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. 2016 Feb 4;184(1):19–28. doi: 10.1111/cei.12745

Novel therapeutic compound tuftsin–phosphorylcholine attenuates collagen‐induced arthritis

T Bashi 1, O Shovman 1, M Fridkin 2, A Volkov 3, I Barshack 3, M Blank 1, Y Shoenfeld 1,✉
PMCID: PMC4778098  PMID: 26618631

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 µg/0.1 ml) or orally (250 µg/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)‐α, interleukin (IL)−17 and IL‐1β (P < 0.0001). TPC significantly expanded the CD4+CD25+ forkhead box protein 3 (FoxP3+) Treg cells and CD19+IL‐10+CD5highCD1dhighT cell immunoglobulin mucin‐1 (TIM‐1+) 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, 2, 3, 4.

Current therapeutic strategies of RA involve mainly methotrexate 5, as well as immune‐based, anti‐inflammatory therapies such as tumour necrosis factor (TNF)‐α, interleukin (IL)−1, CD‐20 targeting agents or Janus kinase 1,2,3 (JAK) inhibitors 6, 7, 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 patients and in animal models improved the clinical score in RA, multiple sclerosis, type I diabetes mellitus and inflammatory bowel disease 11, 12, 13, 14, 15, 16, 17, 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, 21, 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, 24, 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‐Lys‐Pro‐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 high‐performance 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. 696/11. Disease induction was carried out by injecting 100 μg 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 −6, 6 days prior to disease induction; PBS was given as control vehicle. TPC was given either orally using a feeding needle (250 µg/0.1 ml per mouse) once a week or subcutaneously (s.c.) (5 µg/0.1 ml per mouse) twice a week, n = 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 = normal, 1 = slight erythema, 2 = slight erythema plus swelling, 3 = moderate oedema and erythema 4 = 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 µg/ml phosphate‐buffered saline (PBS)] overnight 4°C and then blocked with 3% bovine serum albumin (BSA). Mouse serum (n = 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 anti‐mouse 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 = 10 mice per group), as well as from vehicle PBS‐treated mice (n = 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 × 106 cells/well) in 24‐well plates (Thermo plates; Nunc Fisher Scientific Inc., Waltham, MA, USA), coated with anti‐CD3 antibodies (2 µg/ml) in the presence of 5 µg/ml TPC or PBS for 72 h in RPMI‐1640 enriched medium at 37°C and 5% CO2. Culture supernatants were then collected. Proinflammatory (IL‐1β, IL‐17, TNF‐α) 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 [CD4+CD25+forkhead box protein 3 (FoxP3+)] cells by flow cytometry

Isolated splenocytes, depleted of red blood cells, were incubated with anti‐CD4+fluorescein isothiocyanate (FITC) anti‐CD25+ allophycocyanin (APC) anti‐FoxP3+phycoerythrin (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 CD4+ T cells.

Analysis of B10 regulatory CD19+IL‐10+T cell immunoglobulin mucin‐1 (TIM‐1+)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 anti‐CD19+FITC anti‐T cell Ig domain and mucin domain protein 1 (TIM‐1)+APC anti‐IL‐10+PE (eBioscience) and anti‐IL‐10+FITC anti‐CD1d+APC anti‐CD5+PE (eBioscience) and were analysed by flow cytometry (Becton Dickinson). The gating was on the CD19+ B cells or IL‐10+B10 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 ± 1.5, while PBS orally treated mice had a mean arthritis score of 12.6 ± 1.14 (P < 0.001). Moreover, TPC s.c.‐treated mice had a mean arthritis score of 1.4 ± 0.84, whereas PBS s.c.‐treated mice had a mean arthritis score of 11 ± 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 TPC‐treated mice exhibited milder symptoms.

Figure 1.

Figure 1

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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 = 10 per group), measured from day (−6) (6 days prior to disease induction) until day 31 mean ± standard deviation (s.d.). Values are mean ± 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.

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 oral‐ and 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 oral‐ and 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.

Figure 2.

Figure 2

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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 phosphate‐buffered 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: ×100.

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 anti‐collagen type II antibodies in the TPC oral‐treated mice was 0.685 ± 0.09, while the mean OD level of the anti‐collagen type II antibodies in PBS oral‐treated mice was 1.09 ± 0.17. Furthermore, the mean OD level of the anti‐collagen type II antibodies in TPC s.c.‐treated mice was 0.615 ± 0.08, whereas the mean OD level of the anti‐collagen type II antibodies in PBS s.c.‐treated mice was 1.03 ± 0.13 (P < 0.001).

Figure 3.

Figure 3

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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 = 10 per group). The data are presented as optical density (OD) at 405 nm. Values are mean ± standard deviation (s.d.), P < 0.0001.

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‐α and IL‐1 30, 31. We evaluated the secretion of proinflammatory and anti‐inflammatory cytokines by splenocytes derived from CIA mice treated with TPC or PBS in vitro. The following cytokine secretions were examined: proinflammatory TNF‐α, IL‐1β, 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).

Figure 4.

Figure 4

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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)‐α, interleukin (IL)−17, IL‐1β and the anti‐inflammatory cytokine IL‐10 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 = 10 per group. (a) In‐vitro analyses of the proinflammatory cytokine TNF‐α concentration; (b) in‐vitro analyses of the proinflammatory cytokine IL‐17 concentration; (c). in‐vitro analyses of the proinflammatory cytokine IL‐1β concentration; (d) in‐vitro analyses of the anti‐inflammatory cytokine IL‐10 concentration.

