Clinical and Experimental Immunology
OR I G INA L A RTI CLE
doi:10.1111/cei.12745
Novel therapeutic compound tuftsin–phosphorylcholine attenuates collagen-induced arthritis
T. Bashi,* O. Shovman,* M. Fridkin,† A. Volkov,‡ I. Barshack,‡ M. Blank* and Y. Shoenfeld* *Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, affiliated to the Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv, Israel, †Department of Organic Chemistry, The Weizmann Institute of Sciences, Rehovot, Israel, Tel-Aviv, Israel, and ‡Institute of Pathology, Sheba Medical Center, affiliated to the Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv, Israel
Accepted for publication 26 November 2015 Correspondence: Y. Shoenfeld, Head of Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, TelHashomer, 52621, Israel. E-mail: [email protected]
Summary Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin–PC (TPC), a novel helminth-based compound in collagen-induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 mg/0.1 ml) or orally (250 mg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme-linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (Treg) and regulatory B (Breg) cell phenotypes by fluorescence-activated cell sorter (FACS). TPC-treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P < 0.0001) in both subcutaneous and orally treated groups at day 31. Moreover, histology analysis demonstrated highly inflamed joints in control mice, whereas TPC-treated mice maintained normal joint structure. Furthermore, TPC decreased the titres of circulating collagen II antibodies in mice sera (P < 0.0001), enhanced expression of IL-10 (P < 0.0001) and inhibited production of tumour necrosis factor (TNF)-a, interleukin (IL)217 and IL-1b (P < 0.0001). TPC significantly expanded the CD41CD251 forkhead box protein 3 (FoxP31) Treg cells and CD191IL-101CD5highCD1dhighT cell immunoglobulin mucin1 (TIM-11) Breg cell phenotypes (P < 0.0001) in treated mice. Our data indicate that treatment with TPC attenuates CIA in mice demonstrated by low arthritic score and normal joints histology. TPC treatment reduced proinflammatory cytokines and increased anti-inflammatory cytokine expression, as well as expansion of Treg and Breg cells. Our results may lead to a new approach for a natural therapy for early rheumatoid arthritis onset. Keywords: collagen-induced arthritis, helminth, phosphorylcholine, rheumatoid arthritis, tuftsin
Introduction Rheumatoid arthritis (RA) is a chronic autoinflammation of the joints, with a prevalence of approximately 1% in Western populations [1–4]. Current therapeutic strategies of RA involve mainly methotrexate [5], as well as immune-based, anti-inflammatory therapies such as tumour necrosis factor (TNF)-a, interleukin (IL)21, CD-20 targeting agents or Janus kinase 1,2,3 (JAK) inhibitors [6–8]. However, such biological agents are still relatively limited, and they have various side
effects. Therefore, there is a demand to develop new small immunomodulatory molecules with minimal side effects. One of the novel approaches for treating autoinflammation is to adopt natural strategy to control the immune system. The modern lifestyle has led to a decrease in the infections burden [9]. Moreover, there is a lower prevalence of RA in helminthes-endemic areas [10]. Helminthes survive within the host by immunomodulating the host innate immunity. Treatment with live helminthes or helminthes products in
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patients and in animal models improved the clinical score in RA, multiple sclerosis, type I diabetes mellitus and inflammatory bowel disease [11–18]. Immune regulation functions of some helminthes are attributed to the phosphorylcholine (PC) moiety [19]. PC is a non-immunogenic small molecule also present in helminthes products [20–22]; we have constructed a chimeric compound composed of PC and tuftsin, referred to as tuftsin–phosphorylcholine (TPC). Tuftsin is a physiological natural immunomodulatory tetrapeptide (Thr-Lys-Pro-Arg), a small part of the immunoglobulin (Ig)G heavy-chain molecule produced by enzymatic cleavage in the spleen. Tuftsin has been shown to have anti-microbial, anti-viral and anti-tumour functions mediated by enhancement of macrophagic activity [23–25]. Recently, we have demonstrated that treatment with TPC attenuated glomerulonephritis in lupus-prone mice and prevented colitis severity in dextran–sulphate–sodium salt (DSS)-induced colitis in mice [26,27]. Our current study addresses TPC therapeutic efficacy in a mouse model of collagen-induced arthritis (CIA). TPC immunomodulatory effect is associated with a significant reduction in arthritic score, prevention of joint damage accompanied by immunomodulation of the cytokines profile and enhanced expansion of T and B regulatory cells.
Materials and methods Tuftsin–phosphorylcholine synthesis (TPC) Tuftsin is a physiological natural immunostimulating tetrapeptide (Thr-Lys-Pro-Arg) fraction of the IgG heavy-chain molecule produced by enzymatic cleavage in the spleen [23]. Tuftsin was extended at its C-terminal, i.e. Thr-LysPro-Arg-Gly-Tyr, and was synthesized manually following solid-phase peptide technology (GLS peptide synthesis; GL Biochem, Shanghai, China). The peptide was coupled to diazotized 4-aminophenyphosphorylchloride to form an azo bond between the tuftsin and PC (Sigma-Aldrich, St Louis, MO, USA) [24,28]. The conjugate was characterized by mass spectra and amino acid analysis as well as by highperformance liquid chromatography (HPLC). TPC was diluted in commercial phosphate-buffered saline (PBS) (Biological Industries, Israel Beit-Haemek Ltd, Kibbutz Beit-Haemek, Israel).
Mice and experimental design Experimental arthritis was induced in DBA/1 male mice at the age of 6–7 weeks (Harlan Laboratories, Kreuzelweg, the Netherlands). The mice were maintained in a conventional animal housing facility at Sheba Medical Center and kept in individually ventilated cages. All experiments were approved and executed according to the protocols of the Ethical Committee of the Israeli Ministry of Health, no. 20
696/11. Disease induction was carried out by injecting 100 lg bovine type II collagen intradermally into the base of the tail (Chondrex, Redmond, WA, USA) in 1 : 1 emulsion with Mycobacterium tuberculosis H37RA in Freund’s incomplete adjuvant (Difco Laboratories, Detroit, MI, USA). A boost injection of bovine type II collagen in PBS at the base of the tail was given 2 weeks later [29]. TPC was first administered at day 26, 6 days prior to disease induction; PBS was given as control vehicle. TPC was given either orally using a feeding needle (250 mg/ 0.1 ml per mouse) once a week or subcutaneously (s.c.) (5 mg/0.1 ml per mouse) twice a week, n 5 30 per group. The mice were sacrificed after 31 days.
Assessment of arthritis Mice were monitored three times a week by two blind observers for signs of arthritis, for which severity scores were derived as follows: 0 5 normal, 1 5 slight erythema, 2 5 slight erythema plus swelling, 3 5 moderate oedema and erythema 4 5 oedema and erythema from the ankle to the entire leg. The total arthritis score was the sum score of the four limbs.
Histopathological assessment Mouse paws were obtained from the sacrificed mice and fixed in 4% formalin (Sigma, St Louis, MO, USA), decalcified, cut, and stained with haematoxylin and eosin (H&E). All histopathological evaluations were performed by two blind pathologists.
Anti-collagen type II antibodies Titres of circulating anti-collagen type II antibodies were determined by enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with collagen type II [10 mg/ml phosphate-buffered saline (PBS)] overnight 48C and then blocked with 3% bovine serum albumin (BSA). Mouse serum (n 5 10 for each treatment group) was diluted to 1 : 200 up to 1 : 400 and was incubated for 2 h at room temperature. The binding was probed with goat antimouse IgG conjugated with alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), followed by the addition of appropriate substrate. The data were read by ELISA reader at optical density (OD) 405 nm.
