Skip to main content
NCBI home page
International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2020 May 27;101(1-2):55–64. doi: 10.1111/iep.12348

Anti‐inflammatory effect of omega unsaturated fatty acids and dialysable leucocyte extracts on collagen‐induced arthritis in DBA/1 mice

Pamela I Pérez‐Martínez 1, Oscar Rojas‐Espinosa 1,✉, Víctor G Hernández‐Chávez 2, Patricia Arce‐Paredes 1, Sergio Estrada‐Parra 1
PMCID: PMC7306903  PMID: 32459025

Summary

Rheumatoid arthritis is a disabling autoimmune disease with a high global prevalence. Treatment with disease‐modifying anti‐arthritic drugs (DIMARDs) has been routinely used with beneficial effects but with adverse long‐term consequences; novel targeted biologics and small‐molecule inhibitors are promising options. In this study, we investigated whether purified omega unsaturated fatty acids (ω‐UFAs) and dialysable leukocyte extracts (DLEs) prevented the development of arthritis in a model of collagen‐induced arthritis (CIA) in mice. We also investigated whether the transcription factor NF‐κB and the NLRP3 inflammasome were involved in the process and whether their activity was modulated by treatment. The development of arthritis was evaluated for 84 days following treatment with nothing, dexamethasone, DLEs, docosahexaenoic acid, arachidonic acid, and oleic acid. Progression of CIA was monitored by evaluating clinical manifestations, inflammatory changes, and histological alterations in the pads’ articular tissues. Both DLEs and ω‐UFAs led to an almost complete inhibition of the inflammatory histopathology of CIA and this was concomitant with the inhibition of NF‐kB and the inhibition of the activation of NLRP3. These data suggest that ω‐UFAs and DLEs might have NF‐κB as a common target and that they might be used as ancillary medicines in the treatment of arthritis.

Keywords: arthritis, CIA, collagen, DLEs, mouse, UFAs

1. INTRODUCTION

Rheumatoid arthritis (RA) is a high‐prevalence articular disease with a wide global distribution. 1 It is regarded as a chronic degenerative autoimmune disease that affecting mainly articular joints and, in some cases, other organs. 2 , 3 The causes of the disease are not completely understood but several external and internal factors have been implicated. Active RA joints show many pathological changes such as dense perivascular cell infiltrate, synovial cell hyperplasia and hypertrophy, increased vascularity, erosion, osteoporosis, pannus that progresses to fibrous and bone ankylosis. 4

Inflammatory diseases have been linked to inflammasome activation, and the activation of nucleotide‐binding domain and leucine‐rich repeat pyrin 3 NLRP3 domain (NLRP3) is reported to be associated with disease activity in RA and osteoarthritis 5 , 6 , 7 and also in rodent models of arthritis, 8 , 9 thus suggesting that the inflammasome may be a therapeutic target in arthritis. 10

The conventional treatment of RA makes use of medicines that range from non‐steroidal (including small molecules and antibodies) and steroidal anti‐inflammatory drugs, to strong disease‐modifying drugs (DMARDs), none of which has definitively curative effects, but several of which have undesirable long‐term effects. 11 , 12

Despite the many efforts to find a curative treatment for RA, this goal has not been reached, and thus scientists have continued to focus their attention on possible new treatments.

Hundreds of reports on the use of alternative medicines have been published, most of them showing beneficial effects, but none of them has been proposed as routine treatment for RA. 13 One of these medicines that is regaining wider acceptance is a transfer factor (TF) that is contained in dialysable leucocyte extracts (DLEs).

Dialysable leucocyte extracts have been proposed as immunomodulatory in several infectious and non‐infectious diseases, but their methodical use in arthritis has seldom been reported, and the results have been variable. 14 , 15 , 16 Another approach to prevent the development of arthritis is based on the accepted anti‐inflammatory effect of omega unsaturated fatty acids (ω‐UFAs), which are abundant in certain foods such as fish oil, olive oil and nuts, and whose effects are opposite to the pro‐inflammatory effects of the saturated fats that are found in red meat and poultry. 17 , 18 , 19 , 20

Collagen‐induced arthritis (CIA) in DBA/1 mice has been regarded as closely resembling RA, 21 , 22 and this model has been widely used for the testing of known and new anti‐inflammatory drugs.

The rational use of DLE and purified omega UFA in the CIA model will validate the potential utility of these remedies for the treatment of RA.

In this study, we explored the potential anti‐inflammatory activity of docosahexaenoic acid (DHA/ω‐3), arachidonic acid (AA/ω‐6), oleic acid (OA/ω‐9) and a DLE in the mouse CIA model; additionally, we explored what effect these treatments may have on the activation of the inflammasome components NLRP3 and apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), and on NF‐κB.

2. MATERIALS AND METHODS

2.1. Chemicals

Unless otherwise specified, all chemicals were purchased from Sigma‐Aldrich.

