Dietary fish oil modulation of macrophage amyloid P component responses in mice

Arthritis-susceptible B10.RIII mice, maintained on either fish oil (FO) or corn oil (CO) diets (5% by weight), and amyloid-susceptible CBA/J mice fed chow diets were given 20 micrograms purified LPS by i.p. injection. Both strains of mice responded to LPS with a 20- to 30-fold increase in plasma amyloid P component (AP) levels. There were no differences in the response between males and females or between FO and CO treatment groups. The data demonstrated that cultured peritoneal macrophages (M phi) respond to LPS stimulation with increased secretion of AP. In contrast to plasma AP levels, the MO response to LPS stimulation, as measured by production of AP, was influenced by both gender and diet. Although M phi from both male and female mice on the CO diet and male mice on the FO diet responded similarly, those from female mice on the FO diet secreted only 25 to 35% as much AP as did the other three groups. There were no dietary effects on the LPS-induced serum amyloid A protein response nor was there detectable serum amyloid A protein produced by the M phi. These results demonstrate that unstimulated, resident peritoneal M phi secrete AP as a normal constituent and in increasing amounts in response to LPS stimulation.     

Gender differences in eicosanoid production from macrophages of arthritis-susceptible mice

Collagen-induced arthritis (CIA) in rodents is an experimental animal model that shares many clinical and pathologic findings with rheumatoid arthritis in man. Our previous findings suggested that the amelioration of CIA in mice by a fish oil diet was associated with macrophage accumulation and metabolism of eicosapentaenoic acid and a subsequently altered prostaglandin (PG) profile. In these experiments, we examined the role of gender and found that macrophages from female arthritis-susceptible B10.RIII or B10.G mice synthesized more PG and thromboxane than macrophages isolated from the males. Compared with males, female mice had higher circulating anti-type II collagen antibodies but were less likely to develop CIA. Females, especially those on a fish oil diet, developed a much less severe disease than the males. This supports our hypothesis that the type and/or amount of eicosanoid produced from the macrophage may alter the course of experimentally induced arthritis.     

Methylacetylenic putrescine (MAP), an inhibitor of polyamine biosynthesis, prevents the development of collagen-induced arthritis

The objective of the present investigation was to examine the effects of an irreversible inhibitor of ornithine decarboxylase (2R,5R)-6-heptyne-2,5,diamine (methylacetylenic putrescine, MAP) on experimentally induced arthritis in mice. MAP (0.5-0.05%) was administered in drinking water to DBA/1 mice immunized with native chick type II collagen (CII). The development of arthritis was inhibited only in those mice receiving 0.5% MAP; lower doses were ineffective. Putrescine and spermidine levels were decreased and spermine levels were increased in spleen and lymph node cells from drug-treated mice compared to control arthritic mice. Furthermore, when control mice were developing arthritis, serum anti-CII antibody levels were lower in the MAP-treated group. MAP inhibited antibody production early in the immune response to CII; there was an association between inhibition of antibody production and inhibition of the development of arthritis. When MAP was discontinued, the nonarthritic, drug-treated mice did not develop the disease. Late administration of MAP (beginning 19 days after CII immunization) did not affect the incidence or the severity of the arthritis. Cyclophosphamide treatment begun at the same time significantly inhibited the development of the disease. In vitro T cell responses to denatured type II collagen (dCII) in untreated and MAP-treated mice were examined 14 days after immunization with CII. This is a time of peak T cell responsiveness in untreated animals. MAP treatment had no effect on the T cell response to dCII. These results indicate that MAP can prevent the development of CII-induced arthritis, possibly by inhibiting the autoantibody response. Therefore, inhibitors of polyamine biosynthesis deserve further investigation as potential immunosuppressive agents.     