Analysis of cytokine production by splenocytes derived from treated mice Spleen cells were originated from TPC s.c. and TPC orally treated mice (n 5 10 mice per group), as well as from vehicle PBS-treated mice (n 5 10 mice per group). The spleen cells were isolated after erythrocytes were lysed by red blood cells lysis buffer (Biological Industries). The spleen cells were seeded (5 3 106 cells/well) in 24-well plates (Thermo plates; Nunc Fisher Scientific Inc., Waltham, MA, USA), coated with anti-CD3 antibodies (2 mg/ml) in the
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presence of 5 mg/ml TPC or PBS for 72 h in RPMI-1640 enriched medium at 378C and 5% CO2. Culture supernatants were then collected. Proinflammatory (IL-1b, IL-17, TNF-a) and IL-10 anti-inflammatory cytokine levels in the culture supernatant were detected by DueSet ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.
Analysis of regulatory T [CD41CD251forkhead box protein 3 (FoxP31)] cells by flow cytometry Isolated splenocytes, depleted of red blood cells, were incubated with anti-CD41fluorescein isothiocyanate (FITC) anti-CD251 allophycocyanin (APC) anti-FoxP31phycoerythrin (PE) (eBioscience, San-Diego, CA, USA) and analysed by flow cytometry, with forward- and side-scatter gates adjusted to include all cells and to exclude debris (Becton Dickinson, Franklin Lakes, NJ, USA). For intracellular staining of FoxP3, the cells were preincubated with a fixation solution, washed and resuspended in permeabilization solution (Serotec, Oxford, UK) and stained intracellularly for FoxP3. The gating was on the CD41 T cells.
Analysis of B10 regulatory CD191IL-101T cell immunoglobulin mucin-1 (TIM-11)CD1dhighCD5high cells by flow cytometry B cells from isolated splenocytes underwent isolation by negative selection using monoclonal antibodies against CD43, CD4 and Ter-119 (B cell isolation kit; Miltenyi Biotec, Auburn, CA, USA). B cells were incubated with antiCD191FITC anti-T cell Ig domain and mucin domain protein 1 (TIM-1)1APC anti-IL-101PE (eBioscience) and anti-IL-101FITC anti-CD1d1APC anti-CD51PE (eBioscience) and were analysed by flow cytometry (Becton Dickinson). The gating was on the CD191 B cells or IL101B10 cells.
Statistical analysis To evaluate the significance of the differences between groups, TPC s.c. to PBS s.c. and TPC oral to PBS oral, Student’s t-test was used. Values of P < 0.05 were considered significant.
Results TPC reduces arthritis severity in mice with CIA The main characteristic of RA is joint deformation due to severe inflammation, therefore we followed the TPC effect on arthritis score in CIA mice. CIA mice were subjected to TPC, orally or s.c., and compared to mice treated with PBS. As detailed in Fig. 1a,b, we observed a significantly lower arthritis score in TPC-treated mice compared with PBS-treated mice (P < 0.05). Arthritis score was lower in both the TPC orally and TPC s.c.-treated groups starting at 2 weeks after
disease induction (day 14, 20 days after treatment initiation), until the mice were sacrificed (day 31). When the mice were sacrificed, both the TPC-treated groups had a mean arthritis score of 1.5 (range from 0 to 4), while both the PBS-treated groups had a mean arthritis score of 11.8 (range from 10 to 14) (P < 0.001). TPC orally treated mice had a mean arthritis score of 1.6 6 1.5, while PBS orally treated mice had a mean arthritis score of 12.6 6 1.14 (P < 0.001). Moreover, TPC s.c.-treated mice had a mean arthritis score of 1.4 6 0.84, whereas PBS s.c.-treated mice had a mean arthritis score of 11 6 1.22 (P < 0.001). Representative pictures of mice joints shown in Fig. 1c demonstrate a significant difference in inflammation in the TPC-treated mice in comparison with the PBS-treated ones. PBS-treated mice developed oedema and erythema from the ankle to the entire leg, while TPCtreated mice exhibited milder symptoms.
Significantly attenuated inflammation in TPC-treated mice On day 31 of the experiment, mice were sacrificed and histological analyses of the paws were performed by H&E. Joint sections from the TPC- and PBS-treated mice demonstrated a significant difference. Examples of histological sections are illustrated in Fig. 2; TPC administration maintained a low inflammatory level in the joints. Both TPC oral- and s.c.-treated mice exhibited normal joints histology. The histological sections obtained from the TPC oraland s.c.-treated mice demonstrated significantly less synovial hyperplasia, normal cartilage layer and muscle structure, typical bone organization and uninflamed fat tissue. Nevertheless, microscopic analysis of the joints in PBS oral- and s.c.-treated mice showed high levels of inflammation. The histological sections obtained from the PBS oraland s.c.-treated mice displayed profound lymphocytic infiltration as well as neutrophils. There were large areas of fibrosis and several spots exhibited necrosis. The synovium was widely extended and the cartilage appeared to have erosion on the surface. Similarly, the muscles and bones exhibited severe destruction and fat tissue was not notable.
TPC reduced the titres of anti-collagen antibodies in mice sera The titres of anti-collagen type II antibodies in mice sera were measured at day 30 while the arthritis was fully blown. The data presented in Fig. 3 depict anti-collagen antibody titres in mice sera at a dilution of 1 : 200 (OD at 405 nm). A statistically significant difference was documented between the TPC oral- and s.c.-treated mice in comparison with PBS oral- and s.c.-treated mice (P < 0.001). The mean OD level of the anticollagen type II antibodies in the TPC oral-treated mice was 0.685 6 0.09, while the mean OD level of the anti-collagen type II antibodies in PBS oral-treated mice was 1.09 6 0.17. Furthermore, the mean OD level of the anti-collagen type II antibodies in TPC s.c.-treated mice was 0.615 6 0.08, whereas
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Fig. 1. Tuftsin–phosphorylcholine (TPC) effect on arthritis score in dilute DBA/1 collagen-induced arthritis (CIA) mice. The data are presented as arthritis score of treated mice with TPC versus phosphate-buffered saline (PBS) (n 5 10 per group), measured from day (26) (6 days prior to disease induction) until day 31 mean 6 standard deviation (s.d.). Values are mean 6 s.d. *P < 0.05; **P < 0.01; ***P < 0.001. (a) Arthritis score of oral-treated mice with TPC versus PBS. (b) Arthritis score of subcutaneously (s.c.)-treated mice with TPC versus PBS. (c) Representative joints photographs: (a) mice treated with PBS orally; (b) mice treated with PBS s.c.; (c) mice treated with TPC orally; (d) mice treated with TPC s.c.
the mean OD level of the anti-collagen type II antibodies in PBS s.c.-treated mice was 1.03 6 0.13 (P < 0.001).
TPC immunomodulation of cytokine expression upon treatment with TPC Cytokines play a major role in synovial inflammation, while the major ones are IL-17, TNF-a and IL-1 [30,31]. We evaluated the secretion of proinflammatory and antiinflammatory cytokines by splenocytes derived from CIA mice treated with TPC or PBS in vitro. The following cytokine secretions were examined: proinflammatory TNF-a, IL-1b, IL-17 and anti-inflammatory IL-10. As depicted in Fig. 4a-c, mean proinflammatory cytokine concentrations in TPC oral- and s.c.-treated mice were inhibited significantly in comparison with control PBS-treated mice (P < 0.0001). Mice treated orally with TPC had mean TNF-a levels of 633 6 91 (pg/ml), mean IL-17 levels of 505 6 26 (pg/ml) 22
and mean IL-1b levels of 183 6 111 (pg/ml), while mice treated orally with PBS had mean TNF-a levels of 1677 6 137 (pg/ml), mean IL-17 levels of 1585 6 90 (pg/ ml) and mean IL-1b levels of 686 6 194 (pg/ml). Moreover, TPC s.c.-treated mice had mean TNF-a levels of 621 6 72 (pg/ml), mean IL-17 levels of 497 6 28 (pg/ml) and mean IL-1b levels of 198 6 133 (pg/ml). In comparison, PBS s.c.-treated mice had mean TNF-a levels of 1586 6 65 (pg/ml), mean IL-17 levels of 1585 6 97 (pg/ ml) and mean IL-1b levels of 770 6 115 (pg/ml), whereas TPC increased the level of anti-inflammatory cytokine IL-10 significantly in comparison with control PBS-treated mice (P < 0.0001), as shown in Fig. 4d. Mice treated orally with TPC had mean IL-10 levels of 3605 6 743 (pg/ml), whereas mice treated orally with PBS had mean IL-10 levels of 384 6 149 (pg/ml). Furthermore, TPC s.c.-treated mice had mean IL-10 levels of 2917 6 810 (pg/ml). In comparison, PBS s.c.-treated mice had mean IL-10 levels of 302 6 49 (pg/ml).