2.2. Ethical approval

This project was reviewed and approved by the Committee of Ethical Procedures in the Managing of Animals for Experimentation of the National School of Biological Sciences of the National Polytechnic Institute (CEI‐ENCB 16/01/2014; NOM‐052‐ZOO‐1999)

2.3. The collagen induced arthritis model

Adult (20‐25 g), female DBA‐1 mice (Harlan) were subcutaneously inoculated with 50 µl of an adjuvant emulsion (6 mg of chicken collagen type II (C‐II) admixed with 1.0 mL of complete Freund's adjuvant (SC‐24018, Santa Cruz Biotechnology, Inc), containing 4.0 mg of killed Mycobacterium tuberculosis) on two sides of the tail base. Two weeks later, the mice received a similar, intraperitoneal injection of C‐II in incomplete Freund's adjuvant. 23 Non‐inoculated DBA‐1 mice were included as a control group.

2.4. Collagen type II

Collagen was isolated from the chicken sternum following a protocol by Inglis. 24 The purity of the C‐II was assessed with polyacrylamide gel electrophoresis under reducing conditions; a single protein band of 180.42 kDa was observed.

2.5. Dialysable leucocyte extracts

Dialyzable leukocyte extracts were prepared from the spleens of healthy BALB/c mice. Briefly, the spleens were excised and disaggregated to release splenocytes. PBSG (0.01 M sodium‐potassium phosphate, 0.15 M sodium chloride, 0.1% glucose, pH 7.4) was used as the suspension and washing solution. Cells were counted and adjusted to 107 cells per mL, and the cell suspension was subjected to 10 cycles of freezing (dry ice) and thawing (37°C). Lysates were then dialysed in physiological saline solution, maintaining a ratio of 106 cells (1 unit) per mL of isotonic saline solution for 48 hours. The dialysate was then collected, and the amount of protein was calculated (NanoDrop, Thermo Scientific) and adjusted to 100 ± 10 µg per mL per unit.

2.6. Omega UFAs

Docosahexaenoic acid (ω‐3, DHA), arachidonic acid (ω‐6, AA) and oleic acid (ω‐9, OA) were purchased with over 90% purity. For treatment, UFAs were dissolved in light mineral oil at a concentration of 30 mg/mL.

2.7. Treatment

Seven groups (4 mice each) of DBA‐1 mice were treated as shown in Table 1.

TABLE 1.

Mouse groups and treatment

Group (n = 4) Daily oral treatment per mouse Starting treatment (days after immunization)
1 HLT (untreated) —
2 C‐II (0.1 mL mineral oil) a Day 1
3 C‐II (DEX 1.0 mg/kg) b Day 1
4 C‐II (ω‐3 DHA 3 mg) b Day 1
5 C‐II (ω‐6 ARA 3 mg) b Day 1
6 C‐II (ω‐9 OA 3 mg) b Day 1
7 C‐II (DLE 0.1U) c Day 1
Open in a new tab

Abbreviation: HLT, healthy group (untreated).

a

C‐II group, mice inoculated with collagen type II.

b

C‐II groups treated with DEX, DHA, ARA or OA.

c

C‐II group treated with 0.1U of DLE. (1U DLE = the amount extracted from 1 × 106 leucocytes or ≈100 peptide µg/mL).

Treatments, in 0.1 mL volumes, were administered daily through a metal intragastric cannula and were started one day after the primary immunization; control mice received plain mineral oil. Although the administration of pyrogen‐free saline solution (PSS) by the intragastric route is the adequate control for DLEs, it was not performed since PSS administered by the intragastric route is unlikely to exert an effect at a distant site (ie the joints). The dosages chosen were based on previous studies where significant anti‐inflammatory effects were observed. 23 , 25 Following the initiation of treatment, the arthritis development was evaluated with a clinical score and by calculating an arthritis index as proposed by Sharma et al. 26 Clinical score was assessed by evaluating the inflammatory changes according to the scale: one inflamed finger/toe = 1 point; one knuckle = 1 point; one wrist = 5 points; and one ankle = 5 points (points per mouse = 60). The arthritis index (AI) was calculated from the pad thickness with the formula: AI = [(limb thickness on day X − limb thickness on day 0)/limb thickness on day 0)] × 100.

2.8. Histopathological changes

At the end of the study (84 days postimmunization), the mice were deeply anaesthetized (Ketalar, PISA Farm) and sacrificed.

Hands and feet were amputated, fixed for three days in 4% neutral formalin and decalcified in EDTA‐PBS for three weeks when the bones were soft enough for paraffin embedding and sectioning. 27 Five‐micron‐thick tissue sections were prepared with a Leica microtome (RM2125RT, Leica Biosystems Nussloch GmbH) and stained with haematoxylin‐eosin for general histology and with Masson's stain for the analysis of bone and cartilage.