Mice treated orally with TPC had mean TNF‐α levels of 633 ± 91 (pg/ml), mean IL‐17 levels of 505 ± 26 (pg/ml) and mean IL‐1β levels of 183 ± 111 (pg/ml), while mice treated orally with PBS had mean TNF‐α levels of 1677 ± 137 (pg/ml), mean IL‐17 levels of 1585 ± 90 (pg/ml) and mean IL‐1β levels of 686 ± 194 (pg/ml). Moreover, TPC s.c.‐treated mice had mean TNF‐α levels of 621 ± 72 (pg/ml), mean IL‐17 levels of 497 ± 28 (pg/ml) and mean IL‐1β levels of 198 ± 133 (pg/ml). In comparison, PBS s.c.‐treated mice had mean TNF‐α levels of 1586 ± 65 (pg/ml), mean IL‐17 levels of 1585 ± 97 (pg/ml) and mean IL‐1β levels of 770 ± 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 ± 743 (pg/ml), whereas mice treated orally with PBS had mean IL‐10 levels of 384 ± 149 (pg/ml). Furthermore, TPC s.c.‐treated mice had mean IL‐10 levels of 2917 ± 810 (pg/ml). In comparison, PBS s.c.‐treated mice had mean IL‐10 levels of 302 ± 49 (pg/ml).

The effect of TPC on expansion of CD4+CD25+FoxP3+ regulatory T cells (Treg) cells in isolated mice splenocytes

The frequency of CD4+CD25+FoxP3+ 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 ± 4.89%), whereas the PBS‐treated group of Treg cells measured in the isolated splenocytes had a mean percentage of 2.37 ± 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 CD4+CD25+FoxP3+ levels in splenocytes derived from TPC‐ and PBS‐treated mice gating on CD4+ T cells are presented in Fig. 5b.

Figure 5.

Figure 5

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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: CD4+CD25+forkhead box protein 3 (FoxP3+) expansion in isolated splenocytes of TPC‐ and PBS‐treated mice (n = 10). Values are mean ± standard deviation (s.d.), P < 0.0001. (b). Representative flow cytometry analyses of Treg cells: CD4+CD25+FoxP3+ (gated on CD4+) in splenocytes derived from the TPC‐ and PBS‐treated mice. TPC: 25.9%; PBS: 2.37%.

The effect of TPC on expansion of CD19+IL‐10+TIM‐1+CD25+CD1d+ regulatory B cells (Breg) cells in isolated mice splenocytes

The mean percentage of Breg cells in isolated splenocytes following treatment with TPC or PBS was measured within two main subsets, CD19+IL‐10+TIM‐1+ 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 CD19+IL‐10+TIM‐1+ Breg cells of TPC‐treated mice was significantly higher (21.63 ± 3.2%), while the control group had a mean percentage of 0.05 ± 0.06% (P < 0.0001). Representative data of CD19+IL‐10+TIM‐1+ levels in splenocytes derived from TPC‐ and PBS‐treated mice gating on CD19+ B cells are presented in Fig. 6b. Furthermore, the mean percentage of IL‐10highCD25highCD1dhigh Breg cells in isolated splencocytes of TPC‐ and PBS‐treated mice are displayed in Fig. 6c. TPC‐treated mice exhibited higher Breg cell expansion (89.91 ± 1.21%), while control mice exhibited a lack of IL‐10highCD25highCD1dhigh Breg cell phenotype, with a mean percentage of 0.005 ± 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.

Figure 6.

Figure 6

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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: CD19+interleukin (IL)−10+ T cell immunoglobulin mucin‐1 (TIM‐1+) expansion in splenocytes of TPC‐ and PBS‐ treated mice (n = 10). Values are mean ± standard deviation (s.d.), P < 0.0001. (b) Representative flow cytometry analyses of Breg cells: CD19+IL‐10+TIM‐1+ (gated on CD19+) 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‐10+CD5highCD1dhigh expansion in splenocytes of TPC‐ and PBS‐treated mice (n = 10). Values are mean  ±  s.d., P < 0.0001. (d) Representative flow cytometry analyses of Breg cells: IL‐10+CD5highCD1dhigh (gated on IL‐10+) in splenocytes derived from the TPC‐ and PBS‐treated mice. TPC: 91.3%; PBS: 0.01%.

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 prevented the destruction of joints, whereas joints of PBS‐treated mice were highly inflamed. Similarly, TPC increased the secretion of anti‐inflammatory cytokine IL‐10 and decreased concentrations of key proinflammatory cytokines TNF‐α, IL‐17 and IL‐1β, resulting in a low inflammatory state. Furthermore, TPC enhanced the expansion of CD4+CD25+FoxP3+ Treg as well as CD19+IL‐10+CD5highCD1dhighTIM‐1+ 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 NZB×W/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‐γ and IL‐17 cytokine production and increase of the anti‐inflammatory IL‐10 and TGF‐β cytokine expression, as well as expansion of the CD4+CD25+FoxP3+ 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‐1β, TNF‐α 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)−4 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 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‐α 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 CD4+ T cell infiltration into the synovial membrane, as well as collagen‐specific IgG autoantibody production by B cells 43, 44, 45. Breg cells regulate inflammatory immune responses. Indeed, transferring CD1dhighCD5+ Breg cells into CD19−/− mice reduced inflammation significantly by increasing the IL‐10 levels 46, 47. Moreover, it was demonstrated that TIM‐1+ B cells induced tolerance by induction of Th2 responses 48. 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 CD4+CD25+FoxP3+ Treg and CD19+IL‐10+CD5highCD1dhighTIM‐1+ 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.

Disclosure

The authors have no disclosures to declare.

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.

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