Aortic VCAM‐1: an early marker of vascular inflammation in collagen‐induced arthritis

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in rheumatoid arthritis (RA). There are limited experimental data on vascular involvement in arthritis models. To study the link between CVD and inflammation in RA, we developed a model of vascular dysfunction and articular inflammation by collagen‐induced arthritis (CIA) in C57Bl/6 (B6) mice. We studied the expression of vascular inflammatory markers in CIA with and without concomitant hyperlipidic diet (HD). Collagen‐induced arthritis was induced with intradermal injection of chicken type‐II collagen followed by a boost 21 days later. Mice with and without CIA were fed a standard diet or an HD for 12 weeks starting from the day of the boost. Arthritis severity was evaluated with a validated clinical score. Aortic mRNA levels of vascular cell adhesion molecule‐1 (VCAM‐1), inducible nitric oxide synthase (iNOS) and interleukin‐17 were analysed by quantitative RT‐PCR. Vascular cell adhesion molecule‐1 localization in the aortic sinus was determined by immunohistochemistry. Atherosclerotic plaque presence was assessed in aortas. Collagen‐induced arthritis was associated with increased expression of VCAM‐1, independent of diet. VCAM‐1 overexpression was detectable as early as 4 weeks after collagen immunization and persisted after 15 weeks. The HD induced atheroma plaque formation and aortic iNOS expression regardless of CIA. Concomitant CIA and HD had no additive effect on atheroma or VCAM‐1 or iNOS expression. CIA and an HD diet induced a distinct and independent expression of large‐vessel inflammation markers in B6 mice. This model may be relevant for the study of CVD in RA.     

Effects of high fat diets and supplemental tart cherry and fish oil on obesity and type 2 diabetes in male and female C57BL/6J and TALLYHO/Jng mice

Obesogenic and diabetogenic high fat (HF) diets can influence genetic factors in disease development with sexual dimorphic responses. We investigated potential protective effects of tart cherry (TC), fish oil (FO) and TC+FO supplementation in TALLYHO/Jng (TH) and C57BL/6J (B6) mice fed HF diets. Male and female TH and B6 mice were weaned onto five different diets; low fat (LF), HF, and HF supplemented with TC, FO, or TC+FO and maintained. For both males and females on LF, TH mice were heavier and fatter than B6, which was accentuated by HF in males, but not in females. TH males, but not others, developed severe glucose intolerance and hyperglycemia on HF, with reduced mRNA levels of Adipoq and Esr1 in adipose tissue. Considering energy balance, locomotor activity was lower in TH mice than B6 for both sexes without diet effects, except B6 females where HF decreased it. Compared to LF, HF decreased energy expenditure, RER, and food intake (in grams) for both sexes without strain differences. In all mice, but B6 males, HF increased plasma IL6 levels compared to LF. No preventive effects of TC, FO or TC+FO were noted for HF-induced obesity or energy imbalance, but FO alleviated glucose intolerance in TH males. Further, TC and FO decreased plasma IL6 levels, especially in females, without additive or synergistic effects of these two. Collectively, obesogenic and diabetogenic impacts of HF diets differed depending on the genetic predisposition. Moreover, sexually dimorphic effects of dietary supplementation were observed for glucose metabolism and inflammatory markers.     

Epicutaneous immunization with type II collagen inhibits both onset and progression of chronic collagen-induced arthritis

Epicutaneous immunization is a potential non-invasive technique for antigen-specific immune-modulation. Topical application of protein antigens to barrier-disrupted skin induces potent antigen-specific immunity with a strong Th2-bias. In this study, we investigate whether the autoimmune inflammatory response of chronic collagen-induced arthritis (CCIA) in DBA/1-TCR-beta Tg mice can be modified by epicutaneous immunization. We show that epicutaneous immunization with type II collagen (CII) inhibited development and progression of CCIA and, importantly, also ameliorated ongoing disease as indicated by clinical scores of disease severity, paw swelling and joints histology. Treated mice show reduced CII-driven T cell proliferation and IFN-gamma production, as well as significantly lower levels of CII-specific IgG2a serum antibodies. In contrast, CII-driven IL-4 production and IgE antibody levels were increased consistent with skewing of the CII response from Th1 to Th2 in treated mice. IL-4 production in treated mice was inversely correlated with disease severity. Moreover, T cells from treated mice inhibited proliferation and IFN-gamma production by T cells from CCIA mice, suggesting induction of regulatory T cells that actively inhibit effector responses in arthritic mice. The levels of CD4(+)CD25(+) T cells were however not increased following epicutaneous CII treatment. Together, these results suggest that epicutaneous immunization may be used as an immune-modulating procedure to actively re-programme pathogenic Th1 responses, and could have potential as a novel specific and simple treatment for chronic autoimmune inflammatory diseases such as rheumatoid arthritis.     