2.9. Inflammasome activation

NLRP3 and NF‐κB activation/inhibition were assessed by immunofluorescence. Tissue sections were treated with HistoReveal (ab 103720, Abcam) for 10 minutes, blocked with 3% bovine albumin in PBS for 30 minutes and stained with anti‐NLRP3 (NBP2‐03948, Novus Biologicals), anti‐ASC (NBP1‐78977, Novus Biologicals) or anti‐NF‐κB (MA5‐16160 Pierce) antibodies for 1 hour at 25°C. After washing thrice with PBS, a secondary TRITC‐labelled monoclonal antibody (65‐6120 ZYMED) was added, and the mixture was incubated for 1 hour. After a final wash with PBS, the tissue sections were treated with the fluorescent nuclear stain Hoechst (H1399, Invitrogen), and the slides, protected with VECTASHIELD (H‐1000‐10, Vector), were subjected to confocal observation and imaging.

2.10. Statistical analysis

Two‐way ANOVA with multiple Tukey's tests was used for the analysis of the data.

3. RESULTS

3.1. Inflammation

Mice with CIA showed clear evidence of inflammation that reached its maximum by day 60 after immunization and remained until the end of the study (Figure 1A,B).

FIGURE 1.

FIGURE 1

Open in a new tab

Collagen‐induced arthritis in mice: clinical score and inflammation index. The figure illustrates the appearance of a normal rear footpad in a healthy DBA‐1 mouse (Panel A) and the inflamed condition of a rear pad in a mouse immunized with collagen type II for 60 d (Panel B). The clinical scores in healthy (HLT) mice, CIA mice and CIA mice treated for 84 d with dexamethasone (DEX), dialysable leucocyte extract (DLE), docosahexaenoic acid (DHA, ω‐3), arachidonic acid (AA, ω‐6) or oleic acid (OA, ω‐9) are depicted in Panel C; at the end of the study, a significant difference was observed between the untreated CIA mice and the mice of the HLT, DEX, DLE, DHA, AA and OA groups (*P < .0001). No significant difference was observed among the treatment groups (**P > .5). The inflammation index for the same groups is depicted in Panel D. Again, a significant difference was observed between the untreated CIA mice and the CIA mice treated with DEX, DLE, DHA, AA or OA (P < .0001), and no significant difference was observed among the treatment groups (**P > .5)

3.2. Inflammatory changes

3.2.1. Clinical score

All treatments were successful in preventing inflammatory changes after collagen administration, and the anti‐inflammatory effect was observed very soon after treatment. At the end of the experiment, on day 84, the difference in the clinical score between the CIA mice and mice treated with DEX, DLE (0.1U) or ω‐UFAs was statistically significant at the level of P < .0001. Although there were some variations, at the end of the experiment no significant differences were observed among the DEX, DLE, ω‐UFAs and the HLT groups (Figure 1C).

3.2.2. Inflammation index

All treatments were effective in controlling the disease. Clear significant differences were observed on day 84 between the CIA mice and the mice of the other groups (DEX, DLE, ω‐UFAs and HLT) (P < .0001), and again, no significant difference was observed in the inflammation index among the latter groups (Figure 1D).

3.3. Histologic features of arthritis in the CIA mice

Several histopathological changes were observed in the articular tissues of mice inoculated with chicken type II collagen. These changes included a predominantly mononuclear cell infiltration in the articular periphery and the adjacent connective tissue and muscle; marked synovial and chondral hyperplasia leading to cartilage and bone degeneration and erosion; oedema in the muscle and soft tissue; pannus (highly vascularized granular tissue on the articular cartilage); loss of synovial space; and bone fusion. Some of these pathological changes are illustrated in Figure 2A‐D.

FIGURE 2.

FIGURE 2

Open in a new tab

Main histologic changes in mice with collagen‐induced arthritis. Articulations in collagen‐induced arthritis in mice showed a complex pathology, including extensive mononuclear cell infiltrate (1) marked degeneration and lysis of articular cartilage (2), loss of chondral matrix) (Panel A), extensive cell infiltrate (1) in the extraarticular tissue affecting peripheral nerves (3) (Panel B), arthrosis (4) and regenerative hypertrophy of chondrocytes (5) with the reduction of synovial spaces (6) (Panel C), complete obliteration of synovial spaces (7), erosion of bone and cartilage (8), extensive mononuclear cell infiltrate and some necrotic debris (9)(Panel D). Representative images of the CIA group

3.4. Histologic articular changes following 84 days of treatment

The following description was prepared by two independent pathologists who analysed the whole set of tissue sections and then described the samples that showed the most representative changes in each group; their observations were as follows:

  • Healthy (HLT) group: No abnormal changes (Figure 3, HLT);

  • Arthritis (CIA) group: Active mononuclear inflammatory cell infiltrate mixed with abundant polymorph nuclear cells; marked synovial and chondral hyperplasia; bone and cartilage degeneration; no signs of reparative fibrosis; and oedema in the muscle and soft tissue (Figure 3, CIA);

