Folia Microbiol. 54 (4), 359–363 (2009)
http://www.biomed.cas.cz/mbu/folia/
Treatment of Adjuvant-Induced Arthritis with the Combination of Methotrexate and Probiotic Bacteria Escherichia coli O83 (Colinfant®) J. ROVENSKÝa *, M. STANČÍKOVÁa, K. ŠVÍKa, J. UTĚŠENÝb, K. BAUEROVÁc, J. JURČOVIČOVÁd aNational Institute of Rheumatic Diseases, 921 01 Piešťany, Slovakia b2nd Pediatric Clinic, Faculty Hospital, Brno, Czech Republic cInstitute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovakia dInstitute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia
Received 11 December 2008 Revised version 10 April 2009
ABSTRACT. A certain relationship was observed between the gastrointestinal system, arthritis and immune system. Patients with rheumatoid arthritis have an altered microflora composition and disturbed intestinal defensive barrier. Effect of probiotic bacteria (Colinfant®; COL) with known favorable effect on intestinal microflora was determined on the methotrexate (MTX) treatment of adjuvant arthritis. Rats with adjuvant arthritis were administered methotrexate 0.5 mg/kg body mass 2-times weekly per os, COL 1 mL/kg body mass every second day per os, and a combination of MTX+COL for a period of 28 d from the immunization. Levels of serum albumin, body mass, changes in hind paw swelling, and arthrogram score were estimated in rats as variables of inflammation and destructive arthritis-associated changes. Treatment with MTX, as well as with the combination treatment with MTX+COL significantly inhibited both inflammation and destructive arthritis-associated changes. The combination treatment inhibited both the hind paw swelling and arthrogram score more remarkably than MTX alone; on the other hand, the difference between combination treatment and MTX alone was not significant. Treatment with COL alone had no effect on adjuvant arthritis in rats. Colinfant® can increase the preventive effect of MTX treatment in rat adjuvant arthritis by improving its antiarthritic effects. Abbreviations AA COL
adjuvant arthritis Colinfant® (Escheria coli O83)
MTX RA
methotrexate rheumatoid arthritis
The gastrointestinal tract functions as a barrier against antigens from microorganisms and food. The generation of immunophysiological regulation in the gut depends on the establishment of indigenous microflora. This had led to the introduction of novel therapeutic interventions based on oral application of probiotics. Probiotics are living microorganisms that, if administered in sufficient quantities, beneficially affect the host by improving its intestinal microbial balance (Reid et al. 2003). They show favorable effects on development and stability of the microflora, inhibit colonization by pathogens, influence the mucosal barrier by their trophic effect on intestinal epithelium and stimulate both specific and nonspecific components of the immune system (Isolauri et al. 2001; Tlaskalová-Hogenová et al. 2004; Hudcovic et al. 2007; Koninkx and Malago 2008). Colinfant® – Escherichia coli O83 binds well to intestinal mucosa and survives therefore for a long time in the gut (Lodinová-Žádníková and Sonnenborn 1997). In pre-term babies, it decreases the presence of pathogens, the number of infections, the need for antibiotics and decreases the incidence of allergies and repeated infections in later life when administered orally after birth (Lodinová-Žádníková et al. 2003; Kocourková et al. 2007). Long-lasting unfavorable composition of intestinal microflora could associate with the occurrence of diseases that may have no relation to the bacteria living in intestines; it may also increase the susceptibility to infectious diseases or worsen the absorption of some drugs. Certain relationship was observed between gastrointestinal systems, arthritis and immune system (Sartor 1997; Cebra 1999). Patients with newly diagnosed RA have their intestinal microflora composition altered (especially in the case of Gram-positive anaerobic bacteria) in comparison with healthy controls (Eerola et al. 1994). It has also been suggested that *Address for correspondence: National Institute of Rheumatic Diseases, Nábrežie I. Krasku 4, 921 12 Piešťany, Slovak Republic; fax +421 337 721 192, e-mail
[email protected] .