Effects of dietary lipid source on reproductive performance and tissue lipid levels of Nile tilapia Oreochromis niloticus (Linnaeus) broodstock

Nile tilapia were fed diets supplemented with one of the following lipid sources at 5% level: cod liver oil, corn oil, soybean oil, a coconut oil-based cooking oil or a combination of cod liver oil and corn oil (1 : 1). The control diet had no lipid supplement and tad fish meal as a sole protein source. A diet with soybean meal as a protein source was also tested. The number of females that spawned, spawning frequency, number of fry per spawning, and total fry production were increased at varying degrees by the supplemental lipid sources except for the cod liver oil. Fish fed the soybean oil diet tad the best overall reproductive performance over a 24-week period. Fish fed the cod liver oil diet had the highest weight gain but the poorest reproductive performance. The suplemental lipids significantly increased crude fat levels in the liver and ovaries. Both males and females Ld the cod liver oil diet had the highest levels of fat in the liver and muscle. The ratio of total n-6/n-3 fatty acid in the liver, ovaries and testes was influenced by the supplemental lipid sources. It was highest in fish fed either the soybean oil diet, the corn oil diet, or the soybean meal diet and lowest in fish fed the control diet or the cod liver oil diet.     

A mechanistic approach to understanding conjugated linoleic acid's role in inflammation using murine models of rheumatoid arthritis

A naturally occurring fatty acid, conjugated linoleic acid (CLA), reduces immune-induced TNF and inducible cyclooxygenase (COX-2) expression; key mediators of inflammation in rheumatoid arthritis (RA). On the basis of previous work, it was hypothesized that dietary CLA would act as an anti-inflammatory agent in select animal models of RA. In the collagen antibody-induced arthritis (CAIA) model, mice fed CLA (mixed isomers of c9, t11, and t10, c12-CLA) for 3 wk before anticollagen antibody injection had reduced lipopolysaccharide-induced plasma TNF levels and had arthritic scores that were 60% of mice fed corn oil (CO). In the collagen-induced arthritis (CIA) model, mice fed mixed isomers of CLA for 21 days before immunization had lower IgG(1) titers, earlier signs of joint inflammation, but similar arthritis scores compared with CO fed mice during the remaining 70-day post-injection period. Beginning on day 80 to 133, CLA-fed mice had arthritic scores 70% that of the CO-fed mice. In a second CIA experiment, CLA was fed only after the booster injection. Plasma IgG(1) levels were not reduced and arthritis onset was delayed 4 days in CLA-fed mice compared with the CO-fed mice. Peak arthritis score was similar between CLA and CO-fed mice from day 35 to 56. Because CLA reduced inflammation in the CAIA model, delayed onset of arthritis in the CIA model (CIA experiment 2) and reduced arthritis score after day 80 in the CIA model (CIA experiment 1), we concluded that dietary CLA exhibited anti-inflammatory activity that was dependent on antibody.     

Interaction of dietary saturated and polyunsaturated triglycerides in regulating the processes that determine plasma low density lipoprotein concentrations in the rat

These studies were undertaken to determine how polyunsaturated (n-3 and n-6) and saturated triglycerides interact to regulate rates of low density lipoprotein (LDL) production and rates of receptor-dependent and receptor-independent LDL transport. Animals were fed diets containing 20% (by wt) hydrogenated coconut oil or diets in which the coconut oil was progressively removed and replaced with safflower oil or fish oil concentrate. Plasma LDL concentrations fell when either of the polyunsaturated triglycerides was substituted for saturated triglycleride in the diet; however, the reduction in LDL concentrations was greater with fish oil than with safflower oil at all ratios of polyunsaturated to saturated triglyceride that were examined. The lower plasma LDL concentrations when coconut oil was replaced with fish oil could be attributed almost entirely to a much greater increase in hepatic LDL receptor activity when fish oil was used as the substitute than when safflower oil was used as the substitute. To examine the effect of polyunsaturated triglycerides when used to supplement a high saturated fat diet rather than to replace saturated fat in the diet, animals were fed a diet containing 15% coconut oil (by wt) with or without an additional supplement of 5% fish oil or safflower oil. The addition of 15% coconut oil to low fat control diet increased the rate of LDL production causing circulating LDL levels to rise by 40%. The further supplementation of this high saturated fat diet with fish oil concentrate markedly increased hepatic LDL receptor activity causing plasma LDL concentrations to return to control values whereas supplementation with safflower oil had little effect. Thus, at least in the rat, supplementation of a high saturated fat diet with a fish oil concentrate lowers plasma LDL concentrations as effectively as removing the saturated fat from the diet, although in the former case, both the production and the receptor-dependent uptake of LDL are greatly increased.     