  • Dexamethasone (DEX) group: Absence of inflammatory infiltrate; no synovial hyperplasia; no chondral or bone regeneration; and mild focal reparative fibrosis (Figure 3, DEX);

  • DLE (0.1 U): Absence of inflammatory infiltrate; no synovial hyperplasia, bone, or chondral degeneration; and mild focal reparative fibrosis (Figure 3, DLE);

  • Docosahexaenoic acid (DHA): Absence of inflammatory infiltrate; occasional and mild synovial hyperplasia; no bone or cartilage involvement; and focal and mild reparative fibrosis (Figure 3, DHA);

  • Arachidonic acid (AA): Mild multifocal inflammatory infiltrate; mild multifocal synovial hyperplasia; no bone or cartilage damage; and no residual fibrosis (Figure 3, AA);

  • Oleic acid (OA): Mild focal inflammatory infiltrate; mild focal synovial hyperplasia; absence of bone or chondral damage; and the absence of regenerative fibrosis (Figure 3, OA).

FIGURE 3.

FIGURE 3

Open in a new tab

Architecture of the carpal articulation bones of healthy, CIA mice and CIA mice treated with nothing, DEX, DLE, DHA, AA or OA. Except for the untreated CIA mice, most animals in each group (HLT, DEX, DLE, DHA, AA and OA) showed an essentially normal articular architecture: an absence of pannus and oedema, smooth bone surfaces, normal synovial spaces, a lack of osteocyte and chondrocyte hypertrophy, etc, similar to those observed in the healthy non‐inoculated group. The articular tissue of the CIA mice presents an altered histologic pattern with a reduction of the articular spaces, extensive damage of the soft tissue due to abundant inflammatory cell infiltrate and residual haematopoiesis in the medullar bone cavity (CIAa and CIAb). These results demonstrate that the anti‐inflammatory effect of DLE and ω‐UFAs is similar to the anti‐inflammatory effect of DEX. Particular alterations, if any, in each experimental group are indicated in Table 1 (haematoxylin‐eosin stain, 10×) (n = 4 mice per group)

3.5. Cartilage and collagen/reticular fibres

The loss of cartilage because of inflammatory cell infiltration and regenerative collagen formation by fibroblasts, osteocytes and chondrocytes was observed in the articular tissues of the untreated CIA mice, and this was no longer evident in mice treated with DEX, DLE, DHA, AA or OA (Figure 4).

FIGURE 4.

FIGURE 4

Open in a new tab

Bone alterations in the untreated CIA mice and the CIA mice treated with DEX, DLE or ω‐UFAs. Tissue sections stained with Masson's stain showed no major alterations in the articular bones of healthy (HLT) mice and the CIA mice treated with DEX, DLE, DHA, AA or OA. Together with bone destruction that resulted from the dominantly mononuclear cell infiltrate, bone regeneration with the hyperplasia of osteocytes and chondrocytes and articular matrix regeneration with the synthesis of new collagen (bluish colour) were noted in the articulations of the untreated CIA mice. Mature bone stains red, while collagen, immature bone and regenerating bone matrix stain blue (Masson's stain, 10×) (n = 4 mice per group). Representative images of each group

All of these findings are summarized in Table 2.

TABLE 2.

Average histologic articular changes in mice with CIA and the CIA mice subjected to diverse treatments (n = 4 mice per group)

Group Active inflammation Synovial hyperplasia Chondral degeneration Bone degeneration or erosion Reparative fibrosis Collagen of neoformation (reparative)
1. HLT NO NO NO NO NO NO
2. (CIA) S (4+) S (4+) S (4+) S (4+) NO 2+
3. (DEX) NO NO NO NO MF (1+) NO
4. (DHA) NO MF (1+) NO NO MF (1+) NO
5. (AA) mMF (2+) mMF (2+) NO NO NO NO
6. (OA) MF (1+) MF (1+) NO NO NO NO
7. (DLE‐0.1U) NO NO NO NO MF (1+) NO
Open in a new tab

Abbreviations: AA, arachidonic acid; CIA, collagen‐induced arthritis; DEX, dexamethasone; DHA, docosahexaenoic acid; DLE, dialysable leucocyte extract at 0.1U; HLT, healthy group; MF, mild and focal; mMF, mild multifocal; NO, no changes; OA, oleic acid; S, severe.

3.6. NF‐κB and NLRP3 activation

NF‐κB and NLRP3 inflammatory markers were expressed in the articular tissue of the CIA mice on day 84 postinoculation. The joints of all four CIA mice showed similar levels of the expression of both NF‐κB and NLRP3 (Figure 5A).

FIGURE 5.

