Conceived and designed the experiments: J. Kovalchin JLS EZ. Performed the experiments: J. Kovalchin J. Krieger MG NK MA KC TB AM TH ID. Analyzed the data: J. Kovalchin JLS EZ. Wrote the paper: J. Kovalchin JLS EZ.
The authors have read the journal's policy and have the following conflicts: The authors from Peptimmune Inc., a for profit organization, are employees: Joseph Kovalchin, Jeffrey Krieger, Michelle Genova, Michael Augustyniak, Kathryn Collins, Troy Bloom, Allyson Masci, Tara Hittinger, Ingrid Dufour and Eric Zanelli. This affiliation is also demarcated with a “number one” on the list of authors. JLS is a tenured professor at Harvard University but serves as a Scientific Advisor to Peptimmune. YFAK is in pre-clinical development and covered by Harvard US patent application 10/406783, published 06/03/2008. This does not alter the authors′ adherence to all the PLoS ONE policies on sharing data and materials.
The random amino acid copolymer poly(Y,E,A,K)n (Copaxone®) is widely used in multiple sclerosis treatment and a second generation copolymer poly(Y,F,A,K)n with enhanced efficacy in experimental autoimmune encephalomyelitis in mice has been described. A major mechanism through which copolymers function to ameliorate disease is the generation of immunosuppressive IL-10-secreting regulatory T cells entering the CNS. In addition, the antigen presenting cell to which these copolymers bind through MHC Class II proteins may have an important role. Here, both CCL22 (a Th2 cell chemoattractant) in large amounts and CXCL13 in much smaller amounts are shown to be secreted after administration of YFAK to mice and to a smaller extent by YEAK parallel to their serum concentrations. Moreover, bone marrow-derived macrophages secrete CCL22
Two random amino acid copolymers have been described, the administration of which ameliorates experimental autoimmune encephalomyelitis (EAE) and several other autoimmune diseases in mice and other rodents using several different models. They are poly(Y,E,A,K)n (called YEAK, Copaxone®, glatiramer acetate, Copolymer-1)
Both copolymers have been thought to exert their primary immunosuppressive action through the generation of immunosuppressive T cells that secrete IL-10 as a major immunomodulatory cytokine, as well as other cytokines
The purpose of the present study was to define further the nature of the antigen presenting cell modified by copolymer treatment and its relationship to the IL-10 secreting T cells that have been described previously.
YFAK is a mixture of random-sequence peptides composed of the amino acids L-tyrosine, L-phenylalanine, L-alanine, and L-lysine in the approximate molar ratios of 1.0∶ 1.3∶ 24.0∶ 6.0, respectively. YFAK is manufactured by solid phase synthesis on pre-loaded Wang resin with base labile Fmoc-groups. The excess amino acid derivatives and coupling reagents are removed by filtration. YFAK is cleaved, N-acetylated, precipitated, washed and dried under vacuum. YEAK (Copaxone®, purchased from Hanna Pharmaceuticals (Wilmington, DE)) was stored at 4°C at a concentration of 20 mg/mL according to the manufacturer's package insert. YFAK or YEAK was diluted in 42 mg/mL mannitol (Sigma, St. Louis, MO) in water at concentrations indicated. Compounds were administered s.c. (subcutaneously) interscapularly at a dose volume of 100 µL/10 g body weight.
All mouse work herein discussed was performed under an approved protocol with the review of Harvard University's Standing Committee on the Use of Animals in Research and Teaching, under the Guidelines for the Use of Vertebrate Animals in Research and Teaching of the Faculty of Arts and Sci-ences of Harvard University, and under the NIH Guide for the Care and Use of Laboratory Animals. Ameliorative steps were taken whenever animals were injected or observed in disease states including the administration of anesthesia, food supplementation, and temperature modification. The HU/FAS animal care and use program maintains full AAALAC accreditation, is assured with OLAW (A3593-01), and is currently registered with the USDA. This work was carried out under Protocol 99-01, ap-proved in latest amendment by IACUC on 05/13/11.