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Fig. 2. Histological analysis. Representative arthritic paws from each studied group of mice were removed and stained with haematoxylin and eosin (H&E). (a) Mice treated with phosphatebuffered saline (PBS) orally; (b) mice treated with PBS subcutaneously (s.c.); (c) mice treated with TPC orally; (d) mice treated with TPC s.c. Magnification presented: 3100.
The effect of TPC on expansion of CD41CD251FoxP31 regulatory T cells (Treg) cells in isolated mice splenocytes
The effect of TPC on expansion of CD191IL101TIM-11CD251CD1d1 regulatory B cells (Breg) cells in isolated mice splenocytes
The frequency of CD41CD251FoxP31 Treg cell subsets in isolated mice splenocytes following treatment with TPC or PBS is elucidated in Fig. 5a,b. A striking increase was observed in the mean percentage of the Treg cells measured in isolated splenocytes of TPC-treated mice (19.82 6 4.89%), whereas the PBS-treated group of Treg cells measured in the isolated splenocytes had a mean percentage of 2.37 6 0.56%. As illustrated in Fig. 5a, TPC treatment promoted the Treg phenotype expansion significantly (P < 0.0001) when compared with PBS-treated mice. Representative data of the CD41CD251FoxP31 levels in splenocytes derived from TPC- and PBS-treated mice gating on CD41 T cells are presented in Fig. 5b.
The mean percentage of Breg cells in isolated splenocytes following treatment with TPC or PBS was measured within two main subsets, CD191IL-101TIM-11 and IL-10highCD25highCD1dhigh. As depicted in Fig. 6a–d, both subsets demonstrated that TPC stimulated the Breg phenotype expansion significantly in isolated mice splenocytes (P < 0.0001) when compared with the PBS-treated mice. As demonstrated in Fig. 6a, the mean percentage of CD191IL-101TIM-11 Breg cells of TPC-treated mice was significantly higher (21.63 6 3.2%), while the control group had a mean percentage of 0.05 6 0.06% (P < 0.0001). Representative data of CD191IL-101TIM-11 levels in splenocytes derived from TPC- and PBS-treated
Fig. 3. Titres of circulating anti-collagen antibodies. The levels of circulating anti-collagen antibodies were determined in the sera of mice treated with tuftsin–phosphorylcholine (TPC) orally, phosphate-buffered saline (PBS) orally, TPC subcutaneously (s.c.) and PBS s.c. by enzyme-linked immunosorbent assay (ELISA) at a dilution of 1 : 200 (n 5 10 per group). The data are presented as optical density (OD) at 405 nm. Values are mean 6 standard deviation (s.d.), P < 0.0001.
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Fig. 4. Cytokine expression by spleen cells from tuftsin–phosphorylcholine (TPC) and phosphate-buffered saline (PBS)-treated mice. In-vitro analyses of the proinflammatory cytokines tumour necrosis factor (TNF)-a, interleukin (IL)217, IL-1b and the anti-inflammatory cytokine IL10 concentration in the culture fluids of splenocytes originated from TPC oral, PBS oral, TPC subcutaneous (s.c.)- and PBS s.c.-treated mice. The data are presented as concentration in pg/ml; n 5 10 per group. (a) In-vitro analyses of the proinflammatory cytokine TNF-a concentration; (b) in-vitro analyses of the proinflammatory cytokine IL-17 concentration; (c). in-vitro analyses of the proinflammatory cytokine IL-1b concentration; (d) in-vitro analyses of the anti-inflammatory cytokine IL-10 concentration.
mice gating on CD191 B cells are presented in Fig. 6b. Furthermore, the mean percentage of IL-10highCD25highCD1dhigh Breg cells in isolated splencocytes of TPC- and PBStreated mice are displayed in Fig. 6c. TPC-treated mice exhibited higher Breg cell expansion (89.91 6 1.21%), while control mice exhibited a lack of IL-10highCD25highCD1dhigh Breg cell phenotype, with a mean percentage of 0.005 6 0.007% (P < 0.0001). Representative data of the IL-10highCD25highCD1dhigh phenotype in splenocytes derived from TPC- and PBS-treated mice gating on IL-10 B cells are presented in Fig. 6d.
Discussion Our findings demonstrate the ability of TPC to attenuate joint inflammation in CIA mice. TPC administered orally and s.c. decreased the titres of circulating anti-collagen antibodies titres. H&E staining demonstrated that TPC 24
prevented the destruction of joints, whereas joints of PBStreated mice were highly inflamed. Similarly, TPC increased the secretion of anti-inflammatory cytokine IL-10 and decreased concentrations of key proinflammatory cytokines TNF-a, IL-17 and IL-1b, resulting in a low inflammatory state. Furthermore, TPC enhanced the expansion of CD41CD251FoxP31 Treg as well as CD191IL-101CD5high CD1dhighTIM-11 Breg in TPC-treated mice, in comparison to the highly inflamed PBS-treated mice status. Our results are in accordance with our previous studies, treating lupus mice with TPC- and DSS-induced colitis mice. TPC shows successful modulation of the murine lupus nephritis. Administration of TPC attenuated glomerulonephritis in lupus-prone NZB3W/F1 mice and prolonged their survival time. The lupus reduction was exhibited by lower proteinuria and reduced immunoglobulin deposition in kidney mesangium [27]. Additionally, TPC treatment of the lupus-prone mice resulted in significantly lower proinflammatory IFN-g and IL-17 cytokine production and
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Fig. 5. Regulatory T cell (Treg) expansion in isolated splenocytes of tuftsin–phosphorylcholine (TPC)- and phosphate-buffered saline (PBS)treated mice. (a) The data are presented as percentage of Treg cells: CD41CD251forkhead box protein 3 (FoxP31) expansion in isolated splenocytes of TPC- and PBS-treated mice (n 5 10). Values are mean 6 standard deviation (s.d.), P < 0.0001. (b). Representative flow cytometry analyses of Treg cells: CD41CD251FoxP31 (gated on CD41) in splenocytes derived from the TPC- and PBS-treated mice. TPC: 25.9%; PBS: 2.37%.
