IVIG ameliorate inflammation in collagen‐induced arthritis: projection for IVIG therapy in rheumatoid arthritis

HE staining of representative arthritic paws, from each study group of CIA mice. IVIG treatment ameliorate inflammation in collagen induced arthritis: A) CIA mouse treated with IVIG; B) healthy mouse without CIA; C) CIA mouse treated with PBS; D) non‐treated CIA mouse.

Summary

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease that leads to joint destruction and disability. Despite a significant progress in administration of biological agents for RA patients, there is still a need for improved therapy. Intravenous immunoglobulins (IVIG), a pooled polyspecific immunoglobulin (Ig)G extracted from 5000 to 20 000 healthy subjects, showed beneficial therapeutic effect in patients with immune deficiency, sepsis and autoimmune diseases. The current study aimed to investigate the beneficial effect of treatment with IVIG in established collagen‐induced arthritis in DBA/1j mice. Murine arthritis was induced in DBA/1j mice. Treatment with IVIG began when the disease was established. The clinical score was followed twice a week until day 48. The mice were bled for plasma and the paws were hematoxylin and eosin (H&E)‐stained. Cytokine profile in the plasma was analyzed by Luminex technology and titers of circulating anti‐collagen antibodies in the plasma was tested by enzyme‐linked immunosorbent assay. Our results show that treatment with IVIG in murine significantly reduced the clinical arthritis score (P < 0·001). Moreover, mode of action showed that IVIG significantly reduced circulating levels of inflammatory cytokines [interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐17, IL‐6, tumor necrosis factor (TNF)‐α, P < 0·001], inhibiting anti‐collagen antibodies (P < 0·001) in the plasma of collagen‐induced arthritis mice. Importantly, histopathological examination revealed that IVIG treatment prevented the migration of inflammatory immune cells into the cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Our results proved for the first time the valuable anti‐inflammatory treatment of IVIG in experimental RA. We propose IVIG therapy for a subgroup of patients with rheumatologically related diseases.

Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease that leads to painful joint destruction and disability [1, 2]. Traditional RA therapy includes disease‐modifying anti‐rheumatic drugs (DMARDs), such as first‐line therapy methotrexate, hydroxychloroquine, sulfasalazine, minocycline and leflunomide [3]. In recent years, emerging novel biological agents have been targeting specific pathways. These targeted inflammatory pathways have included: anti‐interleukin (IL)‐1 (anakinra), anti‐IL‐6 (tocilizumab), tumor necrosis factor α (TNF‐α) blockers [infliximab, etanercept (Enbrel), adalimumab (Humira), certolizumab (Cimzia) and golimumab (symoni)], Janus kinase (JNK) inhibitors (tofacitinib, baricitinib) and anti‐CD20 (rituximab) [4, 5, 6, 7]. Despite the significant progress that has been achieved with the administration of biological therapies in changing the natural history of RA, such medications induce immune suppression, which is non‐selective to the pathogenesis of the disease, resulting in higher rates of common and opportunistic infections. Thus, different strategies are used to overcome these issues; for example, processing of antibodies under specific high‐dilution technology to produce drugs that change the conformation state of the antigen and have specific target‐modification activity [8, 9], or produce domain antibodies such as nanobodies, recently approved by the Food and Drug Administration (FDA) for the first time [10]. Another highly promising approach is the usage of intravenous immunoglobulins (IVIG) as a therapy for RA.

IVIG is a blood product, predominantly IgG (> 95%), isolated from 5000 to 20 000 healthy donors. The first mentioned triumph of the use of IVIG therapy was in primary immunodeficiency diseases in the 1950s. IVIG has a good proven beneficial and safety profile, and is one of the first biological therapies which was introduced in 1981 by Imbach et al. [11] for immune thrombocytopenic purpura (ITP). To date, the FDA has approved the use of IVIG as a first‐line therapy in B cell chronic lymphocytic leukemia, primary humoral immunodeficiency, ITP, Kawasaki syndrome and multi‐focal motor neuropathy [12]. Despite FDA off‐labeling, IVIG therapy has been expanded for diverse autoimmune diseases such as: specific subgroups of RA patients, juvenile chronic arthritis (JCA), Guillain–Barré syndrome, chronic inflammatory demyelinating polyneuropathy occurring in the context of rheumatic disease, systemic lupus erythematosis (SLE), idiopathic inflammatory myopathies, systemic sclerosis, anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitides, Still’s disease and more [13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. The mode of action of IVIG encompasses various mechanisms attributed to the F(ab)₂ or Fc portions of the molecule [6, 27, 28, 29, 30]. The Fab part of the IVIG is related to the neutralization of inflammatory cytokines, anti‐idiotypic activity, blocking of cellular receptors, antibody‐dependent cellular cytotoxicity (ADCC) and anaphylatoxin scavenging, whereas the Fc‐related activities encompass regulation of FcγR, immunomodulation of the function of dendritic cells, blocking activating receptors, expansion of T regulatory cells and saturation of FcRn.

