Many studies provided compelling evidence that extracellular vesicles (EVs) are involved in the regulation of the immune response, acting as both enhancers and dampeners of the immune system, depending on the source and type of vesicle. Research, including ours, has shown anti-inflammatory effects of milk-derived EVs, using human breast milk as well as bovine colostrum and store-bought pasteurized cow milk, in in vitro systems as well as therapeutically in animal models. Although it is not completely elucidated which proteins and miRNAs within the milk-derived EVs contribute to these immunosuppressive capacities, one proposed mechanism of action of the EVs is via the modulation of the crosstalk between the (intestinal) microbiome and their host health. There is increasing awareness that the gut plays an important role in many inflammatory diseases. Enhanced intestinal leakiness, dysbiosis of the gut microbiome, and bowel inflammation are not only associated with intestinal diseases like colitis and Crohn’s disease, but also characteristic for systemic inflammatory diseases such as lupus, multiple sclerosis, and rheumatoid arthritis (RA). Strategies to target the gut, and especially its microbiome, are under investigation and hold a promise as a therapeutic intervention for these diseases. The use of milk-derived EVs, either as stand-alone drug or as a drug carrier, is often suggested in recent years. Several research groups have studied the tolerance and safety of using milk-derived EVs in animal models. Due to its composition, milk-derived EVs are highly biocompatible and have limited immunogenicity even cross species. Furthermore, it has been demonstrated that milk-derived EVs, when taken up in the gastro-intestinal tract, stay intact after absorption, indicating excellent stability. These characteristics make milk-derived EVs very suitable as drug carriers, but also by themselves, these EVs already have a substantial immunoregulatory function, and even without loading, these vesicles can act as therapeutics. In this review, we will address the immunomodulating capacity of milk-derived EVs and discuss their potential as therapy for RA patients.Review criteriaThe search terms “extracellular vesicles”, “exosomes”, “microvesicles”, “rheumatoid arthritis”, “gut-joint axis”, “milk”, and “experimental arthritis” were used. English-language full text papers (published between 1980 and 2021) were identified from PubMed and Google Scholar databases. The reference list for each paper was further searched to identify additional relevant articles.

Scope: Data from the Osteoarthritis Initiative shows that females who drink milk regularly have less joint cartilage loss and OA progression, but the biologic mechanism is unclear. Bovine milk is a rich source of extracellular vesicles (EVs), which are small phospholipid bilayer bound structures that facilitate intercellular communication. In this study, the authors aim to evaluate whether these EVs may have the capacity to protect cartilage from osteoarthritis patients, ex vivo, by directly effecting chondrocytes. Methods and Results: Human cartilage explants are exposed to cow's milk-derived EVs (CMEVs), which results in reduced sulfated glycosaminoglycan release and inhibition of metalloproteinase-1 expression. Incubation of articular chondrocytes with CMEVs also effectively reduces expression of cartilage destructive enzymes (ADAMTS5, MMPs), which play key roles in the disease progression. In part, these findings are attributed to the presence of TGF𝜷 on these vesicles, and in addition, a possible role is reserved for miR-148a, which is functionally transferred by CMEVs. Conclusion: These findings highlight the therapeutic potential of local CMEV delivery in osteoarthritic joints, where inflammatory and catabolic mediators are responsible for joint pathology. CMEVs are carriers of both TGF𝜷 and miR-148a, two essential regulators for maintaining chondrocyte homeostasis and protection against cartilage destruction.

Introduction: The pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFβ signaling. TGFβ is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFβ in serum of SSc patients and investigated if this correlates with integrin expression on monocytes.Methods: A TGFβ/SMAD3-or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFβ and BMP levels respectively. A pan-specific TGFβ1/2/3 neutralizing antibody was used to confirm TGFβ signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFβ1 or a small molecule inhibitor of TGFBR1: SB-505124.Results: SSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFβ levels were not different. Addition of a pan-specific TGFβ1/2/3 neutralizing antibody fully inhibited SMAD3reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3, ITGB5, and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFβ1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFβ receptor inhibitor SB-505124 had the opposite effect.Conclusion: Total TGFβ serum levels are not different between SSc patients and controls, but TGFβ activity is. This coincides with a reduced expression of TGFβ-activating integrins in monocytes of SSc patients. Because TGFβ regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.

TGF-β1 is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β1 as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β1 inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. Murine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. Flow cytometry analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction. SB-505124 potently reduced murine Th17 differentiation by decreasing Il17a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 numbers, while increased numbers of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction. Blocking TGF-β1 signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, local SB-505124 treatment does not suppress experimental arthritis.

Objective High levels of IL-22 are present in serum and synovial fluid of patients with RA. As both pro- and anti-inflammatory roles for IL-22 have been described in studies using animal models of RA, its exact function in arthritis remains poorly defined. With this study we aimed to further unravel the mechanism by which IL-22 exerts its effects and to decipher its therapeutic potential by overexpression of IL-22 either locally or systemically during experimental arthritis. Methods CIA was induced in DBA-1 mice by immunization and booster injection with type II collagen (col II). Before arthritis onset, IL-22 was overexpressed either locally by intra-articular injection or systemically by i.v. injection using an adenoviral vector and clinical arthritis was scored for a period of 10 days. Subsequently, joints were isolated for histological analysis of arthritis severity and mRNA and protein expression of various inflammatory mediators was determined in the synovium, spleen and serum. Results Local IL-22 overexpression did not alter arthritis pathology, whereas systemic overexpression of IL-22 potently reduced disease incidence, severity and pathology during CIA. Mice systemically overexpressing IL-22 showed strongly reduced serum cytokine levels of TNF-α and macrophage inflammatory protein 1α that correlated significantly with the enhanced expression of the negative immune regulator SOCS3 in the spleen. Conclusion With this study, we revealed clear anti-inflammatory effects of systemic IL-22 overexpression during CIA. Additionally, we are the first to show that the protective effect of systemic IL-22 during experimental arthritis is likely orchestrated via upregulation of the negative regulator SOCS3.

