Effects of the novel immunosuppressant FTY720 in a murine rheumatoid arthritis model

We investigated the effects and mechanisms by which FTY720 (FTY) inhibits arthritis development in the SKG mouse rheumatoid arthritis (RA) model. FTY (1 mg/kg/day) administration suppressed the progression of laminarin-induced arthritis in SKG mice. FTY treatment decreased IL-6 and TNF-α expression in synovial fibroblast cells and the number of inflammatory cells overall. Bone destruction was also suppressed by treatment with FTY. The numbers of CD4⁺ and CD8⁺ T cells were significantly increased in the thymus and decreased in the spleen in FTY-treated SKG mice. FTY enhanced IL-4 production by CD4⁺ T cells stimulated by allogeneic spleen cells and inhibited prostaglandin E₂ (PGE₂) production by a TNF-α-stimulated synovial fibroblast cell line. These results indicate that FTY can inhibit arthritis in SKG mice via sequestration of autoimmune CD4⁺ T cells in the thymus, enhancement of Th2 immune responses, and inhibition of PGE₂ production by synovial cells.     

Autoimmune arthritis induces paired immunoglobulin‐like receptor B expression on CD4+ T cells from SKG mice

The chronic, destructive autoimmune arthritis in SKG mice, which closely resembles human rheumatoid arthritis, is the result of self‐reactive T cells escaping thymic deletion. Since the inhibitory receptor LIR‐1 is up‐regulated on auto‐reactive T cells in human rheumatoid arthritis, the role of its murine ortholog PIR‐B was investigated. Peripheral CD4⁺ T cells from SKG mice were found to frequently express PIR‐B, and this population produces more frequently IL‐17 upon in vitro stimulation compared to PIR‐B^? cells. A much larger fraction of PIR‐B⁺ T cells, however, was found to secret no IL‐17, but IFN‐γ. With regards to the clinical course of the disease, high frequencies of PIR‐B⁺ CD4⁺ T cells were found to be associated with a milder course of arthritis, suggesting that the net effect of PIR‐B expression is suppression of autoreactive T cells. Our results indicate that overexpression of PIR‐B on IL‐17‐producing SKG CD4⁺ T cells might represent an effective counter‐regulatory mechanism against the destructive potential of those cells. More importantly, a major population of PIR‐B⁺ T cells in SKG mice appears to play an inhibitory role by way of their IFN‐γ production, since high frequencies of those cells ameliorate the disease.     

CD8α⁺ dendritic cells improve collagen-induced arthritis in CC chemokine receptor (CCR)-2 deficient mice

Objective

Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3⁺ regulatory T cells (T_reg), which in turn exert immunosuppression by increasing TGFβ-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient (Ccr2^−/−) mice had impaired DCs migration and reduced CD8α⁺ DCs in the C57Bl/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2^−/− mice, we tested the hypothesis that CD8α⁺ DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8α⁺ DCs in Ccr2^−/− and SKG mice.

Methods

To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant fms-like tyrosine kinase 3 ligand (Flt3L) injections to expand endogenous DCs populations or adoptive transfers of CD8α⁺ DCs.

Results

Flt3L-mediated expansion of endogenous CD8α⁺ DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8α⁺ DCs ameliorated arthritis in Ccr2^−/− mice and enhanced TGFβ1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype.

Conclusion

CD8α⁺ DCs were tolerogenic to the development of arthritis. CD8α⁺ DCs deficiency heightened the sensitivity to arthritis in Ccr2^−/− mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2^−/− mice was T cell-independent.     

Treatment and transfer of emphysema by a new bone marrow transplantation method from normal mice to Tsk mice and vice versa

We have recently established a new bone marrow transplantation (BMT) method in which bone marrow cells are injected into the intrabone marrow (IBM). In the present study, we used an animal model for emphysema (tight-skin [Tsk] mouse) to examine whether IBM-BMT could be used to treat emphysema in Tsk mice. IBM-BMT was carried out from C3H mice into Tsk mice (8-10 weeks old) that had already shown emphysema. Six months after transplantation, the lungs of all the Tsk mice treated with IBM-BMT [C3H-->Tsk] showed similar structures to those of normal mice, whereas the [Tsk-->Tsk] mice showed emphysema, as seen in age-matched Tsk mice. Next, we attempted to transfer emphysema from Tsk mice to C3H mice by IBM-BMT. Six months after IBM-BMT, the [Tsk-->C3H] mice showed emphysema. These results strongly suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow.     