One of the frequent murine models used in preclinical studies to evaluate potential anti‐rheumatic agents is collagen‐induced arthritis (CIA), which imitates human RA. CIA‐treated mice share several pathological features with RA, including generation of autoantibodies, synovial inflammatory cell infiltration, synovial hyperplasia, cartilage destruction and bone erosion [31].

Materials and methods

Mice and experimental design

Experimental arthritis was induced in 7–8‐week‐old DBA/1J male mice (Envigo, Blackthorn, UK). 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 ethics committee of the Israeli Ministry of Health (no. 1221/19) and fulfill the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines version 2.0 for animal research. The CIA model was performed as previously described by us [31, 32, 33]: bovine type II collagen (Chondrex, Redmond, WA, USA) was emulsified 1 : 1 with Mycobacterium tuberculosis H37RA in Freund’s incomplete adjuvant (Difco Laboratories, Detroit, MI, USA). DBA/1J males were subcutaneously injected into the base of the tail with 100 μg emulsion. A booster injection of bovine type II collagen in phosphate‐buffered saline (PBS) at the base of the tail was given 21 days later. Intraperitoneal injections of IVIG (OMRIX Ltd, Rehovot, Israel), 3 mg/0.1 ml/mouse, started at a score of 2–3 after the booster injection and repeated on a weekly base. PBS as vehicle (0·1 ml/mouse) and non‐treated mice were used as controls, n = 10 per each group. The mice were euthanized after 48 days.

Arthritis scoring

The clinical scores and the hind paw widths of the mice were monitored daily during the course of each experiment. The development of arthritis was assessed daily, and the severity of arthritis was scored for each paw on a three‐point scale, in which 0 = normal appearance, 1 = localized edema/erythema over one surface of the paw, 2 = edema/ erythema involving more than one surface of the paw and 3 = marked edema/erythema involving the whole paw. The scores of all four paws were added for a composite score, with a maximum score of 12 per mouse. Ankle thickness of the hind paws was measured in millimeters (mm) at the widest point (the malleoli), with the legs fully extended with digital calipers (Manostat, Herisau, Switzerland).

Histopathological assessment

The paws of the mice were obtained from the euthanized mice and fixed in 4% formalin (Sigma‐Aldrich, St Louis, MO, USA), decalcified and cut and stained with hematoxylin and eosin (H&E). Samples were examined by light microscopy (×200 magnification). All histological evaluations performed by pathologists were double‐blinded.

Quantification of cytokines with Luminex

At the end of the experiment, blood was collected into an ethylenediamine tetraacetic acid (EDTA)‐containing Eppendorf tube, then centrifuged (1800 g for 10 min) to obtain plasma samples.

Luminex technology (mouse high‐sensitivity T cell magnetic bead panel (MHSTCMAG‐70K); Merck GmbH, Darmstadt, Germany) was used to measure interferon (IFN)‐γ, IL‐1β, IL‐6, IL‐17, and TNF‐α in the plasma. All quantifications were performed according to the protocols provided by the manufacturer.

Anti‐collagen type II antibodies determination

Circulating anti‐collagen type II antibodies were determined by home‐made enzyme‐linked immunosorbent assay (ELISA). ELISA plates were coated with collagen type II [10 µg/ml phosphate‐buffered saline (PBS)]. Following overnight incubation at 4°C, the plates were blocked with 3% bovine serum albumin (BSA). Mouse plasma at dilution of 1 : 800 were added to the blocked ELISA plates and incubated for 2 h at room temperature (n = 10 for each treatment group). 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 and presented as OD at 405 nm.