increase of the anti-inflammatory IL-10 and TGF-b cytokine expression, as well as expansion of the CD41CD251FoxP31 Treg phenotype [27]. Moreover, treatment with TPC prevented the severity of disease in DSS-induced colitis mice. The prevention of colitis was manifested as a reduction in colitis score, which entails the daily activity index (DAI) as well as keeping the colon length [26]. Furthermore, TPC treatment of mice with DSS-induced colitis resulted in reduced colon proinflammatory IL-1b, TNF-a and IL-17 cytokine production and an increase of anti-inflammatory IL-10 cytokine expression [26]. The helminthes therapeutic approach in arthritis dates back to 1975. The first study, associating helminthes infection and joint disease, showed the surprising observation that rats infected with the nematode Syphacia obvelata developed milder complete Freund’s adjuvant (CFA)induced arthritis [32]. In addition, Schistosoma mansoni was reported to reduce the severity of arthritis in CIA mice by suppressing systemic and local proinflammatory mediators, thus leading to significantly less synovial hyperplasia [13]. Furthermore, a rodent filarial nematode, Acanthocheilonema viteae, secretes a glycoprotein, ES-62, which is an
immunomodulatory glycoprotein surrounded by PC moiety attached to the protein by N. glycans. The PC moiety was shown to be responsible for the immunomodulatory activity and to have a beneficial effect in the CIA mice model [18,19,33,34]. The quest for a drug that possesses beneficial immunomodulation capabilities, as do helminthes, to enable novel treatment approaches in autoimmunity with low side effects led to the creation of small immunomodulating molecules. Harnett et al. designed a sulphone-containing PC analogue (11a,12b) and provided proof of concept to the therapeutic capabilities of small molecule analogues in mice with CIA model [35,36]. We hypothesize that TPC immunomodulatory activity comprises PC and tuftsin biological functions. PC immunomodulation effect was suggested to mediate Toll-like receptor (TLR)24 signal transduction, which depends upon the TLR adaptor myeloid differentiation primary response gene 88 (MyD88), as demonstrated in TLR-4 knock-out mice [19,37,38]. Tuftsin reduced phospho-signal transducer and activator of transcription-1 (STAT-1) expression, while it increased
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Fig. 6. Regulatory B cell (Breg) expansion in isolated splenocytes of tuftsin–phosphorylcholine (TPC)- and phosphate-buffered saline (PBS)treated mice. (a) The data are presented as a percentage of Breg cells: CD191interleukin (IL)2101 T cell immunoglobulin mucin-1 (TIM-11) expansion in splenocytes of TPC- and PBS- treated mice (n 5 10). Values are mean 6 standard deviation (s.d.), P < 0.0001. (b) Representative flow cytometry analyses of Breg cells: CD191IL-101TIM-11 (gated on CD191) in splenocytes derived from the TPC- and PBS-treated mice. TPC: 23.4%; PBS: 0.13%. (c) The data are presented as a percentage of Breg cells: IL-101CD5highCD1dhigh expansion in splenocytes of TPC- and PBStreated mice (n 5 10). Values are mean 6 s.d., P < 0.0001. (d) Representative flow cytometry analyses of Breg cells: IL-101CD5highCD1dhigh (gated on IL-101) in splenocytes derived from the TPC- and PBS-treated mice. TPC: 91.3%; PBS: 0.01%.
the synthesis of IL-10 and IL-4. The effect was in comparison with control homogenates [39]. RA is associated with a T helper type 1 (Th1)/Th17 response and a general rise of proinflammatory cytokines such as IL-1, IL-6, IL-17 and TNF-a [3,40,41]. Furthermore, B cells are considered to play several roles in the pathogenesis of RA, such as antigen presentation supporting the activation of autoreactive T cells, autoantibody production and cytokine release [41,42]. Moreover, in the CIA model, collagen immunization induces chronic inflammatory arthritis due to CD41 T cell infiltration into the synovial membrane, as well as collagen-specific IgG autoantibody production by B cells [43–45]. Breg cells regulate inflammatory immune responses. Indeed, transferring CD1dhighCD51 Breg cells into CD192/2 mice reduced inflammation significantly by increasing the IL-10 levels [46,47]. Moreover, it was demonstrated that TIM-11 B cells induced tolerance by induction of Th2 responses [48]. 26
In addition, it was found that Breg cells regulate experimental autoimmune encephalomyelitis (EAE) clinical recovery by interacting with Treg cells, causing them to mobilize and migrate into the central nervous system (CNS), resulting in disease resolution via their increased production of IL-10 [49,50]. In this study, prophylactic treatment with TPC induced the expansion of CD41CD251FoxP31 Treg and CD191IL101CD5highCD1dhighTIM-11 Breg cells in isolated splenocytes, thus modulating the cytokine profile and decreasing the level of inflammation and synovial hyperplasia. Prominent Treg and Breg cells enabled the reduction in proinflammatory cytokines and increased the anti-inflammatory cytokines in stimulated isolated splenocytes taken from TPC-treated mice. In conclusion, in this study we have shown that treatment of CIA mice with TPC can prevent the development of arthritis.
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Acknowledgements This study was supported by the Moris Family Foundation, Los Angeles USA and TEVA Ltd, grant no. 147333, Israel. This work was performed as partial fulfilment of the requirements for Tomer Bashi PhD, Sackler Faculty of Medicine, Tel Aviv University, Israel.
Disclosure The authors have no disclosures to declare.
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15 Anthony RM, Rutitzky LI, Urban JF Jr, Stadecker MJ, Gause WC. Protective immune mechanisms in helminth infection. Nat Rev Immunol 2007; 7:975–87. 16 Summers RW, Elliott DE, Urban JF Jr, Thompson R, Weinstock JV. Trichuris suis therapy in Crohn’s disease. Gut 2005; 54:87–90. 17 Marshall FA, Grierson AM, Garside P, Harnett W, Harnett MM. ES-62, an immunomodulator secreted by filarial nematodes, suppresses clonal expansion and modifies effector function of heterologous antigen-specific T cells in vivo. J Immunol 2005; 175:5817–26. 18 Harnett W, Harnett MM, Leung BP, Gracie JA, McInnes IB. The anti-inflammatory potential of the filarial nematode secreted product, ES-62. Curr Top Med Chem 2004; 4:553–9. 19 Harnett MM, Kean DE, Boitelle A et al. The phosphorycholine moiety of the filarial nematode immunomodulator ES-62 is responsible for its anti-inflammatory action in arthritis. Ann Rheum Dis 2008; 67:518–23. 20 Al-Riyami L, Harnett W. Immunomodulatory properties of ES62, a phosphorylcholine-containing glycoprotein secreted by Acanthocheilonema viteae. Endocr Metab Immune Disord Drug Targets 2012; 12:45–52. 21 Poltl G, Kerner D, Paschinger K, Wilson IB. N-glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues. FEBS J 2007; 274:714–26. 22 Pery P, Petit A, Poulain J, Luffau G. Phosphorylcholine-bearing components in homogenates of nematodes. Eur J Immunol 1974; 4:637–9. 23 Siemion IZ, Kluczyk A. Tuftsin: on the 30-year anniversary of Victor Najjar’s discovery. Peptides 1999; 20:645–74. 24 Fridkin M, Najjar VA. Tuftsin: its chemistry, biology, and clinical potential. Crit Rev Biochem Mol Biol 1989; 24:1–40. 25 Najjar VA, Nishioka K. ‘Tuftsin’: a natural phagocytosis stimulating peptide. Nature 1970; 228:672–3. 26 Ben-Ami Shor D, Bashi T, Lachnish J et al. Phosphorylcholine– tuftsin compound prevents development of dextransulfatesodium-salt induced murine colitis: implications for the treatment of human inflammatory bowel disease. J Autoimmun 2015; 56:111–7. 27 Bashi T, Blank M, Ben-Ami Shor D et al. Successful modulation of murine lupus nephritis with tuftsin–phosphorylcholine. J Autoimmun 2015; 59:1–7. 28 Koch Y, Wilchek M, Fridkin M, Chobsieng P, Zor U, Lindner HR. Production and characterization of an antiserum to synthetic gonadotropin-releasing hormone. Biochem Biophys Res Commun 1973; 55:616–22. 29 Brand DD, Latham KA, Rosloniec EF. Collagen-induced arthritis. Nat Protoc 2007; 2:1269–75. 30 Choy E. Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. Rheumatology 2012; 51 (Suppl. 5):v3–11. 31 McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007; 7:429–42. 32 Pearson DJ, Taylor G. The influence of the nematode Syphacia oblevata on adjuvant arthritis in the rat. Immunology 1975; 29: 391–6. 33 Pineda MA, Rodgers DT, Al-Riyami L, Harnett W, Harnett MM. ES-62 protects against collagen-induced arthritis by resetting interleukin-22 toward resolution of inflammation in the joints. Arthritis Rheumatol 2014; 66:1492–503.