Facilitation of hematopoietic recovery by bone grafts with intra-bone marrow-bone marrow transplantation

We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow-bone marrow transplantation: IBM-BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2-3 days old) donor mice is also effective in the IBM-BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM-BMT+bone grafts, but not in the mice that had received IBM-BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM-BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.     

Prevention of graft-versus-host disease by intrabone marrow injection of donor T cells: involvement of bone marrow stromal cells

We have developed a new and effective method for bone marrow transplantation (BMT): bone marrow cells (BMCs) are injected directly into the bone marrow (BM) cavity of recipient mice. The intrabone marrow injection of BMCs (IBM-BMT) greatly facilitates the engraftment of donor-derived cells, and IBM-BMT can attenuate graft-versus-host reaction (GVHR), in contrast to conventional intravenous BMT (i.v.-BMT). Here, we examine the mechanisms underlying the inhibitory effects of IBM-BMT on GVHR using animal models where GVHR is elicited. Recipient mice (C57BL/6) were irradiated and splenic T cells (as donor lymphocyte infusion: DLI) from major histocompatibility complex-disparate donors (BALB/c) were injected directly into the BM cavity (IBM-DLI) or injected intravenously (i.v.-DLI) along with IBM-BMT. The BM stromal cells (BMSCs) from these recipients were collected and related cytokines were examined. The recipient mice that had been treated with IBM-BMT + i.v.-DLI showed severe graft-versus-host disease (GVHD), in contrast to those treated with IBM-BMT + IBM-DLI. The suppressive activity of BMSCs in this GVHD model was determined. The cultured BMSCs from the recipients treated with IBM-BMT + IBM-DLI suppressed the proliferation of responder T cells remarkably when compared with those from the recipients of IBM-BMT + i.v.-DLI in mixed leucocyte reaction. Furthermore, the level of transforming growth factor-beta and hepatocyte growth factor in cultured BMSCs from IBM-BMT + IBM-DLI increased significantly when compared with those from the recipients of IBM-BMT + i.v.-DLI. Thus, the prevention of GVHD observed in the recipients of IBM-BMT + IBM-DLI was attributable to the increased production of immunosuppressive cytokines from BMSCs after interaction with host reactive T cells (in DLI).     

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra-bone-marrow (IBM) route (IBM-BMT) over the intravenous route (IV-BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM-BMT or IV-BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor-derived cells in total and of c-kit(+) cells were observed at 2 through 6 days after IBM-BMT. In parallel, significantly higher numbers of colony-forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM-BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM-BMT over IV-BMT. Disclosure of potential conflicts of interest is found at the end of this article.     

Prevention of senile osteoporosis in SAMP6 mice by intrabone marrow injection of allogeneic bone marrow cells

The SAMP6 mouse (a substrain of senescence-accelerated mice) spontaneously develops osteoporosis early in life and is, therefore, a useful model for examining the mechanisms underlying osteoporosis. We have recently established a new bone marrow transplantation (BMT) method: the bone marrow cells (BMCs) of normal allogeneic mice are directly injected into the bone marrow (BM) cavity of irradiated (5.5 Gy x 2) recipients (IBM-BMT). Using IBM-BMT, we attempted to prevent osteoporosis in SAMP6 mice. The hematolymphoid system was completely reconstituted with donor-type cells after IBM-BMT. Thus-treated SAMP6 mice showed marked increases in trabecular bones even at 12 months of age, and the bone mineral density remained similar to that of normal B6 mice. In concordance with these findings, urinary deoxypyridinoline also remained continuously low until 10 months of age, indicating that IBM-BMT was effective in the prevention of bone absorption. In addition to the above, BM stromal cells in the treated SAMP6 mice were replaced with donor stromal cells, and the message level of interleukin-11 (IL-11), which is produced by the BM stromal cells and is known as an important factor in the regulation of bone remodeling, was restored to a level similar to that observed in normal B6 mice. Furthermore, the message level of IL-6, which is known to enhance osteoclastogenesis, was also restored to normal. These results indicate that the BM microenvironment was normalized after IBM-BMT and that the increased production of IL-11 and IL-6 ameliorated the imbalance between bone absorption and formation, resulting in the prevention of osteoporosis in SAMP6 mice.     

