Targeting of tolerogenic dendritic cells to heat-shock proteins in inflammatory arthritis.

BACKGROUND: Autologous tolerogenic dendritic cells (tolDC) are a promising therapeutic strategy for inflammatory arthritis (IA) as they can regulate autoantigen-specific T cell responses. Here, we investigated two outstanding priorities for clinical development: (i) the suitability of using heat-shock proteins (HSP), abundant in inflamed synovia, as surrogate autoantigens to be presented by tolDC and (ii) identification of functional biomarkers that confirm tolDC regulatory activity. METHODS: Cell proliferation dye-labelled human peripheral blood mononuclear cells of IA (rheumatoid arthritis (RA) and psoriatic arthritis (PsA)) patients or healthy donors were cultured with HSP40-, HSP60- and HSP70-derived peptides or recall antigens (e.g. tuberculin purified protein derivative (PPD)) in the presence or absence of tolDC or control DC for 9 days. Functional characteristics of proliferated antigen-specific T-cells were measured using flow cytometry, gene expression profiling and cytokine secretion immunoassays. Repeated measures analysis of variance (ANOVA) with Bonferroni correction for comparisons between multiple groups and paired Student t test for comparisons between two groups were used to determine significance. RESULTS: All groups showed robust CD4+ T-cell responses towards one or more HSP-derived peptide(s) as assessed by a stimulation index > 2 (healthy donors: 78%, RA: 73%, PsA: 90%) and production of the cytokines IFNγ, IL-17A and GM-CSF. Addition of tolDC but not control DC induced a type 1 regulatory (Tr1) phenotype in the antigen-specific CD4+ T-cell population, as identified by high expression of LAG3, CD49b and secretion of IL-10. Furthermore, tolDC inhibited bystander natural killer (NK) cell activation in a TGFß dependent manner. CONCLUSIONS: HSP-specific CD4+ T-cells are detectable in the majority of RA and PsA patients and can be converted into Tr1 cells by tolDC. HSP-loaded tolDC may therefore be suitable for directing T regulatory responses to antigens in inflamed synovia of IA patients. Tr1 markers LAG3, CD49b and IL-10 are suitable biomarkers for future tolDC clinical trials.

Matured Tolerogenic Dendritic Cells Effectively Inhibit Autoantigen Specific CD4+ T Cells in a Murine Arthritis Model.

Tolerogenic dendritic cells (tolDCs) are a promising treatment modality for diseases caused by a breach in immune tolerance, such as rheumatoid arthritis. Current medication for these diseases is directed toward symptom suppression but no real cure is available yet. TolDC-based therapy aims to restore immune tolerance in an antigen-specific manner. Here we used a mouse model to address two major questions: (i) is a maturation stimulus needed for tolDC function in vitro and in vivo and is maturation required for functioning in experimental arthritis and (ii) can tolDCs modulate CD4+ T cell responses? To answer these questions, we compared matured and immature dexamethasone/vitamin D3-generated tolDCs in vitro. Subsequently, we co-transferred these tolDCs with naïve or effector CD4+ T cells to study the characteristics of transferred T cells after 3 days with flow cytometry and Luminex multiplex assays. In addition, we tested the suppressive capabilities of tolDCs in an experimental arthritis model. We found that tolDCs cannot only modulate naïve CD4+ T cell responses as shown by fewer proliferated and activated CD4+ T cells in vivo, but also effector CD4+ T cells. In addition, Treg (CD4+CD25+FoxP3+) expansions were seen in the proliferating cell population in the presence of tolDCs. Furthermore, we show that administered tolDCs are capable to inhibit arthritis in the proteoglycan-induced arthritis model. However, a maturation stimulus is needed for tolDCs to manifest this tolerizing function in an inflammatory environment. Our data will be instrumental for optimization of future tolDC therapies for autoimmune diseases.

Macrophage proliferation distinguishes 2 subgroups of knee osteoarthritis patients.

Osteoarthritis (OA) is a leading cause of disability, globally. Despite an emerging role for synovial inflammation in OA pathogenesis, attempts to target inflammation therapeutically have had limited success. A better understanding of the cellular and molecular processes occurring in the OA synovium is needed to develop novel therapeutics. We investigated macrophage phenotype and gene expression in synovial tissue of OA and inflammatory-arthritis (IA) patients. Compared with IA, OA synovial tissue contained higher but variable proportions of macrophages (P < 0.001). These macrophages exhibited an activated phenotype, expressing folate receptor-2 and CD86, and displayed high phagocytic capacity. RNA sequencing of synovial macrophages revealed 2 OA subgroups. Inflammatory-like OA (iOA) macrophages are closely aligned to IA macrophages and are characterized by a cell proliferation signature. In contrast, classical OA (cOA) macrophages display cartilage remodeling features. Supporting these findings, when compared with cOA, iOA synovial tissue contained higher proportions of macrophages (P < 0.01), expressing higher levels of the proliferation marker Ki67 (P < 0.01). These data provide new insight into the heterogeneity of OA synovial tissue and suggest distinct roles of macrophages in pathogenesis. Our findings could lead to the stratification of OA patients for suitable disease-modifying treatments and the identification of novel therapeutic targets.