Background:TGFβ is an important growth factor that promotes the differentiation of T helper 17 (Th17) as well as regulatory T-cells (Treg). Due to its dual role, the potential of TGFβ as therapeutic target is unclear.Objectives:In this study we aimed to investigate the effect of inhibition of TGFβ signaling with the ALK5 inhibitor SB-505124 on human Th17 differentiationin vitro, on cytokine production by human rheumatoid arthritis (RA) synovial explants, and study the effects of local SB-505124 treatmentin vivoduring innate immune and Th17-driven experimental arthritis models.Methods:Magnetic sorted naïve human T cells were differentiated into Th17 cells with CD3/CD28 activation beads, IL-2, TGFβ, IL-1β, IL-23, αIFNƳ and αIL-4 for 6 days. Human RA synovial biopsies were cultured for 24h w/o 5µM SB-505124, and supernatant was analyzed by Luminex. T cell-independent SCW arthritis and Th17-driven IL-1/mBSA arthritis were induced in C57Bl6, and mice were treated with SB-505124 by daily intra-articular injections from day 0-4.Results:SB-505124 potently reduced human Th17 differentiationin vitroby decreasing IL-17 and RORƳt gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 and TNFα protein production by human RA synovial explants. In addition, SB-505124 did not affect acute joint inflammation during SCW-arthritis (T-cell independent model). Interestingly, SB-505124 reduced Th17 levels in draining lymph nodes (dLN) during IL-1/mBSA arthritis while increased levels of Tregs were observed. Surprisingly, despite this skewed Th17/Treg balance, this did not result in suppression of joint inflammation and destruction in this Th17-driven arthritis model, whereas anti-IL-17 antibody treatment showed significant therapeutic effects.Conclusion:We revealed suppressive effects of SB-505124 on human Th17 differentiation and the Th17/Treg balance in arthritic mice. However, SB-505124 did not suppress joint inflammation and destruction. This indicates that despite the importance of TGFβ in Th17 differentiation, targeting TGFβ signaling is not enough to suppress experimental arthritis.Disclosure of Interests:None declared

Background:High interleukin-22 (IL-22) levels are detected in serum and synovial fluid of rheumatoid arthritis (RA) patients. The increased IL-22 serum levels in RA patients correlated positively with multiple clinical disease parameters like disease activity score (DAS)28 and serum levels of rheumatoid factor. The role of IL-22 in autoimmunity and inflammation appears to be greatly contradictory, being both pro- and anti-inflammatory. Especially the anti-inflammatory properties of IL-22 are not well understood.Objectives:We aimed to investigate the anti-inflammatory and immune-suppressive effect of IL-22 during experimental arthritis.Methods:Collagen-induced arthritis was induced in DBA1 mice by immunization and booster with bovine collagen type II (CII). After booster, but before arthritis onset, IL-22 was overexpressed either locally or systemically using an adenoviral construct (AdIL-22) or Luciferase as control (AdLuc). 1x107 plaque-forming units (PFU) of the adenoviruses were injected intra-articularly for local overexpression, or 3x108 PFU was injected intravenously for systemic overexpression in immunized mice, and mice were sacrificed 10 days later. Macroscopic scoring and histological analysis was performed, and mRNA expression and protein production of various pro- and anti-inflammatory mediators was determined in synovial tissue, spleen, and serum.Results:Local overexpression of IL-22 by injection of AdIL-22 in the knee joint of CII-immunized mice resulted in an unaltered arthritis incidence and severity as compared to the control virus AdLuc. Accordingly, no changes in mRNA expression or protein production were observed in CIA mice locally overexpressing IL-22. In contrast, systemic overexpression of IL-22 potently reduced disease incidence and severity, which was also confirmed by histological analysis. Systemic levels of IL-1β, IL-17, GM-CSF and MCP1 were unaltered in mice overexpressing IL-22 systemically. However, these mice showed significantly lower serum levels of IFNγ, TNFα, MIP1α, and IL-10. Interestingly, the significantly enhanced splenic SOCS3 expression was found to negatively correlate to serum TNFα and MIP1α levels, which is in line with our hypothesis that that the observed reduction in the cytokine levels is mediated in a SOCS3-dependent manner.Conclusion:With this study, we revealed clear anti-inflammatory effects of IL-22 overexpression during collagen-induced arthritis, which are completely dependent on the systemic route of administration. Additionally, we were the first to show that this protective effect of IL-22 during experimental arthritis is likely orchestrated via up-regulation of the negative regulator SOCS3.Disclosure of Interests:None declared

ObjectivesTGF-β is an important growth factor to promote the differentiation of T helper 17 (Th17) as well as regulatory T cells (Treg). Due to its dual role, the potential of TGF-β as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. In this study, we investigated the effect of TGF-β inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. MethodsMurine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 on Th17 differentiation was studied. RA synovial biopsies were cultured for 24h in the presence or absence of SB-505124. Experimental arthritis models were induced in C57Bl6 mice, and were treated daily with SB-505124. FACS analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction.ResultsSB-505124 potently reduced murine Th17 differentiation by decreasing Il7a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by RA synovial explants. In the Th17-driven arthritis model, SB-505124 reduced Th17 levels, while increased levels of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction in this model.ConclusionsBlocking TGF-β signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, SB-505124 treatment does not suppress experimental arthritis, and is therefore not an adequate way to target Th17-driven inflammation.