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T. Bashi et al. 34 McInnes IB, Leung BP, Harnett M, Gracie JA, Liew FY, Harnett W. A novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholinecontaining glycoprotein ES-62. J Immunol 2003; 171:2127–33. 35 Rzepecka J, Coates ML, Saggar M et al. Small molecule analogues of the immunomodulatory parasitic helminth product ES-62 have anti-allergy properties. Int J Parasitol 2014; 44:669–74. 36 Al-Riyami L, Pineda MA, Rzepecka J et al. Designing antiinflammatory drugs from parasitic worms: a synthetic small molecule analogue of the Acanthocheilonema viteae product ES62 prevents development of collagen-induced arthritis. J Med Chem 2013; 56:9982–10002. 37 Goodridge HS, Marshall FA, Else KJ et al. Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholinecontaining secreted product, ES-62. J Immunol 2005; 174:284–93. 38 Goodridge HS, McGuiness S, Houston KM et al. Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells. Parasite Immunol 2007; 29:127–37. 39 Wu M, Nissen JC, Chen EI, Tsirka SE. Tuftsin promotes an antiinflammatory switch and attenuates symptoms in experimental autoimmune encephalomyelitis. PLOS ONE 2012; 7:e34933 40 Sarkar S, Cooney LA, Fox DA. The role of T helper type 17 cells in inflammatory arthritis. Clin Exp Immunol 2010; 159:225–37. 41 Takemura S, Klimiuk PA, Braun A, Goronzy JJ, Weyand CM. T cell activation in rheumatoid synovium is B cell dependent. J Immunol 2001; 167:4710–8. 42 Moura RA, Graca L, Fonseca JE. To B or not to B the conductor of rheumatoid arthritis orchestra. Clin Rev Allergy Immunol 2012; 43:281–91.
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43 Flores-Borja F, Bosma A, Ng D et al. CD191CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci Transl Med 2013; 5:173ra23 44 Yoshizaki A, Miyagaki T, DiLillo DJ et al. Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions. Nature 2012; 491:264–8. 45 Yanaba K, Bouaziz JD, Matsushita T, Magro CM, St Clair EW, Tedder TF. B lymphocyte contributions to human autoimmune disease. Immunol Rev 2008; 223:284–99. 46 DiLillo DJ, Matsushita T, Tedder TF. B10 cells and regulatory B cells balance immune responses during inflammation, autoimmunity, and cancer. Ann NY Acad Sci 2010; 1183: 38–57. 47 Yanaba K, Bouaziz JD, Haas KM, Poe JC, Fujimoto M, Tedder TF. A regulatory B cell subset with a unique CD1dhiCD51 phenotype controls T cell-dependent inflammatory responses. Immunity 2008; 28:639–50. 48 Ding Q, Yeung M, Camirand G et al. Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice. J Clin Invest 2011; 121:3645–56. 49 Ray A, Basu S, Williams CB, Salzman NH, Dittel BN. A novel IL-10-independent regulatory role for B cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR ligand. J Immunol 2012; 188:3188–98. 50 Mann MK, Maresz K, Shriver LP, Tan Y, Dittel BN. B cell regulation of CD41CD251 T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis. J Immunol 2007; 178:3447–56.
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OR I G INA L A RTI CLE
doi:10.1111/cei.12745
Novel therapeutic compound tuftsin–phosphorylcholine attenuates collagen-induced arthritis
T. Bashi,* O. Shovman,* M. Fridkin,† A. Volkov,‡ I. Barshack,‡ M. Blank* and Y. Shoenfeld* *Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, affiliated to the Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv, Israel, †Department of Organic Chemistry, The Weizmann Institute of Sciences, Rehovot, Israel, Tel-Aviv, Israel, and ‡Institute of Pathology, Sheba Medical Center, affiliated to the Sackler Faculty of Medicine Tel-Aviv University, Tel-Aviv, Israel
Accepted for publication 26 November 2015 Correspondence: Y. Shoenfeld, Head of Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, TelHashomer, 52621, Israel. E-mail: [email protected]
Summary Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin–PC (TPC), a novel helminth-based compound in collagen-induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 mg/0.1 ml) or orally (250 mg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme-linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (Treg) and regulatory B (Breg) cell phenotypes by fluorescence-activated cell sorter (FACS). TPC-treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P < 0.0001) in both subcutaneous and orally treated groups at day 31. Moreover, histology analysis demonstrated highly inflamed joints in control mice, whereas TPC-treated mice maintained normal joint structure. Furthermore, TPC decreased the titres of circulating collagen II antibodies in mice sera (P < 0.0001), enhanced expression of IL-10 (P < 0.0001) and inhibited production of tumour necrosis factor (TNF)-a, interleukin (IL)217 and IL-1b (P < 0.0001). TPC significantly expanded the CD41CD251 forkhead box protein 3 (FoxP31) Treg cells and CD191IL-101CD5highCD1dhighT cell immunoglobulin mucin1 (TIM-11) Breg cell phenotypes (P < 0.0001) in treated mice. Our data indicate that treatment with TPC attenuates CIA in mice demonstrated by low arthritic score and normal joints histology. TPC treatment reduced proinflammatory cytokines and increased anti-inflammatory cytokine expression, as well as expansion of Treg and Breg cells. Our results may lead to a new approach for a natural therapy for early rheumatoid arthritis onset. Keywords: collagen-induced arthritis, helminth, phosphorylcholine, rheumatoid arthritis, tuftsin
Introduction Rheumatoid arthritis (RA) is a chronic autoinflammation of the joints, with a prevalence of approximately 1% in Western populations [1–4]. Current therapeutic strategies of RA involve mainly methotrexate [5], as well as immune-based, anti-inflammatory therapies such as tumour necrosis factor (TNF)-a, interleukin (IL)21, CD-20 targeting agents or Janus kinase 1,2,3 (JAK) inhibitors [6–8]. However, such biological agents are still relatively limited, and they have various side
effects. Therefore, there is a demand to develop new small immunomodulatory molecules with minimal side effects. One of the novel approaches for treating autoinflammation is to adopt natural strategy to control the immune system. The modern lifestyle has led to a decrease in the infections burden [9]. Moreover, there is a lower prevalence of RA in helminthes-endemic areas [10]. Helminthes survive within the host by immunomodulating the host innate immunity. Treatment with live helminthes or helminthes products in
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patients and in animal models improved the clinical score in RA, multiple sclerosis, type I diabetes mellitus and inflammatory bowel disease [11–18]. Immune regulation functions of some helminthes are attributed to the phosphorylcholine (PC) moiety [19]. PC is a non-immunogenic small molecule also present in helminthes products [20–22]; we have constructed a chimeric compound composed of PC and tuftsin, referred to as tuftsin–phosphorylcholine (TPC). Tuftsin is a physiological natural immunomodulatory tetrapeptide (Thr-Lys-Pro-Arg), a small part of the immunoglobulin (Ig)G heavy-chain molecule produced by enzymatic cleavage in the spleen. Tuftsin has been shown to have anti-microbial, anti-viral and anti-tumour functions mediated by enhancement of macrophagic activity [23–25]. Recently, we have demonstrated that treatment with TPC attenuated glomerulonephritis in lupus-prone mice and prevented colitis severity in dextran–sulphate–sodium salt (DSS)-induced colitis in mice [26,27]. Our current study addresses TPC therapeutic efficacy in a mouse model of collagen-induced arthritis (CIA). TPC immunomodulatory effect is associated with a significant reduction in arthritic score, prevention of joint damage accompanied by immunomodulation of the cytokines profile and enhanced expansion of T and B regulatory cells.