Amelioration of cognitive ability in senescence-accelerated mouse prone 8 (SAMP8) by intra-bone marrow-bone marrow transplantation

Bone marrow cells (BMCs) can increase the number of activated microglias, which play a central role in the inflammatory response in Alzheimer's disease (AD). Senescence-accelerated mouse (SAM) prone 8 (SAMP8) are widely used in various experiments because of cognitive deficits observed with age. In the present study, 4-month-old SAMP8 were reconstituted with BMCs of C57BL/6 mice by intra-bone marrow-bone marrow transplantation (IBM-BMT), which can reconstitute both donor-derived hemopoietic stem cells and mesenchymal stem cells. Three months after IBM-BMT, the impairment of spatial memory in SAMP8 was found to be ameliorated after analyzing the results of the water maze test. Although IL-1β, IL-6 and iNOS increased and TGF-β decreased in 7 M SAMP8, IL-1β, IL-6 and iNOS decreased while TGF-β increased after IBM-BMT by RT-PCR. Moreover, oxidative stress-related heme oxygenase-1 (HO-1) increased in 7 M SAMP8, but significantly decreased after IBM-BMT. In conclusion, this is the first report suggesting that the impaired cognitive ability of SAMP8 is ameliorated by IBM-BMT. It seems likely that decreases in IL-1β, IL-6, iNOS and HO-1 are a result of the development of donor-derived BMCs.     

Enhancement of allogeneic hematopoietic stem cell engraftment and prevention of GVHD by intra-bone marrow bone marrow transplantation plus donor lymphocyte infusion

We examined the effect of intra-bone marrow (IBM)-bone marrow transplantation (BMT) in conjunction with donor lymphocyte infusion (DLI) on the engraftment of allogeneic bone marrow cells (BMCs) in mice. Recipients that had received 6 Gy of radiation completely rejected donor BMCs, even when IBM-BMT was carried out. However, when BMCs were IBM injected and donor peripheral blood mononuclear cells (PBMNCs) were simultaneously injected intravenously (DLI), donor cell engraftment was observed 7 days after BMT and complete donor chimerism continued thereafter. It is of interest that the cells of recipient origin did not recover, and that the hematolymphoid cells, including progenitor cells (Lin-/c-kit+ cells) in the recipients, were fully reconstituted with cells of donor origin. The cells in the PBMNCs responsible for the donor BMC engraftment were CD8+. Recipients that had received 6 Gy of radiation, IBM-BMT, and DLI showed only a slight loss of body weight, due to radiation side effects, and had no macroscopic or microscopic symptoms of graft-versus-host disease. These findings suggest that IBM-BMT in conjunction with DLI will be a valuable strategy for allogeneic BMT in humans.     

Intra-bone marrow-bone marrow transplantation facilitates hemopoietic recovery including dendritic cells

In this report, we provide evidence using a serial bone marrow transplantation (BMT) protocol that intra-bone marrow (IBM)-BMT (IBM-BMT) can efficiently reconstitute the hemopoietic system with cells of donor origin, in contrast to conventional intravenous (IV)-BMT (IV-BMT). Furthermore, the hematolymphoid system of secondary recipients that had received bone marrow cells (BMCs) from primary recipients treated with IBM-BMT recovered earlier than that of the secondary recipients of BMCs from primary recipients treated with IV-BMT. This was the case when the Lin-/c-kit+ progenitor cells of the secondary and tertiary recipients were examined. These findings indicate that IBM-BMT can facilitate the development of not only cells of various lineages but also the effective generation and, more importantly, the maintenance of the progenitor cells. Furthermore, we show that IBM-BMT can reconstitute the dendritic cell (DC) subsets (myeloid and lymphoid DCs), which are critical for the initiation of both adaptive and innate immune responses. The frequency of both myeloid and lymphoid DC subsets was approximately equal to that of normal age-matched untreated controls and, after second and third BMT, this ratio was close to that observed in the normal controls. However, the lymphoid DCs were clearly reduced in the secondary and tertiary recipients of BMCs from mice that had received IV-BMT. Therefore, the development of DC subsets is also normally maintained in the IBM-BMT group.     