FIGURE 5

Open in a new tab

Panel A: NF‐κB and NLRP3 are activated in collagen‐induced arthritis in mice. Upper row: the expression of activated NF‐κB in the inflamed carpal articulation of a DBA‐1 mouse that was immunized with C‐II. Clear NF‐κB expression (red) appears in the articular bones of this CIA mouse. Middle row: the expression of NLRP3 (red) in the articular tissue of a mouse immunized with C‐II. Coincident with the expression of activated NF‐κB, activated NLRP3 appears in the articular tissue of this CIA mouse. Lower row: the expression of the ASC component of inflammasome NLRP3 (green) in the articular tissue of a mouse immunized with C‐II. In all cases, tissues were stained with specific antibodies (red or green) and were counterstained with Hoechst reagent (blue). Confocal microscopy, n = 4 animals per group. Panel B: DEX, DLE and ω‐UFAs inhibit the activation of NLRP3. Treatment of the CIA mice with DEX, DLE, DHA, AA or OA inhibited the activation of NLRP3. Only merged (Hoechst + anti‐NLRP3 antibody) images are shown. Confocal microscopy, n = 4 mice per group

While activated NF‐κB and NLRP3 appeared to be strongly expressed in the articular tissue of the CIA mice, these proteins were not significantly expressed in the articular tissue of healthy mice or in the articular tissue of the CIA mice that were treated for 84 days with DEX, DLE or ω‐UFAs (Figure 5B). This result was coincident with the lack of inflammatory changes in mice treated with these substances, demonstrating their anti‐inflammatory efficacy.

4. DISCUSSION

Many reports on the beneficial effects of DLEs in diverse infectious and non‐infectious diseases have been published. 28

In the case of RA, however, scientifically validated reports on the use of DLE are scarce and the results have been inconsistent. 14 , 15 , 16 , 28 , 29 , 30 In the present investigation, a clear anti‐inflammatory effect of DLE was demonstrated in a well‐controlled arthritis model in mice. Mice treated with DLE (0.1 U) exhibited a suppressed development of CIA at a level that was comparable to that observed with DEX, a potent anti‐inflammatory compound used in the treatment of RA. The mechanism of action of DLEs is not known; however, some in vitro studies in humans and in small animals suggest that the TF in DLE promotes the release of IFNγ, IL‐2 and TNF, as well as IL‐10 and other cytokines, 31 , 32 while some others suggest that TF downregulates NF‐κB, a critical transcription factor for several proinflammatory cytokines. 33 This observation is in agreement with our present results, where the inhibition of inflammation as a result of the treatment with DLE was correlated with the virtual absence of NF‐κB activity.

Concerning the effects of ω‐UFAs in RA, several papers have been published, but most of them are clinical trials whose effectiveness is evaluated‐only on clinical grounds. 34 , 35 , 36 , 37 , 38 Some reports, however, indicate that ω‐3 UFAs are incorporated into the cell membrane, modifying its structure and function and interfering with signalling pathways that are linked to cell to surface and intracellular receptors. 17 , 18 , 19 , 20 One consequence of this effect is the interference of TNF/IL‐1/TLR signalling and the inhibition of NF‐κB, a key family of transcription factors for several proinflammatory cytokines. 39 , 40 Additionally, the incorporation of arichadonic acid in the cell membrane occurs, which is a source of pro‐inflammatory mediators including prostaglandins, thromboxanes and leucotrienes. The metabolism of ω‐3 UFAs through cyclooxygenase‐2 and lipoxygenases will, on the other hand, give rise to anti‐inflammatory compounds, such as lipoxins, resolvins, protectins and maresins 17 that antagonize the activity of arachidonic‐derived mediators and inhibit the recruitment and function of polymorphonuclear leucocytes, inducing their apoptosis and promoting efferocytosis by macrophages. 41 An additional, more immunologic anti‐inflammatory mechanism of UFA was proposed by Kim et al, 42 who found that ω‐3 UFAs restore the Th17 and Treg balance in CIA. In our study, we observed that DHA (ω‐3), AA (ω‐6), OA (a monounsaturated, ω‐9, acid) and DLE showed significant anti‐inflammatory activity. Kawahito et al 43 and Carregaro et al 44 also found that an AA derivative (15‐deoxy‐D12,14‐PGJ2) exerted a marked anti‐inflammatory effect in rodent arthritis and this effect was related to an increase in the activity of regulatory T cells (Treg) and a diminution in the differentiation of IL‐17‐producing cells (Th17). These reports support our results showing the anti‐inflammatory effect of AA in CIA.

Regarding OA, the anti‐inflammatory effect of OA has been widely documented both in vivo and in vitro (reviewed by Carrillo et al 45 ), and it seems that its anti‐inflammatory effect is also related to the inhibition of the NF‐κB pathway. 46 Its beneficial effect in the CIA model has already been reported. 23 , 47

Finally, the histopathological findings correlated with the anti‐inflammatory effects of DLE and ω‐UFAs, as these substances significantly limited the disease signs, including inflammatory infiltrate, synovial hyperplasia, arthrosis, and bone and cartilage degeneration.