Complement deficiency ameliorates collagen-induced arthritis in mice

Collagen-induced arthritis (CIA) is an experimental animal model of human rheumatoid arthritis being characterized by synovitis and progressive destruction of cartilage and bone. CIA is induced by injection of heterologous or homologous collagen type II in a susceptible murine strain. DBA/1J mice deficient of complement factors C3 (C3(-/-)) and factor B (FB(-/-)) were generated to elucidate the role of the complement system in CIA. When immunized with bovine collagen type II emulsified in CFA, control mice developed severe arthritis and high CII-specific IgG Ab titers. In contrast, the C3(-/-) and FB(-/-) were highly resistant to CIA and displayed decreased CII-specific IgG Ab response. A repeated bovine collagen type II exposure 3 wk after the initial immunization led to an increase in the Ab response in all mice and triggered arthritis also in the complement-deficient mice. Although the arthritic score of the C3(-/-) mice was low, the arthritis in FB(-/-) mice ranked intermediate with regard to C3(-/-) and control mice. We conclude that complement activation by both the classical and the alternative pathway plays a deleterious role in CIA.     

Collagen-induced arthritis

The collagen-induced arthritis (CIA) mouse model is the most commonly studied autoimmune model of rheumatoid arthritis. Autoimmune arthritis is induced in this model by immunization with an emulsion of complete Freund's adjuvant and type II collagen (CII). This protocol describes the steps necessary for acquisition, handling and preparation of CII, as well as selection of mouse strains, proper immunization technique and evaluation of the arthritis incidence and severity. Typically, the first signs of arthritis appear in this model 21-28 days after immunization, and identification of the arthritic limbs is not difficult. Using the protocol described, the investigator should be able to reproducibly induce a high incidence of CIA in various strains of genetically susceptible mice as well as learn how to critically evaluate the pathology of the disease. The total time for the preparation of reagents and the immunization of ten mice is about 1.5 h.     

Biomechanical bone strength and bone mass in young male and female rats fed a fish oil diet

The study objective was to determine if male and female rats fed a diet rich in fish oil had femurs and vertebrae that were stronger and more resistant to fracture than rats not fed omega-3 long chain polyunsaturated fatty acids. Weanling rats were randomized to a control or a fish oil diet for 5 weeks. Feeding fish oil to males had no effect on biomechanical strength properties of femurs and vertebrae as measured by three point bending and compression, respectively. In contrast, females fed fish oil had reduced length growth and a lower vertebral peak load. These effects may have been partly mediated by a lower food intake but were not associated with differences in serum IGF-I, estradiol or urinary calcium. The effect of consuming a fish oil diet into later adulthood should be investigated to determine if femur strength is also affected among females.     

The contrasting effects of ad libitum and restricted feeding of a diet very high in saturated fats on sex ratio and metabolic hormones in mice

Skewing of the sex ratio towards males occurs among pups born to mice fed a very high saturated fat (VHF) diet. In the present study, we tested whether the fat content of the VHF diet rather than the number of calories consumed is responsible for this effect. Eight-week-old NIH Swiss mice were placed on the VHF diet either ad libitum (VHF) or in a restricted manner (VHF-R). The VHF-R mice gained weight at a similar rate to controls fed a standard chow diet. Mice were bred at 15 wk and subsequently at 26 wk and 35 wk of age. Overall, the VHF, VHF-R, and control groups delivered 244, 242, and 274 pups, respectively, with male proportions of 0.60, 0.43, and 0.48, respectively. The pup sex ratios of the VHF group (favoring males) and VHF-R group (favoring females) each differed from 0.5 (P < 0.01). The sex ratios also differed (P < 0.0001) between the VHF and control groups, and between the VHF and VHF-R groups. Within the diet groups, maternal body weight had no effect on sex ratio. Serum leptin concentrations among the dams were similar in the VHF and VHF-R groups but higher than in the control group, while the IGF1 and corticosterone levels were comparable in all three groups. Therefore, the atypical sex ratios of offspring born to dams on the VHF diet seem to be influenced by the amount of fat consumed. Since males fed the VHF diet had neither more Y-sperm nor sired more sons than daughters, the dietary effects are manifested exclusively through the female.     