The pharmacokinetic (PK) assays (validated in CD-1 male mice obtained from Avogadro, Fontenilles, France and bred at Charles River Laboratories, Wilmington, MA) for YFAK and YEAK are direct competition ELISAs
YFAK/YEAK biotinylated antibodies were Protein A purified from rabbit polyclonal antiserum generated against the appropriate antigen. Briefly, rabbits were immunized with YFAK or YEAK. The rabbit polyclonal antiserum was diluted 1∶1 with pH 8.0 buffer, added to a Protein A column (column was equilibrated with the pH 8.0 buffer), incubated one hour at room temperature, then the unbound proteins were washed off the column with the pH 8.0 buffer. The specific antibody was eluted with pH 2.8 buffer, dialyzed against PBS at 4°C overnight and the protein concentration was determined using Coomassie blue. The dialyzed antibody was then incubated two hours at room temperature with a ten molar excess of biotin and dialyzed against PBS at 4°C overnight. The dialyzed biotinylated antibody was concentrated using a desalting column and the protein concentration was determined using Coomassie. The mole-to-mole ratio of biotin to protein was determined using the HABA method (Pierce Biotechnology, Rockford, IL).
Male CD-1 mice (Charles River Laboratories) at 8–12 weeks of age, female SJL mice (Charles River Laboratories) at 7–9 weeks of age, and MHC II −/− mice (B6.SJL(129)-
RAW264.7 cells (ATCC, Manassas, VA) were plated in 96 well U-bottom tissue culture plates at 5×105 cells per well with and without compounds incubated in 200 µl culture medium (10%FBS (Thermo Scientific-Hyclone, Waltham, MA) / DMEM with 1%PSG (Invitrogen-Gibco, Carlsbad, CA)) for 24 hours at 37°C with 5% CO2 in a humidified environment. Cell-free culture supernatant was collected and immediately frozen at −80°C for future testing with a commercial CCL22 ELISA kit following manufacturer's protocol (R&D Systems, Minneapolis, MN).
Femurs were excised and bone marrow cells were collected from twenty-five naïve female SJL mice (Charles River Laboratories; 9–12 weeks of age) by snipping off the ends and flushing the marrow with HL-1 media (Lonza-Biowhittaker, Walkersville, MD) using a 1 mL syringe equipped with a 30 gauge needle. Single-cell suspensions from femur-derived bone marrow cells were depleted of T and B cells by negative selection using a combination of CD90.2 and CD19 magnetic beads with the method described in the manufacturer's package insert (Miltenyi Biotec, Auburn, CA). The residual cells, which are enriched for myeloid progenitors, were re-suspended and plated at 1×106 cells per well to 24 well tissue culture plates in 2 mL CM+ media, defined as HL-1 media containing 10 µM HEPES (Invitrogen-Gibco), 1 mM Sodium Pyruvate (Invitrogen-Gibco), 2 mM L-glutamine (Invitrogen-Gibco), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen-Gibco), 1% nonessential amino acids (NEAA) (Invitrogen-Gibco), and 50 µM 2-Mercaptoethanol (Sigma)) supplemented with 10% FBS (Thermo Scientific-Hyclone), 10 ng/mL IL-3 (R&D Systems), and 2.5 ng/mL TNF-α (R&D Systems) to obtain bone marrow –derived macrophages
Bone marrow cells were analyzed by flow cytometry on day 1, both pre- and post- depletion, and on day 9. At each day, cells were washed twice in staining buffer containing 2% FBS and 0.09% sodium azide at pH 7.4 (BD Pharmingen, San Diego, CA). To decrease non-specific cell staining, Fc receptors were blocked by incubating cells for 5 minutes on ice with an optimal concentration of rat anti-mouse CD16/CD32 (BD Pharmingen) diluted in staining buffer. Cells were then stained for 30 minutes on ice in the dark by the addition of an antibody conjugate panel selected to enable the phenotypic analysis of monocytes and to confirm the absence of T and B cells (F4/80: FITC- eBioscience, San Diego, CA, and the remainder from BD Pharmingen: B220: PE-, CD11b: PerCP-Cy5.5-, CD11c: PE-Cy7-, CD3: APC-, and GR-1: APC-Cy7). After staining, cells were washed twice with staining buffer, then red blood cells (RBC) were lysed and cells were fixed (FACS Lyse, BD Pharmingen) on day 1, or cells were fixed without lysis (CytoFix, BD Pharmingen) on day 9. Following 2 washes with staining buffer, cells were resuspended to staining buffer and acquired on a Becton Dickinson FACS CANTO II flow cytometer with FACSDiva software.