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in arthritis the intestinal defensive barrier is disturbed (Malin et al. 1997). Short-term therapy with Lactobacillus GG showed a tendency to increase the IgA secretion and thus improve the mucosal barrier mechanism (Hatakka et al. 2003). The positive alterations in intestinal flora in RA can be induced also by uncooked, lactobacilli-rich, vegan food. Using the vegan diet, a decrease of RA activity (Peltonen at al. 1997) and reduction of the subjective symptoms were observed (McDougall et al. 2002). In our previous study, we showed that the probiotic bacteria Enterococcus faecium have a beneficial effect on MTX treatment in adjuvant arthritis (Rovenský et al. 2002, 2005). MTX (an inhibitor of dihydrofolate reductase) is the most common and one of the first prescribed disease-modifying antirheumatic drug to treat RA in the world. The animal model of AA is a widely used model for testing drugs with anti-inflammatory and antiarthritic effects. The aim of our study was to evaluate the effect of COL on clinical symptoms and certain laboratory markers of AA during basal treatment with MTX. MATERIALS AND METHODS Materials. MTX injection solution 10 mg/mL in sterile saline (Medac Company, Germany) was used. Escherichia coli strain O83 (Colinfant®) was purchased from Dyntec (Czech Republic; 1 mL of Colinfant® contains 8 × 108 bacteria); Mycobacterium butyricum was purchased from Difco Laboratories (USA), and incomplete Freund’s adjuvant from Sigma-Aldrich Chemie (Germany). Animals. Male Lewis rats (160–180 g) obtained from Charles River Wiga (Germany) were maintained during the experiment in standard animal facilities that comply with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. The animals were fed pelleted food and had free access to food and water. The experimental protocol and all procedures were approved by the State Veterinary Committee for Control of Animal Experimentation and by Animal Ethics Committee of the National Institute of Rheumatic Diseases. Induction of arthritis. The rats were injected with 0.1 mL suspension of heat-killed M. butyricum (12 mg/mL) in incomplete Freund’s adjuvant intradermally into the tail base (Wooley 1991). Treatment. MTX and COL were administered in corresponding doses from day 0 (at immunization) to day 28. MTX in sterile saline in dose 0.5 mg/kg body mass was applied per os twice a week. COL was administered orally every second day in a dose of 1 mL/kg per os. The untreated groups received vehiculum (sterile saline) in the same manner daily for 28 d. The animals were divided into the following 5 groups of eight: group 1 – non-arthritic untreated controls; group 2 – untreated rats with AA; group 3 – AA rats treated with MTX; group 4 – AA rats treated with COL; group 5 – AA rats treated with combination of MTX+COL. The following parameters of the treatment effect were determined: Body mass of rats was measured every week. Hind paw swelling: The volume of the hind paw swelling was determined plethysmographically on days 14, 21 and 28. Arthrogram score: The severity of arthritis was quantified by scoring each paw from 0 to 5, based on increasing levels of swelling and periarticular erythema. The sum of the scores for the limbs was calculated as the arthritic index with a maximum possible score of 20 per rat. Arthrogram score was evaluated on days 14, 21 and 28. Serum albumin levels were measured on days 14, 21 and 28 in the rat serum by spectrophotometric method using SYS 1 kit (BM/Hitachi, Boehringer Mannheim, Germany) on a Hitachi 911 automatic biochemical analyzer. Statistical analysis. One-way analysis of variance (ANOVA) was used for statistical analysis; p < 0.05 was taken as the significance limit. RESULTS Body mass. In the first 7 d the increment in body mass was similar in all groups of rats (Table I). However, on day 14 the body mass of arthritic control rats (group 2) and rats treated with COL alone (4) was significantly lower compared with healthy controls (1) and with arthritic rats treated with MTX (3) or with
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combination of MTX+COL (5). The body mass in rats treated with combination MTX+COL was similar to that treated with MTX alone. Table I. Effect of MTX, COL, and their combination on body mass of rats (in g) on days 1–28a Group of rats Healthy controls (group 1) Arthritic untreated controls (2) Arthritic rats treated with MTX (3) Arthritic rats treated with COL (4) Arthritic rats treated with MTX+COL (5)
1
7
167 ± 6 170 ± 8 173 ± 6 170 ± 4 171 ± 5
219 ± 13 217 ± 12 227 ± 7 216 ± 8 228 ± 9
14
21
224 ± 13*** 186 ± 11 228 ± 17*** 190 ± 11 241 ± 9***
239 ± 14*** 179 ± 18 236 ± 14*** 182 ± 12 246 ± 14***
28 250 ± 15*** 199 ± 13 247 ± 15*** 208 ± 14 249 ± 18***
aMean values ± SD; groups of 8 rats; significantly different from arthritic control rats at ***p < 0.001.