Intrauterine growth restriction in rats is associated with hypertension and renal dysfunction in adulthood

Epidemiological studies have produced evidence that unfavorable intrauterine environments during fetal life may lead to adverse outcomes in adulthood. We have previously shown that a low-sodium diet, given to pregnant rats over the last week of gestation, results in intrauterine growth restriction (IUGR). We hypothesize that pups born with IUGR are more susceptible to the development of hypertension in adulthood. IUGR fetuses and rats aged 1 wk were characterized for organ growth and renal morphogenesis. The adults (12 wk) were evaluated for weight, systolic blood pressure, activity of the renin-angiotensin-aldosterone system (RAAS), and renal function; hearts and kidneys underwent a histological examination. Brain and cardiac ventricle-to-body ratios were increased in IUGR fetuses compared with age-matched controls, whereas the kidney-to-body ratio was unchanged. Systolic blood pressure was elevated in both IUGR male and female adults. Plasma aldosterone levels were not correlated with increased plasma renin activity. Moreover, urinary sodium was decreased, whereas plasma urea was elevated in both males and females, and creatinine levels were augmented only in females, suggesting a glomerular filtration impairment in IUGR. In our model of IUGR induced by a low-sodium diet given to pregnant rats, high blood pressure, alteration of the RAAS, and renal dysfunction are observed in adult life. Differences observed between male and female adults suggest the importance of gender in outcomes in adulthood after IUGR.     

Decreased IL-12 production underlies the decreased ability of male lymph node cells to induce experimental autoimmune encephalomyelitis

Myelin basic protein (MBP)-specific T lymphocytes from male SJL mice were shown to be less encephalitogenic than MBP-specific T lymphocytes from females. Mechanisms underlying this gender difference in the induction phase of EAE were examined. Following immunization with MBP, draining lymph nodes contained fewer cells, and Ag-specific proliferative responses were decreased in males as compared with females. These gender differences in the proliferative response were not unique to MBP-specific responses since they were also observed after immunization with hen eggwhite lysozyme. Short-term MBP-specific T cell lines derived from females and males mapped with identical specificity, indicating no defect in the ability of male APCs to process Ag. Interestingly, IL-12 and IFN-gamma production was decreased following Ag-specific stimulation of draining lymph node cells (LNC) from males as compared with females, but IL-10 and IL-4 were no different. While male-derived LNCs were less encephalitogenic than female derived LNCs, cotransfer and coculture of male LNCs with female LNCs demonstrated that male LNCs were not immunosuppressive. Administration of IL-12 to LNCs from male mice enhanced encephalitogenicity. These data indicate that deficient endogenous IL-12 production within draining LNCs of male SJL mice is central to gender differences in the induction phase of experimental autoimmune encephalomyelitis.     

Leptin levels and body composition of mice selectively bred for high voluntary locomotor activity

Selective breeding produced four replicate lines of high-runner (HR) mice that run on wheels for approximately 2.7 times more revolutions per day than four unselected control lines. Previous studies found that HR mice of both sexes have lower body fat (isotope dilution at 15 wk of age) and that males (females not studied) have smaller retroperitoneal fat pads (17 wk). HR mice also exhibit elevated plasma corticosterone and insulin-stimulated glucose uptake by some hindlimb muscles but apparently do not differ in circulating insulin or glucose levels (males at 18 wk). Given their lower body fat and higher activity levels, we hypothesized that HR mice would have lower circulating leptin levels than controls. Female mice were given wheel access for 6 d at 7 wk of age, as part of the routine wheel testing for the selective breeding protocol, and then were killed after one additional week without wheels to reduce possible acute effects of activity on leptin. As hypothesized, serum leptin levels were significantly lower in HR mice. ANCOVA indicated that leptin was strongly positively correlated with both total body fat (measured by ether extraction) and body mass change from weaning, but HR mice still had significantly lower adjusted leptin levels (ANCOVA). Within HR lines but not within control lines, individual variation in leptin levels was negatively correlated with amount or speed of wheel running measured a week before being killed. Growth from weaning to euthanasia and body dry mass were lower in HR mice than in controls, but absolute dry masses of the ventricles, liver, gut, and uterus plus ovaries did not significantly differ, nor did percentage of the total dry mass as fat. HR mice offer a novel model for studying the causes and consequences of physiologically relevant variations in serum leptin.     