Materials and methods Tuftsin–phosphorylcholine synthesis (TPC) Tuftsin is a physiological natural immunostimulating tetrapeptide (Thr-Lys-Pro-Arg) fraction of the IgG heavy-chain molecule produced by enzymatic cleavage in the spleen [23]. Tuftsin was extended at its C-terminal, i.e. Thr-LysPro-Arg-Gly-Tyr, and was synthesized manually following solid-phase peptide technology (GLS peptide synthesis; GL Biochem, Shanghai, China). The peptide was coupled to diazotized 4-aminophenyphosphorylchloride to form an azo bond between the tuftsin and PC (Sigma-Aldrich, St Louis, MO, USA) [24,28]. The conjugate was characterized by mass spectra and amino acid analysis as well as by highperformance liquid chromatography (HPLC). TPC was diluted in commercial phosphate-buffered saline (PBS) (Biological Industries, Israel Beit-Haemek Ltd, Kibbutz Beit-Haemek, Israel).
Mice and experimental design Experimental arthritis was induced in DBA/1 male mice at the age of 6–7 weeks (Harlan Laboratories, Kreuzelweg, the Netherlands). The mice were maintained in a conventional animal housing facility at Sheba Medical Center and kept in individually ventilated cages. All experiments were approved and executed according to the protocols of the Ethical Committee of the Israeli Ministry of Health, no. 20
696/11. Disease induction was carried out by injecting 100 lg bovine type II collagen intradermally into the base of the tail (Chondrex, Redmond, WA, USA) in 1 : 1 emulsion with Mycobacterium tuberculosis H37RA in Freund’s incomplete adjuvant (Difco Laboratories, Detroit, MI, USA). A boost injection of bovine type II collagen in PBS at the base of the tail was given 2 weeks later [29]. TPC was first administered at day 26, 6 days prior to disease induction; PBS was given as control vehicle. TPC was given either orally using a feeding needle (250 mg/ 0.1 ml per mouse) once a week or subcutaneously (s.c.) (5 mg/0.1 ml per mouse) twice a week, n 5 30 per group. The mice were sacrificed after 31 days.
Assessment of arthritis Mice were monitored three times a week by two blind observers for signs of arthritis, for which severity scores were derived as follows: 0 5 normal, 1 5 slight erythema, 2 5 slight erythema plus swelling, 3 5 moderate oedema and erythema 4 5 oedema and erythema from the ankle to the entire leg. The total arthritis score was the sum score of the four limbs.
Histopathological assessment Mouse paws were obtained from the sacrificed mice and fixed in 4% formalin (Sigma, St Louis, MO, USA), decalcified, cut, and stained with haematoxylin and eosin (H&E). All histopathological evaluations were performed by two blind pathologists.
Anti-collagen type II antibodies Titres of circulating anti-collagen type II antibodies were determined by enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with collagen type II [10 mg/ml phosphate-buffered saline (PBS)] overnight 48C and then blocked with 3% bovine serum albumin (BSA). Mouse serum (n 5 10 for each treatment group) was diluted to 1 : 200 up to 1 : 400 and was incubated for 2 h at room temperature. The binding was probed with goat antimouse IgG conjugated with alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), followed by the addition of appropriate substrate. The data were read by ELISA reader at optical density (OD) 405 nm.
Analysis of cytokine production by splenocytes derived from treated mice Spleen cells were originated from TPC s.c. and TPC orally treated mice (n 5 10 mice per group), as well as from vehicle PBS-treated mice (n 5 10 mice per group). The spleen cells were isolated after erythrocytes were lysed by red blood cells lysis buffer (Biological Industries). The spleen cells were seeded (5 3 106 cells/well) in 24-well plates (Thermo plates; Nunc Fisher Scientific Inc., Waltham, MA, USA), coated with anti-CD3 antibodies (2 mg/ml) in the
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presence of 5 mg/ml TPC or PBS for 72 h in RPMI-1640 enriched medium at 378C and 5% CO2. Culture supernatants were then collected. Proinflammatory (IL-1b, IL-17, TNF-a) and IL-10 anti-inflammatory cytokine levels in the culture supernatant were detected by DueSet ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.
Analysis of regulatory T [CD41CD251forkhead box protein 3 (FoxP31)] cells by flow cytometry Isolated splenocytes, depleted of red blood cells, were incubated with anti-CD41fluorescein isothiocyanate (FITC) anti-CD251 allophycocyanin (APC) anti-FoxP31phycoerythrin (PE) (eBioscience, San-Diego, CA, USA) and analysed by flow cytometry, with forward- and side-scatter gates adjusted to include all cells and to exclude debris (Becton Dickinson, Franklin Lakes, NJ, USA). For intracellular staining of FoxP3, the cells were preincubated with a fixation solution, washed and resuspended in permeabilization solution (Serotec, Oxford, UK) and stained intracellularly for FoxP3. The gating was on the CD41 T cells.
Analysis of B10 regulatory CD191IL-101T cell immunoglobulin mucin-1 (TIM-11)CD1dhighCD5high cells by flow cytometry B cells from isolated splenocytes underwent isolation by negative selection using monoclonal antibodies against CD43, CD4 and Ter-119 (B cell isolation kit; Miltenyi Biotec, Auburn, CA, USA). B cells were incubated with antiCD191FITC anti-T cell Ig domain and mucin domain protein 1 (TIM-1)1APC anti-IL-101PE (eBioscience) and anti-IL-101FITC anti-CD1d1APC anti-CD51PE (eBioscience) and were analysed by flow cytometry (Becton Dickinson). The gating was on the CD191 B cells or IL101B10 cells.
Statistical analysis To evaluate the significance of the differences between groups, TPC s.c. to PBS s.c. and TPC oral to PBS oral, Student’s t-test was used. Values of P < 0.05 were considered significant.
Results TPC reduces arthritis severity in mice with CIA The main characteristic of RA is joint deformation due to severe inflammation, therefore we followed the TPC effect on arthritis score in CIA mice. CIA mice were subjected to TPC, orally or s.c., and compared to mice treated with PBS. As detailed in Fig. 1a,b, we observed a significantly lower arthritis score in TPC-treated mice compared with PBS-treated mice (P < 0.05). Arthritis score was lower in both the TPC orally and TPC s.c.-treated groups starting at 2 weeks after
disease induction (day 14, 20 days after treatment initiation), until the mice were sacrificed (day 31). When the mice were sacrificed, both the TPC-treated groups had a mean arthritis score of 1.5 (range from 0 to 4), while both the PBS-treated groups had a mean arthritis score of 11.8 (range from 10 to 14) (P < 0.001). TPC orally treated mice had a mean arthritis score of 1.6 6 1.5, while PBS orally treated mice had a mean arthritis score of 12.6 6 1.14 (P < 0.001). Moreover, TPC s.c.-treated mice had a mean arthritis score of 1.4 6 0.84, whereas PBS s.c.-treated mice had a mean arthritis score of 11 6 1.22 (P < 0.001). Representative pictures of mice joints shown in Fig. 1c demonstrate a significant difference in inflammation in the TPC-treated mice in comparison with the PBS-treated ones. PBS-treated mice developed oedema and erythema from the ankle to the entire leg, while TPCtreated mice exhibited milder symptoms.
Significantly attenuated inflammation in TPC-treated mice On day 31 of the experiment, mice were sacrificed and histological analyses of the paws were performed by H&E. Joint sections from the TPC- and PBS-treated mice demonstrated a significant difference. Examples of histological sections are illustrated in Fig. 2; TPC administration maintained a low inflammatory level in the joints. Both TPC oral- and s.c.-treated mice exhibited normal joints histology. The histological sections obtained from the TPC oraland s.c.-treated mice demonstrated significantly less synovial hyperplasia, normal cartilage layer and muscle structure, typical bone organization and uninflamed fat tissue. Nevertheless, microscopic analysis of the joints in PBS oral- and s.c.-treated mice showed high levels of inflammation. The histological sections obtained from the PBS oraland s.c.-treated mice displayed profound lymphocytic infiltration as well as neutrophils. There were large areas of fibrosis and several spots exhibited necrosis. The synovium was widely extended and the cartilage appeared to have erosion on the surface. Similarly, the muscles and bones exhibited severe destruction and fat tissue was not notable.