The lack of inflammatory infiltrates in the CIA mice treated with ω‐UFAs may support the local production of protectins, resolvins and maresins that inhibit neutrophil diapedesis and migration, lymphocyte proliferation and the synthesis of proinflammatory cytokines. 17 , 20 , 47 Dialysable leucocyte extract in turn acts on chondrocytes and osteocytes, favouring the production of proteoglycans and collagen, thus counteracting bone and cartilage degradation. 48

As in other inflammatory diseases, inflammasomes play a central role in RA. Guo et al 9 studied the activation of the NLRP3 inflammasome in RA and CIA. Treatment of CIA with MCC950 (a selective inhibitor of NLRP3) resulted in less severe joint inflammation and bone destruction and reduced the production of IL‐1β, a potent proinflammatory cytokine, compared to those in the controls. Inflammation in CIA may activate inflammasomes via the release of prostaglandins and leucotrienes from the membrane lipids or through collagen‐induced danger signals (DAMPs). Synovial fibroblasts and endothelial cells seem to be the main source of NLRP3 in RA. 49 In the present study, we found that the activation of the NLRP3 inflammasome was linked to the activation of NF‐κB, and there was a deactivation of these molecules following treatment with DEX, DHA, OA, AA and DLE.

It is known that ω UFAs block phosphorylation of IκBα, thus impeding NF‐κB translocation to the nucleus and its union with DNA. 17 , 45 Blockage of NF‐κB activity and activation of NLRP3 suppress the synthesis of proinflammatory mediators (cytokines, prostaglandins and leucotrienes) and their consequential effects. 46 , 50

4.1. Conclusion

We report the clear beneficial anti‐inflammatory effects of both DLEs and unsaturated fatty acids in a mouse model of CIA and confirm the participation of NF‐κB and the NLRP3 inflammasome in the pathology of CIA and the inhibition of these proteins by DLE and ω‐UFAs. Pending a therapeutic study, we envisage DLE and ω‐UFAs usage as ancillary treatments for RA with few side effects, if any.

CONFLICT OF INTEREST

None.

AUTHORS' CONTRIBUTIONS

OR‐E designed and directed the investigation; PIP‐M performed the experiments; PA‐P supervised the study and made the statistical analysis of data; and VGH‐Ch and SE‐P described the histopathological changes observed.

ACKNOWLEDGEMENTS

This investigation received financial support from SIP (Projects 2018‐2019), COFAA, EDI (IPN) and SNI (CONACYT), México.

Pérez‐Martínez PI, Rojas‐Espinosa O, Hernández‐Chávez VG, Arce‐Paredes P, Estrada‐Parra S. Anti‐inflammatory effect of omega unsaturated fatty acids and dialysable leucocyte extracts on collagen‐induced arthritis in DBA/1 mice. Int J Exp Path. 2020;101:55–64. 10.1111/iep.12348