Statistical analysis

Statistical analysis was made using the repeated‐measures analysis of variance (anova) procedure; while the weights data fitted all the required assumptions, the arthritis score data did not meet the linear and sphericity assumption, thus increasing type 1 error for the model. Furthermore, violation for this assumption is not known to bias the post‐hoc analysis [34]. Therefore, we aggressively adjusted for multiple comparisons using Bonferroni’s adjustment. For both models, time was set as the within‐subject effect, while the treatment group was set as the between‐subjects effect. The significance level was set at 5%. Statistical analysis and graph plotting were made with IBM release 2015 spss Statistics for Windows, version 23.0 (IBM Corporation Armonk, NY, USA). Cytokine and anti‐collagen concentration were first assessed for distribution. As normal distribution was not met, non‐parametric tests were chosen throughout the statistical analysis. Comparisons of the mean concentrations began with the Kruskal–Wallis H‐test, followed by Dunnett’s test for pairwise comparisons. Post‐hoc comparisons, including plotted comparisons, were adjusted for multiple comparisons following the Bonferroni procedure. Both plots and statistical analysis were conducted using the R Core Team language and environment for statistical computing (2013) (R Foundation for Statistical Computing, Vienna, Austria; URL http://www.R‐project.org/).

Results

IVIG treatment significantly abrogates the arthritis clinical score

CIA is a mouse model which imitates rheumatoid arthritis in genetically prone human RA patients, in which inflammation leads to the joint destruction. We investigated the effect of IVIG treatment in CIA mice and compared its efficacy with CIA mice treated with PBS (vehicle) or non‐treated CIA mice.

Both IVIG and PBS treatments started when the baseline arthritic score was almost 3. We observed a significantly lower marginal arthritis score in IVIG‐treated mice compared to marginal arthritis score of PBS‐treated mice or non‐treated mice (P < 0·001), as illustrated in Fig. 1. Repeated‐measures anova analysis revealed a significant difference between the treatment groups mean arthritis score over time (F = 30·224, P < 0·0001, lower bound‐adjusted). Marginal arthritis scores of the different studied groups were as follows: the IVIG group had the lowest arthritis score marginal mean of 4·128 [95% confidence interval (CI) = 3·995, 4·262], in comparison to 5·78 (95% CI = 5·56, 6·01) for non‐treated CIA mice and 5·62 (95% CI = 5·45, 5·77) for PBS‐treated CIA mice, P < 0·0001 (Bonferroni‐adjusted). The significantly lower arthritis score lasted until the mice were euthanized at day 48 (Fig. 1). No significant change in mice body weight was documented over time in the three studied groups, P > 0·05 (data not shown).

Fig. 1.

IVIG effect on arthritis score in collagen‐induced arthritis (CIA) mice. The data are presented as arthritis score over time in DBA/j CIA mice, upon treatment with intravenous immunoglobulin (IVIG), phosphate‐buffered saline (PBS) or non‐treated mice; n = 10 per each studied group of mice, P < 0·001.

Analyzing the clinical score at day 27 after the boost administration (day 48 from day 0 of disease induction) showed a score of non‐treated CIA mice, respectively (Fig. 2).

Fig. 2.

Intravenous immunoglobulin (IVIG) effect on arthritis score at the last point of the experiment. This arthritis score was evaluated at day 48 (27 days since the beginning of treatment with IVIG). Kruskal–Wallis, χ² ₍₂₎ = 38·65, P < 0·001.

Representative pictures of the mice joints are illustrated in Fig. 3a,b. A significant difference in the joints can be seen between CIA mice and IVIG treatment. Mice treated with IVIG showed no arthritis picture, similar to the healthy non‐collagen‐injected mice (Fig. 3aAB). PBS and CIA‐non‐treated mice showed developed edema and erythema from ankle to the entire leg (Fig. 3aC). H&E staining of the joints tissue sections from the IVIG‐treated mice as described in Fig. 3bA demonstrates significantly less synovial hyperplasia, no inflammatory process or neutrophil infiltration, normal cartilage layer and muscle structure, typical bone organization and uninflamed fat tissue, as in healthy non‐injected mice (Fig. 3bD). In CIA mice treated with PBS (Fig. 3bC) and in non‐treated CIA mice (Fig. 3bD), a massive infiltration of neutrophils can be observed as well as bone and joint destruction.

Fig. 3.

Prototypical clinical improvement of the mice joints. (a) Joints upon intravenous immunoglobulin (IVIG) treatment; (b) H&E staining of the joints, magnification x200. (abA) CIA mouse treated with IVIG; (abB) healthy mouse without CIA; (abC) CIA mouse treated with PBS; (abD) non‐treated CIA mouse.

IVIG treatment significantly reduced the production of anti‐collagen antibodies in CIA mice

IVIG is known to consist neutralizing antibodies targeting the Fab portion of pathogenic autoantibodies, a phenomenon previously described as an anti‐idiotypic activities. Therefore, we analyzed the effect of IVIG in amelioration of anti‐collagen antibodies in the plasma of CIA mice ± treatment (Fig. 4).

Fig. 4.