Hind paw swelling, arthrogram score. The clinical signs of arthritis hind paw swelling and arthrogram score reflect both arthritic and inflammatory changes occurring in rats with AA. The degree of hind paw swelling in untreated arthritic rats was significantly higher on days 14, 21 and 28, as supported by the mean value for two hind paws (Table II); statistically significant reduction of hind paw swelling and arthrogram score were observed after immunization in the arthritic rats treated with MTX. The combination treatment MTX+COL reduced these parameters more significantly than MTX treatment alone. The treatment with COL alone had no effect on hind paw swelling and arthrogram score in AA rats. Table II. The effect of MTX, COL, and their combination on hind paw swelling (mL), on the arthrogram score, and on serum albumin concentration (g/L) in AA ratsa Group of rats
Day 14
Day 21
Day 28
Hind paw swelling Healthy controls (1) Arthritic untreated controls (2) Arthritic rats treated with MTX (3) Arthritic rats treated with COL (4) Arthritic rats treated with MTX+COL (5)
1.36 ± 0.04*** 2.25 ± 0.20 1.85 ± 0.23** 2.20 ± 0.28 1.72 ± 0.22***
1.40 ± 0.02*** 2.51 ± 0.18 2.16 ± 0.23 ** 2.55 ± 0.15 2.03 ± 0.20***
1.41 ± 0.05*** 2.34 ± 0.15 2.04 ± 0.14** 2.28 ± 0.16 1.95 ± 0.13***
17.22 ± 1.99 13.33 ± 2.16** 16.10 ± 2.27 11.87 ± 2.10***
14.89 ± 1.96 11.00 ± 1.55** 14.63 ± 2.62 10.02 ± 1.17***
39.92 ± 2.45*** 30.91 ± 2.10 35.33 ± 1.79*** 31.93 ± 0.99 36.24 ± 3.56***
42.15 ± 2.14*** 34.28 ± 1.22 38.03 ± 2.19*** 33.75 ± 1.91 38.75 ± 3.04***
Arthrogram scoresb Arthritic untreated controls (2) Arthritic rats treated with MTX (3) Arthritic rats treated with COL (4) Arthritic rats treated with MTX+COL (5)
13.44 ± 1.81 8.50 ± 1.52** 12.75 ± 2.76 6.88 ± 1.73*** Serum albumin
Healthy controls (1) Arthritic untreated controls (2) Arthritic rats treated with MTX (3) Arthritic rats treated with COL (4) Arthritic rats treated with MTX+COL (5)
42.00 ± 2.70*** 27.68 ± 1.08 35.01 ± 3.36*** 28.90 ± 1.04 37.51 ± 3.02***
aMean values ± SD for groups of 8 rats; significantly different from arthritic control rats at **p < 0.01, ***p < 0.001. bThe severity of arthritis was quantified by scoring each paw from 0 to 5, based on increasing levels of swelling and periarticular ery-
thema; the sum of the scores for the limbs was calculated as the arthritic index with a maximum possible score of 20 per rat.