REFERENCES

  • 1. Cross M, Smith E, Hoy D, et al. The burden of rheumatoid arthritis: estimates from the global burden of disease 2010 study. Ann Rheum Dis. 2014;73:1316‐1322. [DOI] [PubMed] [Google Scholar]
  • 2. Bellucci E, Terenzi R, La Paglia GM, et al. One year in review 2016: pathogenesis of rheumatoid arthritis. Clin Exp Rheumatol. 2016;34:793‐801. [PubMed] [Google Scholar]
  • 3. Angelotti F, Parma A, Cafaro G, Capecchi R, Alunno A, Puxeddu I. One year in review 2017: pathogenesis of rheumatoid arthritis. Clin Exp Rheumatol. 2017;35:368‐378. [PubMed] [Google Scholar]
  • 4. Shankar V. Rheumatoid arthritis. Pathology Outlines. http://www.pathologyoutlines.com/topic/jointsra.html. Accessed January 24, 2018.
  • 5. Choulaki C, Papadaki G, Repa A, et al. Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis. Arthritis Res Ther. 2015;19:257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Kastbom A, Verma D, Eriksson P, Skogh T, Wingren G, Soderkvist P. Genetic variation in proteins of the cryopyrin inflammasome influences susceptibility and severity of rheumatoid arthritis (the Swedish TIRA project). Rheumatology (Oxford). 2008;47:415‐417. [DOI] [PubMed] [Google Scholar]
  • 7. Hamad M, Cornelis F, Marzouk S, Chabchoub G, Bahloul Z, Rebai A. Association study of CARD8 (p.C10X) and NLRP3 (p.Q705K) variants with rheumatoid arthritis in French and Tunisian populations. Int J Immunogenet. 2012;39(2):131‐136. [DOI] [PubMed] [Google Scholar]
  • 8. Zhang Y, Zheng Y, Li H. NLRP3 inflammasome plays an important role in the pathogenesis of collagen‐induced arthritis. Mediators Inflamm. 2016;2016:1‐9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Guo C, Fu R, Wang S, et al. NLRP3 inflammasome activation contributes to the pathogenesis of rheumatoid arthritis. Clin Exp Immunol. 2018;194:231‐243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Nabih ES. NLRP3 inflammasome: a novel therapeutic target in arthritis. J Arthritis. 2018;7:118. [Google Scholar]
  • 11. Singh JA, Saag KG, Bridges SL Jr, et al. American College of Rheumatology guideline for the treatment of rheumatoid arthritis. Arthritis Care Res. 2015;68:1‐26. [DOI] [PubMed] [Google Scholar]
  • 12. Ferro L, Elefante E, Luciano N, Talarico R, Todoerti M. One year in review 2017: novelties in the treatment of rheumatoid arthritis. Clin Exp Rheumatol. 2017;35:721‐734. [PubMed] [Google Scholar]
  • 13. Efthimiou P, Kukar M. Complementary and alternative medicine use in rheumatoid arthritis: proposed mechanisms of action and efficacy of commonly used modalities. Rheumatol Int. 2010;30:571‐586. [DOI] [PubMed] [Google Scholar]
  • 14. Kass E, Froland SS, Natvig JB, Blichfeldt P, Hoyeraal HM, Munthe E. A new principle of immunotherapy in rheumatoid arthritis: treatment with transfer factor. Scand J Rheumatol. 1974;3:113‐117. [DOI] [PubMed] [Google Scholar]
  • 15. Hoyeraal HM, Froland SS, Salvensen CF, et al. No effect of transfer factor in juvenile rheumatoid arthritis by double‐blind trial. Ann Rheum Dis. 1978;37:175‐179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Georgescu C. Effect of long‐term therapy with transfer factor in rheumatoid arthritis. Med Interne. 1985;23:135‐140. [PubMed] [Google Scholar]
  • 17. Duvall MG, Levy BD. DHA and EPA‐derived resolvins, protectins and maresins in airway inflammation. Eur J Pharmacol. 2016;15(785):144‐155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Yaqoob P, Newsholme EA, Calder PC. The effect of dietary lipid manipulation on rat lymphocyte subsets and proliferation. Immunology. 1994;82:603‐610. [PMC free article] [PubMed] [Google Scholar]
  • 19. Calder PC. Omega‐3 fatty acids and inflammatory processes. Nutrients. 2010;2:355‐374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Gorjao R, Cury MA, Martins T, Curi R. Regulation of human lymphocyte proliferation by fatty acids. Cell Biochem Funct. 2007;25:305‐315. [DOI] [PubMed] [Google Scholar]
  • 21. Williams RO. Collagen‐induced arthritis as a model for human rheumatoid arthritis. Methods Mol Med. 2004;98:2017‐2216. [DOI] [PubMed] [Google Scholar]
  • 22. Brand DD, Kang AH, Rosloniec EF. The mouse model of collagen‐induced arthritis. Methods Mol Med. 2004;102:295‐312. [DOI] [PubMed] [Google Scholar]
  • 23. Perez PI, Hernandez VG, Rodriguez O, Arce P, Rojas O. Differential anti‐inflammatory effects of three purified omega unsaturated fatty acids on collagen‐induced arthritis in mouse. Modern Res Inflamm. 2016;5:31‐44. [Google Scholar]
  • 24. Inglis J, Simelyte E, McCann F, Criado G, Williams R. Protocol for the induction of arthritis in C57BL/6 mice. Nat Prot. 2008;4:612‐618. [DOI] [PubMed] [Google Scholar]
  • 25. Fabre RA, Perez TM, Aguilar LD, et al. Transfer factors as immunotherapy and supplement of chemotherapy in experimental pulmonary tuberculosis. Clin Exp Immunol. 2004;136:215‐223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Sharma S, Arif M, Kumar R, Gupta R, Chand S. Cumulative therapeutic effects of phytochemicals in Arnica montana flower extract alleviated collagen induced arthritis: inhibition of both proinflammatory mediators and oxidative stress. J Sci Food Agric. 2016;96:1500‐1510. [DOI] [PubMed] [Google Scholar]
