). Reflecting these changes in immune cell populations, various inflammatory cytokines in the paws were significantly decreased by intravenous injection of SA-IL-10, which levels were comparable with those in healthy mice (Fig. 5b).

Fig. 5. Albumin-fused IL-10 suppressed inflammatory responses within the paws.

Fig. 5.

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Arthritis (CAIA) was induced by passive immunization of anti-collagen antibodies, followed by intraperitoneal injection of LPS. On the day of LPS injection (defined as Day 3), PBS, wt IL-10 or SA-IL-10 were intravenously injected into the arthritic mice. (a) Single cells were extracted from the hind paws on day 11, followed by flow cytometric analysis. Graphs depict the frequency of CD45+ cells, B cells (B220+ cells within CD45+ lymphocytes), dendritic cells (CD11c+ cells within CD45+ lymphocytes), monocytes (CD11b+ cells within CD45+ lymphocytes), granulocytic MDSC/neutrophils (Ly6G+ Ly6C+ CD11b+ CD45+), monocytic MDSC (Ly6G- Ly6C+ CD11b+ CD45+), macrophages (F4/80+ CD11b+ CD45+), M2 macrophages (CD206+ F4/80+ CD11b+ CD45+), and M1 macrophages (MHC II+ F4/80+ CD11b+ CD45+). (mean ± SEM; n = 7) (b) Cytokine levels in hind paws on day 11 (n = 5–7). Statistical analyses were done using analysis of variance (ANOVA) with Tukey’s test except for %CD11c+ in (a). For analysis of %CD11c+ in (a), Kruskal-Wallis test followed by Dunn’s multiple comparison test was employed. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Albumin-fused IL-10 shows no toxicity and no immunosuppression after injection

Finally, safety assessments were performed to investigate whether engineered IL-10 demonstrates any adverse effects. Representative blood parameters measured by a hematology analyzer and spleen weights did not show any significant changes among the treatment groups (Fig. S4a). Various biochemical markers in serum were also investigated using a biochemistry analyzer (Fig. S4b). For the engineered IL-10-treated groups, most markers, except for amylase (which was not increased, rather slightly decreased), showed similar levels compared with PBS-treated group. Furthermore, in a model of vaccination to ovalbumin (OVA), neither injection of SAIL-10 nor αTNF-α affected anti-OVA IgG titers, whereas FTY720, a clinically approved drug (fingolimod) for treating multiple sclerosis, showed some immunosuppressive trend (although not statistically significant) under these experimental conditions (Fig. S5). These results indicate that engineered IL-10 possesses high safety and lack of broad immunosuppression after systemic administration.

Discussion

Current treatment for RA is based on symptomatic therapy by relieving pain, controlling synovitis and suppressing joint injury. Antibody drugs or competitive soluble receptors that neutralize inflammatory cytokines, especially TNF-α, have provided high therapeutic efficacy for patients with RA (4). These biotherapeutics mainly act at the inflamed joints to capture the inflammatory cytokines. However, these inhibitory drugs are known to increase the risk of infection, because their targets are pleiotropic in immune function and these drugs are repeatedly administered to provide their anti-inflammatory effect at disease site (21–24). In addition, administration of antibody drugs can also cause the induction of neutralizing anti-drug antibodies, which decreases therapeutic efficiency (25). Therefore, development of an alternative approach with a structurally different molecular class and with a different immuno-suppressive molecular mechanism such as tolerance is desired.

Here, we explore a novel approach to treat RA through enhanced lymph node trafficking using engineered IL-10, which is a representative anti-inflammatory cytokine and modulates the phenotypes of RA-relating immune cells toward immunosuppressive states. Clinical trials using recombinant IL-10 have been already performed to treat autoimmune diseases including RA (6–8, 26). One of the drawbacks of IL-10 is its short half-life in the blood (8). Here, we genetically fused SA to IL-10 to extend its retention in the secondary lymphoid organs. We evaluated SA-IL-10, in comparison to wt IL-10, for amelioration of arthritis in two models. CAIA is a macrophage- and neutrophil-mediated acute RA model, whereas CIA is a T cell-, and especially Th17-mediated RA model. Given that RA is a heterogeneous disease in the clinic and the models complement each other, it is encouraging to show and SA-fused IL-10 suppresses disease severity in both models. SA fusion to IL-10 is crucial for obtaining a marked therapeutic effect, which was comparable or relatively high to treatment with αTNF-α antibody, a common therapy in the clinic. Additionally, to the best of our knowledge, this study is the first to show a therapeutic effect of IL-10 in the CAIA model.