Serum albumin levels. Lower levels of serum albumin (as a biochemical variable of inflammation) corresponded to higher levels of inflammatory activity. Both MTX alone and the combination treatment with MTX+COL induced similar (significant) increase in serum albumin levels when compared with the untreated AA animals, suggesting lower inflammatory activity (Table II).
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DISCUSSION The results of our investigation confirmed the previously reported effect of MTX treatment in rats with AA (Welles et al. 1985). MTX at a dose of 1 mg/kg body mass per week suppressed but did not prevent arthritis development. The reduction of hind paw swelling and arthrogram score were more pronounced in rats treated with combination of MTX+COL than in those treated with MTX alone (p < 0.001 vs. p < 0.01). Serum albumin acts as a negative acute-phase protein also in rat arthritis and its decrease reflects the changes in the synthesis of this protein in the liver secondary to the activation of hepatic cells by inflammatory cytokines, mainly IL-1 (Conolly et al. 1988a,b). We found that the MTX treatment in a dose 1 mg/kg body mass per week as well as the combination treatment MTX+COL significantly reduced the decrease of serum albumin levels observed in untreated rats with AA. The COL treatment had no effect on this inflammatory marker. So et al. (2008) reported that Lactobacillus casei suppresses experimental arthritis by down-regulating effector functions of CD4+ T cells. Oral administration of L. casei also suppressed collagen-induced arthritis, reduced hind paw swelling, lymphocyte infiltration and destruction of cartilage tissue and pro-inflammatory cytokines. In our experiments, administration of E. coli O83 alone had no significant beneficial effect on evaluated parameters (body mass, hind paw swelling, arthrogram score and serum albumin). On the other hand, it potentiates the beneficial effect of MTX on clinical markers of AA similarly as E. faecium (Rovenský et al. 2002) which can be explained by the altered intestinal absorption of MTX after colonization of gut by this probiotic agent. During the experiment no diarrhea was observed in rats. Gastrointestinal side effects often occur in rheumatic patients during oral application of MTX as nausea, abdominal pain, stomatitis and diarrhea (Hoekstra et al. 2003). Enterocolitis is the major toxicity of methotrexate-based cancer chemotherapy, which limits its clinical applications (Papaconstantinou et al. 2001). Mao et al. (1996) showed that administration of lactobacilli, especially L. plantarum, is helpful in reducing the severity of the methotrexate-induced enterocolitis in rats. Oral administration of probiotic bacteria E. coli O83 can protect mice against (dextran and disodium sulfate)-induced colitis and may significantly decrease the average symptom score in BALB/c mice (Kokešová et al. 2006). As COL not only increases the efficacy of MTX treatment but may also favorably influence the intestinal flora and protect against related infectious diseases we can see rationale for the use of this probiotic agent in the MTX treatment of experimental arthritis. The focus of this investigation is the preventive treatment of arthritic rats; similar, more throughput studies contribute to explaining the role of COL in MTX therapy regimen and help in the possible therapeutic use of this probiotic agent in the treatment of arthritis in humans. This work was supported by the Slovak Grant Agency APVV (grant no. APVV-21-055205). REFERENCES CEBRA J.J.: Influences of microbiota on intestinal immune system development. Am.J.Clin.Nutr. 69, 1046–1051(1999). CONNOLLY K.M., STECHER V.J., DANIS E., PRUDEN D.J., LABRIE T.: Alteration of interleukin-1 production and the acute phase response following medication of adjuvant arthritic rats with cyclosporin-A or methotrexate. Internat.J.Immunopharmacol. 10, 717–728 (1988a). CONNOLLY K.M., STECHER V.J., LABRIE T., FLUNO C.: Modulation of the acute phase response and in vitro measurement of interleukin-1 activity following administration of dexamethasone to adjuvant-arthritic rats. 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