Circulating anti‐collagen antibodies. Mouse anti‐collagen type II circulating antibodies, were tested in the plasma of the three groups of mice at dilution of 1 : 800. Data are presented at optical density (OD) at 405 nm. Kruskal–Wallis, χ² ₍₂₎ = 30·7, P < 0·001.

As illustrated in Fig. 4, ELISA examination of antibodies against collagen in the IVIG‐CIA group showed OD of 1·15 ± 0·28 at 405 nm, compared to 1·56 ± 0·31 OD and 1·76 ± 0·7 OD in PBS or untreated CIA mice, respectively, P < 0·001.

IVIG treatment, regulated circulating cytokines levels in CIA mice

A direct effect of the cytokines on the destruction of the joint or another mechanism besides recruitment of inflammatory cells has been shown [1, 2, 35]. We evaluated the circulating inflammatory cytokine (IFN‐γ, IL‐1β, IL‐17, IL‐6, TNF‐α) levels in the CIA mice plasma upon treatment with IVIG, vehicle or non‐treatment (Fig. 5a–e). The inflammatory cytokine levels in IVIG‐treated CIA mice were significantly lower in comparison to the control PBS or non‐treated mice for all the tested cytokines, P < 0·001. For example, IL‐6 levels in the plasma of CIA mice treated with IVIG were found to be 896 ± 168 pg/ml in comparison to 13 999 ± 917 pg/ml and 13 481 ± 869 pg/ml for non‐treated CIA mice and PBS‐treated CIA mice, respectively, P < 0·001. The IL‐17 levels in the plasma of IVIG‐treated CIA mice were 1173 ± 497 pg/ml, whereas in the non‐treated or PBS‐treated CIA mice the levels were 10 318 ± 1521 pg/ml and 11 487 ± 371 pg/ml, respectively, P < 0.001. The TNF‐α levels in the CIA mice treated with IVIG were 2 164.28 ± 652 pg/ml compared to 11 099 ± 4176 pg/ml and 13 969 ± 3044 pg/ml in the non‐treated CIA mice or PBS‐treated CIA mice, respectively, P < 0·001.

Fig. 5.

Levels of circulating cytokines in collagen‐induced arthritis (CIA) mice upon treatment with intravenous immunoglobulin (IVIG). We analyzed the concentrations of the inflammatory cytokines interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐17, IL‐6 and tumor necrosis factor (TNF)‐α in the plasma of CIA mice ± treatment with IVIG, phosphate‐buffered saline (PBS) or on‐treated. The data are presented as concentration in pg/ml; n = 10 per group. (a) IFN‐γ Kruskal–Wallis, χ² ₍₂₎ = 42·76; (b) IL‐1β Kruskal–Wallis, χ² ₍₂₎ = 42·77; (c) IL‐6 Kruskal–Wallis, χ² ₍₂₎ = 43·22; (d) IL‐17 Kruskal‐Wallis, χ² ₍₂₎ = 42·42; (e) TNF‐α Kruskal–Wallis, χ² ₍₂₎ = 38·6. P < 0·001 for all studied groups of mice.

Discussion

RA is a systemic autoimmune inflammatory disease that leads to painful joint destruction and disability [11]. IVIG, with a good proven beneficial and safety profile, is one of the first biological therapies which was introduced in 1981 by Imbach et al. for immune thrombocytopenic purpura (ITP) [11]. Since then, IVIG has been employed successfully in a wide range of conditions, such as immune deficiency, sepsis, autoimmunity (e.g. specific subgroups of RA patients, juvenile chronic arthritis (JCA), Still’s disease, Guillain–Barré syndrome and chronic inflammatory demyelinating polyneuropathy occurring in the context of rheumatic disease, as well as in systemic lupus erythematosus (SLE), idiopathic inflammatory myopathies, systemic sclerosis and ANCA‐associated vasculitides, Still’s disease and more [13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24].

We show herein a beneficial effect of IVIG in the treatment in established murine CIA. IVIG inhibited the arthritis clinical score inflammatory condition, exemplified by the reduced levels of circulating inflammatory cytokines. IVIG prevents infiltration of immune cells by histopathological examination and inhibits joint destruction, compared with the untreated or PBS‐subjected CIA mice. The lower clinical score was accompanied by reduced levels of anti‐collagen antibodies (P < 0·001). Similarly, the diminished levels of circulating inflammatory cytokines (IFN‐γ, IL‐1β, IL‐6, IL‐17, TNF‐α) were recorded (P < 0·001). Our data support and strengthen our previous observation that prophylactic treatment with IVIG inhibited the severity of disease inflammation in murine CIA. In that study, IVIG affinity purified on curillin peptides (ACPA‐specific IVIG) and, more significantly, citrullinated peptide‐specific IVIG, was found to be 200 times more efficient in the reduction of CIA inflammation activity, inhibiting the inflammatory cytokine production ex vivo by spleen cells and enhanced expansion of spleen T regulatory cells compared to regular IVIG treatment [36]. Moreover, the immunomodulatory make‐up of IVIG in this study was attributed to its anti‐idiotypic activity via the Fab portion of the molecules, although we cannot exclude the Fc contribution via elevation of T regulatory cell numbers.