Donor bone marrow cells are essential for iNKT cell‐mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance

Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti‐CD40 ligand (anti‐CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub‐lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3‐positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)‐γ‐producing CD8⁺T cells, expand host Ki67⁺CD4⁺CD25⁺Foxp3⁺ Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC‐recipients accepted skin grafts in an allo‐specific manner. Myeloid‐derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.     

A new strategy for treatment of malignant tumor: intra-bone marrow-bone marrow transplantation plus CD4- donor lymphocyte infusion

Donor lymphocyte infusion (DLI) is clinically used for the treatment of malignant tumors. We have found recently that intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to treat various autoimmune diseases, even when radiation doses are reduced. In addition, recently we have found that IBM-BMT can prevent not only graft failure but also graft-versus-host disease (GvHD). Based on these findings, we attempted to prevent and treat the progression of a tumor (Meth-A cell line: BALB/c-derived fibrosarcoma) by DLI plus IBM-BMT. When the tumors had grown to approximately 10 x 10 mm, the tumor-bearing BALB/c (H-2(d)) mice were irradiated with 5 Gy, and whole spleen cells from C57BL/6J (B6) (H-2(b)) mice (as DLI) were then intravenously injected into the BALB/c mice. Simultaneously, bone marrow cells (BMCs) from B6 mice were injected directly into the bone marrow cavity of the BALB/c mice (IBM-BMT). The tumors decreased in size, but the mice died of GvHD. However, when CD4(+) T-cell-depleted spleen cells were used for DLI, the recipients showed only mild GvHD and survived longer, due to the slow growth of the tumor. In contrast, when CD8(+) T-cell-depleted spleen cells were used for DLI, the recipients showed more severe GvHD than those injected with whole spleen cells. These results suggest that IBM-BMT plus DLI (the depletion or reduction of a certain cell population like CD4(+) T cells) could be helpful to suppress both GvHD and tumor growth.     

Intra-bone marrow injection of donor bone marrow cells suspended in collagen gel retains injected cells in bone marrow, resulting in rapid hemopoietic recovery in mice

We have recently developed an innovative bone marrow transplantation (BMT) method, intra-bone marrow (IBM)-BMT, in which donor bone marrow cells (BMCs) are injected directly into the recipient bone marrow (BM), resulting in the rapid recovery of donor hemopoiesis and permitting a reduction in radiation doses as a pretreatment for BMT. However, even with this IBM injection, some of the injected BMCs were found to enter into circulation. Therefore, we attempted to modify the method to allow the efficient retention of injected BMCs in the donor BM. The BMCs of enhanced green fluorescent protein transgenic mice (C57BL/6 background) were suspended in collagen gel (CG) or phosphate-buffered saline (PBS), and these cells were then injected into the BM of irradiated C57BL/6 mice. The numbers of retained donor cells in the injected BM, the day 12 colony-forming units of spleen (CFU-S) counts, and the reconstitution of donor cells after IBM-BMT were compared between the CG and PBS groups. The number of transplanted cells detected in the injected BM in the CG group was significantly higher than that in the PBS group. We next carried out CFU-S assays. The spleens of mice in the CG group showed heavier spleen weight and considerably higher CFU-S counts than in the PBS group. Excellent reconstitution of donor hemopoietic cells in the CG group was observed in the long term (>100 days). These results suggest that the IBM injection of BMCs suspended in CG is superior to the injection of BMCs suspended in PBS.     

Stem cell transplantation for autoimmune diseases: what can we learn from experimental models?