SA fusion to IL-10 resulted in enhanced accumulation within LNs after intravenous injection and kept high IL-10 concentrations in the LNs for prolonged durations compared with wt IL-10 (Fig. 2a). So far, LN trafficking of SA or albumin-binding nanoparticles has been mainly achieved by intradermal or subcutaneous administration, where the LN is accessed via the afferent lymphatic vessel downstream of the collecting lymphatics at the injection site (27–30). In the context of a biodistribution study of an inflammatory cytokines, one paper showed high localization of human SA-fused IL-2 after intravenous injection into spleen, liver and LNs, where IL-2 receptor-expressing T cells exist, but the precise mechanism for this high localization has not been elucidated (31). Here, we reveal enhanced trafficking of SA-fused IL-10 into LNs after intravenous injection, where the SA enters the LN via the blood vasculature. SA-IL-10 exhibited high binding affinity (micromolar Kd, as expected) to FcRn (Fig. 1b), which provides prolonged blood circulation properties to proteins via recycling mediated via FcRn expressed in vascular endothelial cells (Fig. S1a). Recycling via transcytosis (from the basal side back to the lumenal side) of IgG via FcRn is a well-established phenomenon, whereas the same phenomenon for SA has been more recently reported (17, 18). In the LN, FcRn-mediated molecular transport would seem to lead to accumulation there. Interestingly, histological analysis revealed the accumulation of SA-IL-10 surrounding the HEVs of the LNs (Fig. 1d). Further experiments are required to reveal a more detailed mechanism of enhanced LN accumulation and the relationship with FcRn, such as LN accumulation analysis using mutated SA-fused IL-10 to abrogate FcRn binding.

SA-IL-10 shows high binding to APCs in the spleen and the joint-draining LN (Fig. 1b). After accumulation within the LNs, SA-IL-10 molecules are taken up by APCs resident within the LNs, leading to the suppression of dendritic cell and M1 macrophage activities and the induction of M2 macrophages (Fig. S2). M2 macrophages can change the differentiation fate of Th0 cells to Treg cells in the LNs (32). Furthermore, the immunosuppressive environment by high concentrations of IL-10 in the LN may cause the further polarization of macrophages to M2 phenotype and the suppression of Th17 differentiation (33, 34), resulting in the decrease of IL-17, GM-CSF or other cytokines in the LNs that we observed (Fig. 2d and 2f). GM-CSF is a cytokine that is a marker for pathogenic Th17, and its inhibitory antibody is currently being tested in clinical trials (35). Thus, the decrease of GM-CSF by SA-IL-10 treatment indicates decreased immunoactivation in the joint-draining LN. Th17 cells reportedly express IL-10 receptor, and IL-10 binding suppresses IL-17 expression and secretion (14, 36). Because Th17 cell antigen recognition primarily occurs in the lymphoid tissue, SA-IL-10 may bind to Th17 cells directly to suppress the IL-17 pathway. These changes in LNs also suppressed the infiltration of immune cells, especially G-MDSC and macrophages, into the paws (Fig. 5a) and also induced an increase of M2 macrophages (Fig. 5a), resulting in the decrease of inflammatory cytokines (Fig. 5b) and the suppression of joint inflammation (Fig. 3 and 4).

SA-IL-10 induced high anti-inflammatory responses after administration by any of the routes tested, namely intravenous, subcutaneous (at a distant site) or footpad (local) injections (Fig. 3c), suggesting that SA-IL-10 can enter the LNs systemically after uptake by a local injection-site draining lymphatics and transit through the lymphatics back into the systemic circulation via the thoracic duct. The high therapeutic effect by subcutaneous injection suggests a particular clinical benefit of SA-IL-10. Therefore, SA-fusion is a simple but effective way for preparation of engineered cytokines to achieve enhanced LN trafficking.

In this study, SA-fusion to IL-10 achieved increased persistence within the LNs, where autoimmunity-relating immune recognition develops and persists. As a result, SA-IL-10 suppressed the main inflammatory pathway of RA progression, yet without inhibition of a pleiotropic inflammatory cytokine such as TNF-α. In addition, SA-IL-10 did not show any remarkable toxicities and immunosuppressive effects in preliminary safety assessments (Fig. S4 and S5). SA-IL-10 exhibited marked therapeutic effects in both CIA and CAIA models. Therefore, our data suggest the potential of SA-IL-10 for clinical application in suppression of RA, and our findings more broadly suggest the ability to modulate immunity through systemic tolerogenic manipulation of the LNs in other autoimmune and inflammatory diseases.

Supplementary Material

Suppl Material

Acknowledgments

We thank the Human Tissue Resource Center of the University of Chicago for histology analysis. We thank the Integrated Light Microscopy Core of the University of Chicago for Imaging. We thank S. Gomes for SDS-PAGE and experimental help and W. Hou for subcutaneous injection in a tail and experimental help. We thank Prof. T. Sano (Univ. Illinois at Chicago) for experimental advice and helpful discussions.

Funding

This work was supported by the University of Chicago (to J.A.H.).

Footnotes

Competing interests

A.I, K.K., A.M., J.I., and J.A.H. are inventors on patent application PCT/US20/19668. E.Y., A.I, E.A.W., J.I., and J.A.H. are inventors on U.S. Provisional Patent Application No. 63/083,722, which covers the technology described in this paper. The other authors declare that they have no competing interests.

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