Stories about acyl chains

The effect of dietary polyunsaturated fatty acids and alpha-tocopherol supplementation on erythrocyte lipid peroxidation and immunocompetent cells in mice was studied comparatively using seven dietary oils (15% oil/diet, w/w) including fish oil rich in eicosapentaenoic acid (EPA, 20:5, n-3) and docosahexaenoic acid (DHA, 22:6, n-3). A 43% increase in spleen weight, about twice as many spleen cells and no change in the subpopulations of spleen cells, as well as a significant depression of mitogen-induced blastogenesis of both T and B cells in the spleen were observed in mice fed fish oil for 30 days in comparison with soybean oil diet-fed mice. In the fish oil diet-fed mice, membranous lipid hydroperoxide (hydroperoxides of phosphatidylcholine and phosphatidylethanolamine) accumulation as a marker of oxidative senescence in red blood cells (RBC) was 2.7-3.5 times higher than that in mice fed soybean oil, although there was no difference in the plasma phosphatidylcholine hydroperoxide concentration. In spite of the supplementation of alpha-tocopherol to up to 10 times the level in the basal diet, the degeneration of spleen cells and the stimulated oxidative senescence of RBC found by the fish oil feeding could not be prevented. The results suggest that oral intake of excess polyunsaturated fatty acids, i.e. EPA and DHA, in a fish oil diet can lead to acceleration of membrane lipid peroxidation resulting in RBC senescence linked to the lowering of immune response of spleen cells, and that supplementation of alpha-tocopherol as antioxidant does not always effectively prevent such oxidative degeneration as observed in spleen cells and RBC in vivo.     

Type II collagen-reactive T cell clones from mice with collagen-induced arthritis

Certain strains of mice develop a symmetrical polyarthritis after immunization with type II collagen. The incidence of arthritis after such immunization is variable. To study the arthritogenic potential of T cells reactive with type II collagen, we isolated draining lymph node cells from mice that had developed arthritis after immunization with bovine type II collagen. From these immune lymph node cells we were able to clone T cells reactive with type II collagen. Two separate sets of T cell clones were isolated. The first set reacted with either native bovine or native chick type II collagen, but did not react with type I collagen. The second set of T cell clones reacted with bovine type II collagen, but did not respond to either native chick type II collagen or type I collagen. These clones will be tested for their influence on the development of arthritis in vivo.     

HLA-DR2-associated Dw subtypes correlate with RFLP clusters: most DR2 IDDM patients belong to one of these clusters

The incidence of arthritis and the antibody response to mouse and to rat type 11 collagen after immunization with native rat type II collagen was studied in different mouse strains, including wild mouse-derived strains belonging to the H-2^p/H-2^q family. High serum levels of antibodies to mouse and rat type II collagen were seen only in H-2^q mice, whereas mice belonging to the p, w3, w5, and w17 haplotypes displayed low type II collagen-specific antibody responses. Mice from three different H-2^q-carrying strains (DBA/1, NFR/N, and B10.G) with different non-major histocompatibility complex genes were all susceptible to collagen arthritis, but they displayed a varying incidence of arthritis and varying clinical features. No arthritis was seen in non-H-2^q mice, except in the B10.CAS2 strain where a few mice developed arthritis despite very low serum levels of type II collagen-specific antibodies. We conclude that small differences in the A chain of class II transplantation antigens are of importance for the development of arthritis and for the stimulation of a high response after immunization with type 11 collagen.Abbreviations used in this paper ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - Ig immunoglobulin - MHC major histocompatibility complex