Using animal models for autoimmune diseases, we have previously shown that allogeneic bone marrow transplantation (allo BMT) can be used to treat autoimmune diseases. Using cynomolgus monkeys, we have recently developed new BMT methods for the treatment of autoimmune diseases. The methods include the perfusion method (PM) for the collection of bone marrow cells (BMCs), and intra-bone marrow (IBM)-BMT for the direct injection of collected whole BMCs into the bone marrow cavity. The PM, in comparison with the conventional aspiration method, can minimize the contamination of BMCs with T cells from the peripheral blood. Therefore, without removing T cells, no graft-versus-host disease (GvHD) develops in the case of the PM. Since BMCs collected by the PM contain not only hemopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs), the injection of both cells directly into the bone marrow cavity (IBM-BMT) facilitates the engraftment of donor hemopoietic cells. In organ allografts with IBM-BMT, no graft failure occurs even if the radiation dose is reduced. In addition, IBM-BMT is applicable to regeneration therapy and various age-associated diseases such as osteoporosis, since it can efficiently recruit donor-derived normal MSCs. We have also found that IBM-BMT in conjunction with donor lymphocyte infusion can prevent GvHD, but suppress tumor growth. We believe that this strategy heralds a revolution in the field of transplantation (BMT and organ allografts) and regeneration therapy.     

Effects of intrabone marrow-bone marrow transplantation plus adult thymus transplantation on survival of mice bearing leukemia

We recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) plus adult thymus transplantation (ATT) from the same donor is effective in mice bearing solid tumors. In the current study, we examined the effects of this strategy on the survival of mice with leukemia. One week after intravenous injection of 1×10(6) leukemic cells (EL-4, H-2(b)) into 8-week-old B6 (H-2(b)) mice, the mice were 8 Gy irradiated and transplanted with 1×10(7) bone marrow cells (BMCs) from 8-week-old BALB/c mice (H-2(d)) by IBM-BMT with or without donor lymphocyte infusion (DLI) or ATT. All the mice without treatment died within 70 days after injection of EL-4. About 40% of those treated with IBM-BMT alone died within 100 days due to tumor relapse. In contrast, those treated with IBM-BMT+DLI or ATT showed the longest survival rate without relapse of leukemia. In addition, the former showed less graft versus host disease (GVHD) than the latter. The mice treated with IBM-BMT+ATT also showed an intermediate percentage of effector memory (EM) and central memory (CM) cells between those treated with BMT alone and those treated with IBM-BMT+DLI. The numbers and functions of T cells increased in those treated with IBM-BMT+ATT with interleukin-2 and interferon-γ production. These results suggest that IBM-BMT+ATT is effective in the treatment of leukemia with strong graft versus leukemia without increased risk of GVHD.     

Treatment of senile osteoporosis in SAMP6 mice by intra-bone marrow injection of allogeneic bone marrow cells

A substrain of the senescence-accelerated mouse, SAMP6 (senescence-accelerated mouse prone 6), spontaneously develops osteoporosis early in life. Therefore, this strain is a useful animal model for developing new strategies for the treatment of osteoporosis in humans. We succeeded in treating osteoporosis in SAMP6 mice after the onset of this disease, using a newly developed method of bone marrow transplantation (BMT): Allogeneic bone marrow cells obtained from normal mouse strains were directly injected into the bone marrow cavity of irradiated SAMP6 mice (intra-bone marrow BMT [IBM-BMT]). After the treatment with IBM-BMT, hematolymphoid cells were completely reconstituted by donor-derived cells, and bone marrow stromal cells were also found to be of donor origin. The treated SAMP6 mice showed histologically-normal trabecular bone. In addition, bone mineral density and urinary deoxypiridinoline, a hallmark of bone destruction, were normalized. When the message levels of cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], IL-11, and receptor activator of nuclear factor-kappa B ligand [RANKL]) were examined, IL-11, RANKL (from bone marrow stromal cells), and IL-6 (from osteoclasts), which regulate bone remodeling, were restored to levels similar to those in normal B6 mice. These findings indicate that not only the hemopoietic system but also the bone marrow microenvironment were normalized after IBM-BMT, resulting in an amelioration of the imbalance between bone absorption and formation.     

Porphyromonas gingivalis infection exacerbates the onset of rheumatoid arthritis in SKG mice

Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti‐cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase‐3 (MMP‐3) and cytokine level in serum by enzyme‐linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP‐3, interleukin (IL)‐2, IL‐6, CXCL1 and macrophage inflammatory protein (MIP)‐1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.