  • 27. Lee G. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed New York, NY: McGraw Hill; 1968. [Google Scholar]
  • 28. Arnaudov A, Kostova Z. Dialysable leukocyte extracts in immunotherapy. Biotechnol Biotechnol Equip. 2015;29:1017‐1023. [Google Scholar]
  • 29. Cozine WS, Stanfield AB, Stephens CAL, et al. Transfer Factor Basic Properties and Clinical Applications. New York, NY: Academic Press; 1976. [Google Scholar]
  • 30. Maini RN, Scott JT, Hamblin A, Roffe L, Dumonde DC. Is the clinical benefit from transfer factor in rheumatoid arthritis a placebo effect? In: Ascher MS, Gottlieb AA, Kirkpatrick CH. (Eds.). Transfer Factor Basic Properties and Clinical Applications (pp. 601‐612). New York, NY: Academic Press; 1976. [Google Scholar]
  • 31. Salinas N, Estrada S, Becerril M, et al. Herpes murine model as a biological assay to test dialyzable leukocyte extracts activity. J Immunol Res. 2015;2015:1‐9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Ojeda MO, van't Veer C, Fernández Ortega CB, Araña Rosainz Mde J, Buurman WA. Dialyzable Leukocyte Extract differentially regulates the production of TNFa, IL‐6, and IL‐8 in bacterial component‐activated leukocytes and endothelial cells. Inflamm Res. 2005;54:74‐81. [DOI] [PubMed] [Google Scholar]
  • 33. Ojeda MO, Fernández Ortega CB, Rosaínz MJ. Dialyzable leukocyte extract suppresses the activity of essential transcription factors for HIV‐1 gene expression in unstimulated MT‐4 cells. Biochem Biophys Res Commun. 2000;273:1099‐1103. [DOI] [PubMed] [Google Scholar]
  • 34. Akbar U, Yang M, Kurian D, Mohan C. Omega‐3 fatty acids in rheumatic diseases: a critical Review. J Clin Rheumatol. 2017;3:330‐339. [DOI] [PubMed] [Google Scholar]
  • 35. Navarini L, Afeltra A, Gallo‐Afflitto G, Margiotta DPE. Polyunsaturated fatty acids: any role in rheumatoid arthritis? Lipids Health Dis. 2017;16:197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Skocznska M, Swierkot J. The role of diet in rheumatoid arthritis. Reumatologia. 2018;56:259‐267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Gioxari A, Kaliora AC, Marantidou F, Panagiotakos DP. Intake of ω‐3 polyunsaturated fatty acids in patients with rheumatoid arthritis: a systematic review and meta‐analysis. Nutrition. 2018;45:114‐124. [DOI] [PubMed] [Google Scholar]
  • 38. Gan RW, Demoruelle MK, Deane KD, et al. Omega‐3 fatty acids are associated with a lower prevalence of autoantibodies in shared epitope‐positive subjects at risk for rheumatoid arthritis. Ann Rheum Dis. 2017;76:147‐152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Liu T, Zhang L, Joo D, Sun S‐C. NFkB signaling in inflammation. Signal Transduc Targ Ther. 2017;2:17023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Scher J, Pillinger M. 15d‐PGJ2: the anti‐inflammatory prostaglandin? Clin Immunol. 2005;114:100‐109. [DOI] [PubMed] [Google Scholar]
  • 41. Headland SE, Norling LV. The resolution of inflammation: principles and challenges. Semin Immunol. 2015;27:149‐169. [DOI] [PubMed] [Google Scholar]
  • 42. Kim JY, Lim K, Kim KH, Kim JH, Choi JS, Shim SC. N‐3 polyunsaturated fatty acids restore Th17 and Treg balance in collagen antibody‐induced arthritis. PLoS ONE. 2018;13(3):e0194331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Kawahito Y, Kondo M, Tsubouchi Y, et al. 15‐deoxy‐delta (12,14)‐PGJ(2) induces synoviocyte apoptosis and suppresses adjuvant‐induced arthritis in rats. J Clin Invest. 2000;106:189‐197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Carregaro V, Napimoga M, Peres R, et al. Therapeutic treatment of arthritic mice with 15‐deoxy Δ12,14‐prostaglandin J2 (15d‐PGJ2) ameliorates disease through the suppression of Th17 cells and the induction of CD4+CD25−FOXP3+ cells. Mediators Inflamm. 2016;2016:1‐13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Carrillo C, Cavia MM, Alonso S. Role of oleic acid in immune system; mechanism of action; a review. Nutr Hosp. 2012;27:978‐990. [DOI] [PubMed] [Google Scholar]
  • 46. Medeiros‐de‐Moraes I, Gonçalves C, Kurz A, et al. Omega‐9 oleic acid, the main compound of olive oil, mitigates inflammation during experimental sepsis. Oxid Med Cell Longev. 2018;2018:1‐13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Aquino MY, Rodríguez L, Arce P, Hérnandez VG, Becerril E, Rojas O. The effect of Alpha asarone, olive oil, and dexamethasone on collagen‐induced arthritis (CIA) in the mouse. Modern Res Inflamm. 2013;2:9‐20. [Google Scholar]
  • 48. Acosta P, Pérez N, Pérez N, et al. Anti‐inflammatory effect of dialysable leucocyte extract in a rat model of osteoarthritis: histopathological and molecular characterization. Scand J Rheumatol. 2016;45:528‐535. [DOI] [PubMed] [Google Scholar]
  • 49. Kolly L, Busso N, Palmer G, Talabot‐Ayer D, Chobaz V, So A. Expression and function of NALP3 inflammasome in rheumatoid arthritis. Immunology. 2009;129:178‐185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Betiati DS, de Oliveira PF, Camargo CQ, Nunes EA, Trindade EB Effects of omega‐3 fatty acids on regulatory T cells in hematologic neoplasms. Rev Bras Hematol Hemoter. 2013;35:119‐125. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from International Journal of Experimental Pathology are provided here courtesy of Wiley

RESOURCES