Cell-free culture supernatants, EDTA plasma, and serum samples were sent to Rules-Based Medicine (Austin, TX) for analysis of cytokine and chemokine production using their Rodent Multi-Analyte Profiling (MAP) Version 1.6. EDTA plasma samples were also tested for CCL22 and CXCL13 and the cell-free culture supernatants for CCL22, CXCL13, and TNF-α using commercially available ELISA kits following manufacturer's protocol (R&D Systems). For IL-3 analysis, female SJL mice were dosed daily for 5 days with 0.25, 2.5, or 25 mg/kg of YFAK or YEAK. Spleens were collected after a one week resting period. Splenocytes were re-stimulated for 3 days with 5 µg/mL of corresponding copolymer after which cell culture supernatants were harvested and tested for IL-3 secretion using commercially available ELISA kits following the manufacturer's protocol (R&D Systems).
Recently, a role for myeloid cells, as well as IL-10-secreting T cells, in the protective response to amino acid copolymers has been demonstrated as the ability of YEAK to stimulate macrophages, termed “M2 regulatory macrophages,” that on adaptive transfer decreased disease severity
Data are shown as mean ± SEM from an experiment in which 3–4 mice were euthanized and samples collected at each time point. (A) Serum YFAK (ng/mL) is significantly greater than YEAK (ng/mL) at multiple time points from 15 to 120 minutes. (B, C) YFAK induced increased plasma CCL22 (pg/mL) and CXCL13 (pg/mL) levels when compared to YEAK as shown. Significance was calculated using an unpaired t-test: YFAK vs. YEAK or Control * p≤0.05, ** p≤0.01.
Evidence of macrophage activation through the release of M2 chemokines
Since binding of amino acid copolymers to MHC class II molecules has been considered to be central to their mechanism of action
Female MHC Class II deficient mice and their littermate controls (n = 9–10) were administered one subcutaneous dose of 50 mg/kg YFAK, 50 mg/kg YEAK, or Vehicle Control. Blood was collected and plasma was prepared 30 minutes post administration and tested for CCL22 (A) and CXCL13 (B). Data is shown as mean (pg/mL) ± SEM. Significance was calculated using an unpaired t-test: YFAK or YEAK vs. Vehicle Control + p≤0.05; + + p≤0.01; + + + p≤0.001; + + + + p≤0.0001; YFAK vs. YEAK * p≤0.05; ** p≤0.01.
A similar phenomenon was observed
(A) RAW264.7 cells were plated on 96 well U-bottom tissue culture plates at 5×105 cells per well and incubated in culture medium (10% FBS/DMEM with 1% PSG) without or with molar equivalent concentrations of copolymers for 24 hours at 37°C with 5% CO2 in triplicate. Cell-free culture supernatant was collected and immediately frozen at −80°C for testing using a commercial CCL22 ELISA kit. Data are represented as a non-linear regression curve fit and shown as mean (pg/mL) ± SEM. (B) Linear correlation be-tween area under the curve of CCL22 production and the length/molecular weight (Da) in the culture supernatants of RAW264.7 cells incubated in culture medium with YEAK, YFAK, or molar equivalent of truncated YFAK (r2 = 0.9637, p<0.0001.
Next, bone marrow derived myeloid cells
These myeloid cells secreted a very high baseline level of CCL22 (26,400 pg/ml), but incubation with YFAK, and less effectively YEAK, induced much higher dose dependent levels peaking at 45,200 pg/ml for YFAK at 6 µM and at 52,500 pg/ml for YEAK at 12 µM (
Cytokine and chemokine production was analyzed from bone marrow-derived myeloid cells (BMMC). Data are from a representative experiment in which samples were run in 4 replicates and are shown as mean (pg/mL) ± SEM. In vitro compound concentrations administered were 1.5, 3.0, 6.0, and 12.0 µM. Significance was calculated using an unpaired t-test: YFAK vs. YEAK * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.