TPC reduced the titres of anti-collagen antibodies in mice sera The titres of anti-collagen type II antibodies in mice sera were measured at day 30 while the arthritis was fully blown. The data presented in Fig. 3 depict anti-collagen antibody titres in mice sera at a dilution of 1 : 200 (OD at 405 nm). A statistically significant difference was documented between the TPC oral- and s.c.-treated mice in comparison with PBS oral- and s.c.-treated mice (P < 0.001). The mean OD level of the anticollagen type II antibodies in the TPC oral-treated mice was 0.685 6 0.09, while the mean OD level of the anti-collagen type II antibodies in PBS oral-treated mice was 1.09 6 0.17. Furthermore, the mean OD level of the anti-collagen type II antibodies in TPC s.c.-treated mice was 0.615 6 0.08, whereas
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Fig. 1. Tuftsin–phosphorylcholine (TPC) effect on arthritis score in dilute DBA/1 collagen-induced arthritis (CIA) mice. The data are presented as arthritis score of treated mice with TPC versus phosphate-buffered saline (PBS) (n 5 10 per group), measured from day (26) (6 days prior to disease induction) until day 31 mean 6 standard deviation (s.d.). Values are mean 6 s.d. *P < 0.05; **P < 0.01; ***P < 0.001. (a) Arthritis score of oral-treated mice with TPC versus PBS. (b) Arthritis score of subcutaneously (s.c.)-treated mice with TPC versus PBS. (c) Representative joints photographs: (a) mice treated with PBS orally; (b) mice treated with PBS s.c.; (c) mice treated with TPC orally; (d) mice treated with TPC s.c.
the mean OD level of the anti-collagen type II antibodies in PBS s.c.-treated mice was 1.03 6 0.13 (P < 0.001).
TPC immunomodulation of cytokine expression upon treatment with TPC Cytokines play a major role in synovial inflammation, while the major ones are IL-17, TNF-a and IL-1 [30,31]. We evaluated the secretion of proinflammatory and antiinflammatory cytokines by splenocytes derived from CIA mice treated with TPC or PBS in vitro. The following cytokine secretions were examined: proinflammatory TNF-a, IL-1b, IL-17 and anti-inflammatory IL-10. As depicted in Fig. 4a-c, mean proinflammatory cytokine concentrations in TPC oral- and s.c.-treated mice were inhibited significantly in comparison with control PBS-treated mice (P < 0.0001). Mice treated orally with TPC had mean TNF-a levels of 633 6 91 (pg/ml), mean IL-17 levels of 505 6 26 (pg/ml) 22
and mean IL-1b levels of 183 6 111 (pg/ml), while mice treated orally with PBS had mean TNF-a levels of 1677 6 137 (pg/ml), mean IL-17 levels of 1585 6 90 (pg/ ml) and mean IL-1b levels of 686 6 194 (pg/ml). Moreover, TPC s.c.-treated mice had mean TNF-a levels of 621 6 72 (pg/ml), mean IL-17 levels of 497 6 28 (pg/ml) and mean IL-1b levels of 198 6 133 (pg/ml). In comparison, PBS s.c.-treated mice had mean TNF-a levels of 1586 6 65 (pg/ml), mean IL-17 levels of 1585 6 97 (pg/ ml) and mean IL-1b levels of 770 6 115 (pg/ml), whereas TPC increased the level of anti-inflammatory cytokine IL-10 significantly in comparison with control PBS-treated mice (P < 0.0001), as shown in Fig. 4d. Mice treated orally with TPC had mean IL-10 levels of 3605 6 743 (pg/ml), whereas mice treated orally with PBS had mean IL-10 levels of 384 6 149 (pg/ml). Furthermore, TPC s.c.-treated mice had mean IL-10 levels of 2917 6 810 (pg/ml). In comparison, PBS s.c.-treated mice had mean IL-10 levels of 302 6 49 (pg/ml).
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Fig. 2. Histological analysis. Representative arthritic paws from each studied group of mice were removed and stained with haematoxylin and eosin (H&E). (a) Mice treated with phosphatebuffered saline (PBS) orally; (b) mice treated with PBS subcutaneously (s.c.); (c) mice treated with TPC orally; (d) mice treated with TPC s.c. Magnification presented: 3100.
The effect of TPC on expansion of CD41CD251FoxP31 regulatory T cells (Treg) cells in isolated mice splenocytes
The effect of TPC on expansion of CD191IL101TIM-11CD251CD1d1 regulatory B cells (Breg) cells in isolated mice splenocytes
The frequency of CD41CD251FoxP31 Treg cell subsets in isolated mice splenocytes following treatment with TPC or PBS is elucidated in Fig. 5a,b. A striking increase was observed in the mean percentage of the Treg cells measured in isolated splenocytes of TPC-treated mice (19.82 6 4.89%), whereas the PBS-treated group of Treg cells measured in the isolated splenocytes had a mean percentage of 2.37 6 0.56%. As illustrated in Fig. 5a, TPC treatment promoted the Treg phenotype expansion significantly (P < 0.0001) when compared with PBS-treated mice. Representative data of the CD41CD251FoxP31 levels in splenocytes derived from TPC- and PBS-treated mice gating on CD41 T cells are presented in Fig. 5b.
The mean percentage of Breg cells in isolated splenocytes following treatment with TPC or PBS was measured within two main subsets, CD191IL-101TIM-11 and IL-10highCD25highCD1dhigh. As depicted in Fig. 6a–d, both subsets demonstrated that TPC stimulated the Breg phenotype expansion significantly in isolated mice splenocytes (P < 0.0001) when compared with the PBS-treated mice. As demonstrated in Fig. 6a, the mean percentage of CD191IL-101TIM-11 Breg cells of TPC-treated mice was significantly higher (21.63 6 3.2%), while the control group had a mean percentage of 0.05 6 0.06% (P < 0.0001). Representative data of CD191IL-101TIM-11 levels in splenocytes derived from TPC- and PBS-treated
Fig. 3. Titres of circulating anti-collagen antibodies. The levels of circulating anti-collagen antibodies were determined in the sera of mice treated with tuftsin–phosphorylcholine (TPC) orally, phosphate-buffered saline (PBS) orally, TPC subcutaneously (s.c.) and PBS s.c. by enzyme-linked immunosorbent assay (ELISA) at a dilution of 1 : 200 (n 5 10 per group). The data are presented as optical density (OD) at 405 nm. Values are mean 6 standard deviation (s.d.), P < 0.0001.
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Fig. 4. Cytokine expression by spleen cells from tuftsin–phosphorylcholine (TPC) and phosphate-buffered saline (PBS)-treated mice. In-vitro analyses of the proinflammatory cytokines tumour necrosis factor (TNF)-a, interleukin (IL)217, IL-1b and the anti-inflammatory cytokine IL10 concentration in the culture fluids of splenocytes originated from TPC oral, PBS oral, TPC subcutaneous (s.c.)- and PBS s.c.-treated mice. The data are presented as concentration in pg/ml; n 5 10 per group. (a) In-vitro analyses of the proinflammatory cytokine TNF-a concentration; (b) in-vitro analyses of the proinflammatory cytokine IL-17 concentration; (c). in-vitro analyses of the proinflammatory cytokine IL-1b concentration; (d) in-vitro analyses of the anti-inflammatory cytokine IL-10 concentration.
mice gating on CD191 B cells are presented in Fig. 6b. Furthermore, the mean percentage of IL-10highCD25highCD1dhigh Breg cells in isolated splencocytes of TPC- and PBStreated mice are displayed in Fig. 6c. TPC-treated mice exhibited higher Breg cell expansion (89.91 6 1.21%), while control mice exhibited a lack of IL-10highCD25highCD1dhigh Breg cell phenotype, with a mean percentage of 0.005 6 0.007% (P < 0.0001). Representative data of the IL-10highCD25highCD1dhigh phenotype in splenocytes derived from TPC- and PBS-treated mice gating on IL-10 B cells are presented in Fig. 6d.