Lee et al., using a prophylactic protocol for the IVIG treatment in CIA mice, showed that IVIG inhibited the development of disease score in CIA mice, reduced the number of T helper type 17 (Th17) cells in the spleen, expansion of spleen regulatory T cells and inflammatory cytokines in the joints [37]. Similarly, IVIG up‐regulated IL‐10 and Fcγ receptor IIB expression by spleen cells [37]. It is worth mention that in our current study, IVIG was found to abrogate the course of disease when the treatment started in established CIA mice. Passive transfer of sera from RA patients to K/BxN mice is an additional murine model of arthritis [38]. IVIG given in a prophylactic protocol or recombinant IgG1 Fc hexamer (Fc‐μTP‐L309C) resulted in attenuation of the arthritis score in the artherogenic K/BxN mice [39].

The IVIG mode of action encompasses numerous biological functions related in part to the F(ab)₂ of the molecule and/or to the Fc activities. The anti‐inflammatory activity of IVIG is based on the following activities: (a) neutralization of inflammatory cytokines, as IVIG targets at least 13 different cytokines or cytokine inhibitors [40, 41, 42, 43, 44]. Due to the fact that IVIG has anti‐inflammatory cytokine activities, it was shown that IVIG is an efficient therapy in the cytokine storm in cases of catastrophic anti‐phospholipid syndrome, severe influenza and currently in COVID‐19 [42, 43, 44, 45, 46, 47]. Specifically, IVIG bind the spike protein on RS‐Cov‐2 as well as the receptor‐binding protein (personal data). (b) IVIG impairs the generation of human monocyte‐derived anti‐inflammatory macrophages by inducing JNK activation and activin, limiting the production of inflammatory macrophage differentiation by inhibiting granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐driven signal transducer and activator of transcription 5 (STAT‐5) activation in vitro [48]. (c) IVIG can function through the Fc portion, thus contributing to the expansion of T regulatory cells, Fc receptor blocking, including the sialic acid content of the Fc portion of the antibodies, and the interaction with ITAM‐bearing Fc receptors [49, 50]. Of note, and as opposed to some common assumptions in the medical scientific community, IVIG was found to have fewer side effects compared to current biological treatments, which might cause severe side effects and loss of efficacy in the long term. Moreover, IVIG might be even cheaper than current expansive biological agents; therefore, it might change the financial burden of biological treatment in RA and other inflammatory/autoimmune diseases.

Conclusion

In summary, in the current study we demonstrate for the first time, to our knowledge, an amelioration of CIA in mice with established arthritis using a treatment protocol. We show herein a powerful anti‐inflammatory potential of IVIG in attenuating the pathogenesis in CIA in mice. The beneficial effect was presented by reducing the clinical score, reducing the titers of anti‐collagen type II antibodies and declining the concentrations of inflammatory circulating cytokines. Our study may pave the way for clinicians to reconsider the use of IVIG as a safe and beneficial optional treatment in a subgroup of patients with rheumatoid arthritis.

Disclosures

G. H., I. K., O. S., S. T., A. V., I. B., M. B. and H. A. declare that they have no conflicts of interest. S. T., K. K. G. and N. P. are employees of OOO NPF Materia Medica Holding. OOO NPF Materia Medica Holding sponsored the study and took part in the design of the experiments and the manuscript writing. OOO NPF Materia Medica Holding does not manufacture IVIG or have any other commercial interest in the results of the experiment. S Tarasov, N Petrova and KK Ganina are eployed by OOO NPF Materia Medica.

Author contributions

All authors were involved in drafting the article and all authors approved the current version of the manuscript. Study design: M. B., S. T,, N. P., H. A.; performed the experiments: G. H., M. B., I. K., O. S.; pathology analysis: A. V., I. B.; statistical analysis: I. K. manuscript writing: K. K. G., N. P., S. T., M. B., G. H, M. T., H. A. The data are available upon request.

Data Availability Statement

The data will be available upon request.