After treatment of the myeloid cells with YFAK, the concentration of CXCL13 secreted increased to 671±51 pg/mL, while treatment with YEAK or PLP139–151 resulted in no change from the baseline level of ∼20 pg/mL (
The effects of YFAK and YEAK on supernatant concentration of CXCL1 and CXCL2 that are associated with neutrophil chemotaxis were also examined. Reciprocally, the concentration of CXCL1 and CXCL2 fell from baseline levels of 5,225±165 pg/mL or 9,193±385 pg/mL respectively to near 0 in both cases while little or no change was observed with YEAK or PLP139–151 (
The concentration of the pro-inflammatory cytokines TNF-α, IL-6, and IL12p70 were all substantially decreased by YFAK, but not by YEAK or PLP139–151 (
These assays were carried out either using the Rules-Based Medicine Rodent MAP version 1.6 panel that examines 69 secreted factors (
Since both YFAK and YEAK stimulate splenocytes and spleen-derived T cell lines to secrete TH2 cytokines including IL-10, IL-13, and IL-4, the latter two of which have been reported to induce M2 macrophage differentiation
To answer this question, female SJL mice were dosed daily for 5 days with 0.25, 2.5, or 25 mg/kg of YFAK or YEAK. Spleens were collected after a one week resting period. Splenocytes were re-stimulated for 3 days with 5 µg/mL of corresponding copolymer after which cell culture supernatants were harvested and tested for IL-3 secretion. Splenocytes from mice treated with 0.25 mg/kg of YEAK or YFAK produced the greatest concentrations of IL-3 (
Female SJL mice were dosed daily for 5 days with 0.25, 2.5, or 25 mg/kg of YFAK or YEAK. Spleens were collected after a one week resting period. Splenocytes were re-stimulated for 3 days with 5 µg/mL copolymer. Splenocytes harvested from Vehicle-treated mice and stimulated with YFAK or YEAK had no detectable level of IL-3. Mean (pg/mL) ± SEM of IL-3 is shown. No significant difference in the release of IL-3 in splenocytes re-stimulated with YFAK or YEAK was evident.
Since the approval of the random amino acid copolymer YEAK (Copaxone®) for the treatment of RR-MS, YFAK, a second generation copolymer with enhanced efficacy in mouse models of EAE has been described. Understanding the mechanism of action of the copolymers has engaged numerous laboratories (reviewed by
YFAK | YEAK | |
|
Solid Phase | Solution Phase |
|
YFAK (Tyrosine 1.0, Phenylalanine 1.2, Alanine 23.5, Lysine 6.0) | YEAK (Tyrosine 1.0, Glutamic Acid 1.5, Alanine 4.5, Lysine 3.6) |
|
Strong Net Positive Charge | Slightly Net Positive Charge |
|
4–5 kDa | 5–9 kDa |
|
52-mer | 20–200-mer |
|
N-terminus | No |
The serum levels reached in mice after s.c. administration (measured using a newly developed antibody-based method) of YFAK and its duration were much larger than those for YEAK (
The detection of YFAK and YEAK in the serum coincided with the appearance in plasma of CCL22 and CXCL13 within minutes of copolymer administration (
Myeloid progenitors differentiated to macrophages
It is not known whether IL-3 is secreted by the same T cells that secrete IL-4 and IL-13.
The induction of these Tregs by YEAK
Some differences in cytokine/chemokine production were observed between cells cultured in the presence of YFAK or YEAK (
Some data suggest that CCL22 and CXCL13 may be produced by different cell populations. The serum level of CXCL13 induced
In addition, YFAK significantly decreased the secretion of the chemokines CXCL1 and CXCL2 and of pro-inflammatory cytokines TNF-α, IL-6, and IL-12p70. The pro-inflammatory properties associated with TNF-α play a major role in autoimmune diseases and interference with TNF- α production is a major treatment modality
The relationship between dose of YFAK and efficacy in EAE is of considerable interest, i.e., 0.75 mg/kg is inefficient at generating efficacy, 2.5 mg/kg gives significant efficacy, and at 7.5 mg/kg efficacy is lost. As the dose of YFAK increased, so did activation of the innate immune response (
Interaction between cells of the innate and adaptive immune system is quite important in the mechanism. Th17 cells are believed to play a major role in autoimmune pathologies and multiple sclerosis in particular. YEAK has been shown to dampen differentiation of Th17 cells through altered production of IL-6 by monocytes
In summary, chemokine release from myeloid cells, which occurs rapidly on administration of amino acid copolymers
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