Discussion Our findings demonstrate the ability of TPC to attenuate joint inflammation in CIA mice. TPC administered orally and s.c. decreased the titres of circulating anti-collagen antibodies titres. H&E staining demonstrated that TPC 24
prevented the destruction of joints, whereas joints of PBStreated mice were highly inflamed. Similarly, TPC increased the secretion of anti-inflammatory cytokine IL-10 and decreased concentrations of key proinflammatory cytokines TNF-a, IL-17 and IL-1b, resulting in a low inflammatory state. Furthermore, TPC enhanced the expansion of CD41CD251FoxP31 Treg as well as CD191IL-101CD5high CD1dhighTIM-11 Breg in TPC-treated mice, in comparison to the highly inflamed PBS-treated mice status. Our results are in accordance with our previous studies, treating lupus mice with TPC- and DSS-induced colitis mice. TPC shows successful modulation of the murine lupus nephritis. Administration of TPC attenuated glomerulonephritis in lupus-prone NZB3W/F1 mice and prolonged their survival time. The lupus reduction was exhibited by lower proteinuria and reduced immunoglobulin deposition in kidney mesangium [27]. Additionally, TPC treatment of the lupus-prone mice resulted in significantly lower proinflammatory IFN-g and IL-17 cytokine production and
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Fig. 5. Regulatory T cell (Treg) expansion in isolated splenocytes of tuftsin–phosphorylcholine (TPC)- and phosphate-buffered saline (PBS)treated mice. (a) The data are presented as percentage of Treg cells: CD41CD251forkhead box protein 3 (FoxP31) expansion in isolated splenocytes of TPC- and PBS-treated mice (n 5 10). Values are mean 6 standard deviation (s.d.), P < 0.0001. (b). Representative flow cytometry analyses of Treg cells: CD41CD251FoxP31 (gated on CD41) in splenocytes derived from the TPC- and PBS-treated mice. TPC: 25.9%; PBS: 2.37%.
increase of the anti-inflammatory IL-10 and TGF-b cytokine expression, as well as expansion of the CD41CD251FoxP31 Treg phenotype [27]. Moreover, treatment with TPC prevented the severity of disease in DSS-induced colitis mice. The prevention of colitis was manifested as a reduction in colitis score, which entails the daily activity index (DAI) as well as keeping the colon length [26]. Furthermore, TPC treatment of mice with DSS-induced colitis resulted in reduced colon proinflammatory IL-1b, TNF-a and IL-17 cytokine production and an increase of anti-inflammatory IL-10 cytokine expression [26]. The helminthes therapeutic approach in arthritis dates back to 1975. The first study, associating helminthes infection and joint disease, showed the surprising observation that rats infected with the nematode Syphacia obvelata developed milder complete Freund’s adjuvant (CFA)induced arthritis [32]. In addition, Schistosoma mansoni was reported to reduce the severity of arthritis in CIA mice by suppressing systemic and local proinflammatory mediators, thus leading to significantly less synovial hyperplasia [13]. Furthermore, a rodent filarial nematode, Acanthocheilonema viteae, secretes a glycoprotein, ES-62, which is an
immunomodulatory glycoprotein surrounded by PC moiety attached to the protein by N. glycans. The PC moiety was shown to be responsible for the immunomodulatory activity and to have a beneficial effect in the CIA mice model [18,19,33,34]. The quest for a drug that possesses beneficial immunomodulation capabilities, as do helminthes, to enable novel treatment approaches in autoimmunity with low side effects led to the creation of small immunomodulating molecules. Harnett et al. designed a sulphone-containing PC analogue (11a,12b) and provided proof of concept to the therapeutic capabilities of small molecule analogues in mice with CIA model [35,36]. We hypothesize that TPC immunomodulatory activity comprises PC and tuftsin biological functions. PC immunomodulation effect was suggested to mediate Toll-like receptor (TLR)24 signal transduction, which depends upon the TLR adaptor myeloid differentiation primary response gene 88 (MyD88), as demonstrated in TLR-4 knock-out mice [19,37,38]. Tuftsin reduced phospho-signal transducer and activator of transcription-1 (STAT-1) expression, while it increased
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Fig. 6. Regulatory B cell (Breg) expansion in isolated splenocytes of tuftsin–phosphorylcholine (TPC)- and phosphate-buffered saline (PBS)treated mice. (a) The data are presented as a percentage of Breg cells: CD191interleukin (IL)2101 T cell immunoglobulin mucin-1 (TIM-11) expansion in splenocytes of TPC- and PBS- treated mice (n 5 10). Values are mean 6 standard deviation (s.d.), P < 0.0001. (b) Representative flow cytometry analyses of Breg cells: CD191IL-101TIM-11 (gated on CD191) in splenocytes derived from the TPC- and PBS-treated mice. TPC: 23.4%; PBS: 0.13%. (c) The data are presented as a percentage of Breg cells: IL-101CD5highCD1dhigh expansion in splenocytes of TPC- and PBStreated mice (n 5 10). Values are mean 6 s.d., P < 0.0001. (d) Representative flow cytometry analyses of Breg cells: IL-101CD5highCD1dhigh (gated on IL-101) in splenocytes derived from the TPC- and PBS-treated mice. TPC: 91.3%; PBS: 0.01%.
the synthesis of IL-10 and IL-4. The effect was in comparison with control homogenates [39]. RA is associated with a T helper type 1 (Th1)/Th17 response and a general rise of proinflammatory cytokines such as IL-1, IL-6, IL-17 and TNF-a [3,40,41]. Furthermore, B cells are considered to play several roles in the pathogenesis of RA, such as antigen presentation supporting the activation of autoreactive T cells, autoantibody production and cytokine release [41,42]. Moreover, in the CIA model, collagen immunization induces chronic inflammatory arthritis due to CD41 T cell infiltration into the synovial membrane, as well as collagen-specific IgG autoantibody production by B cells [43–45]. Breg cells regulate inflammatory immune responses. Indeed, transferring CD1dhighCD51 Breg cells into CD192/2 mice reduced inflammation significantly by increasing the IL-10 levels [46,47]. Moreover, it was demonstrated that TIM-11 B cells induced tolerance by induction of Th2 responses [48]. 26
In addition, it was found that Breg cells regulate experimental autoimmune encephalomyelitis (EAE) clinical recovery by interacting with Treg cells, causing them to mobilize and migrate into the central nervous system (CNS), resulting in disease resolution via their increased production of IL-10 [49,50]. In this study, prophylactic treatment with TPC induced the expansion of CD41CD251FoxP31 Treg and CD191IL101CD5highCD1dhighTIM-11 Breg cells in isolated splenocytes, thus modulating the cytokine profile and decreasing the level of inflammation and synovial hyperplasia. Prominent Treg and Breg cells enabled the reduction in proinflammatory cytokines and increased the anti-inflammatory cytokines in stimulated isolated splenocytes taken from TPC-treated mice. In conclusion, in this study we have shown that treatment of CIA mice with TPC can prevent the development of arthritis.
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Acknowledgements This study was supported by the Moris Family Foundation, Los Angeles USA and TEVA Ltd, grant no. 147333, Israel. This work was performed as partial fulfilment of the requirements for Tomer Bashi PhD, Sackler Faculty of Medicine, Tel Aviv University, Israel.
Disclosure The authors have no disclosures to declare.
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