Targeting of tolerogenic dendritic cells towards heat-shock proteins: a novel therapeutic strategy for autoimmune diseases?

Tolerogenic dendritic cells (tolDCs) are a promising therapeutic tool to restore immune tolerance in autoimmune diseases. The rationale of using tolDCs is that they can specifically target the pathogenic T-cell response while leaving other, protective, T-cell responses intact. Several ways of generating therapeutic tolDCs have been described, but whether these tolDCs should be loaded with autoantigen(s), and if so, with which autoantigen(s), remains unclear. Autoimmune diseases, such as rheumatoid arthritis, are not commonly defined by a single, universal, autoantigen. A possible solution is to use surrogate autoantigens for loading of tolDCs. We propose that heat-shock proteins may be a relevant surrogate antigen, as they are evolutionarily conserved between species, ubiquitously expressed in inflamed tissues and have been shown to induce regulatory T cells, ameliorating disease in various arthritis mouse models. In this review, we provide an overview on how immune tolerance may be restored by tolDCs, the problem of selecting relevant autoantigens for loading of tolDCs, and why heat-shock proteins could be used as surrogate autoantigens.

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

Minimum information about tolerogenic antigen-presenting cells (MITAP): a first step towards reproducibility and standardisation of cellular therapies.

Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application.

DEC205+ Dendritic Cell-Targeted Tolerogenic Vaccination Promotes Immune Tolerance in Experimental Autoimmune Arthritis.

Previous studies in mouse models of autoimmune diabetes and encephalomyelitis have indicated that the selective delivery of self-antigen to the endocytic receptor DEC205 on steady-state dendritic cells (DCs) may represent a suitable approach to induce Ag-specific immune tolerance. In this study, we aimed to examine whether DEC205(+) DC targeting of a single immunodominant peptide derived from human cartilage proteoglycan (PG) can promote immune tolerance in PG-induced arthritis (PGIA). Besides disease induction by immunization with whole PG protein with a high degree of antigenic complexity, PGIA substantially differs from previously studied autoimmune models not only in the target tissue of autoimmune destruction but also in the nature of pathogenic immune effector cells. Our results show that DEC205(+) DC targeting of the PG peptide 70-84 is sufficient to efficiently protect against PGIA development. Complementary mechanistic studies support a model in which DEC205(+) DC targeting leads to insufficient germinal center B cell support by PG-specific follicular helper T cells. Consequently, impaired germinal center formation results in lower Ab titers, severely compromising the development of PGIA. Overall, this study further corroborates the potential of prospective tolerogenic DEC205(+) DC vaccination to interfere with autoimmune diseases, such as rheumatoid arthritis.

The immunostimulatory effect of CpG oligodeoxynucleotides on peripheral blood mononuclear cells of healthy dogs and dogs with atopic dermatitis.

Synthetic oligodeoxynucleotides containing cytosine phosphatidyl guanine-rich DNA sequences (CpG ODN) can promote T-helper type 1 (Th1) responses, reduce T-helper type 2 (Th2) responses and/or favour regulatory T cell (Treg) responses in vitro and in vivo in humans and animals, by acting via Toll-like receptor 9 (TLR9). Since CpG ODN can be used as immune-modulators for canine atopic dermatitis (AD), the aim of the current study was to investigate their immunostimulatory potential on peripheral blood mononuclear cells (PBMC) and their subsets, from AD and healthy dogs. Expression of TLR9 and cytokine mRNA in CpG ODN-stimulated and unstimulated cells was assessed by real-time quantitative PCR. Stimulation of PBMC with CpG class C ODN upregulated mRNA expression of interleukin (IL)-6, interferon (IFN)-γ and IL-12p40 in AD dogs (P<0.05). It also stimulated IFN-γ protein secretion by PBMC of atopic and healthy dogs as measured by ELISA. In healthy dogs only, CpG class C ODN stimulated IFN-α mRNA production by CD21(+) cells, and IL-10, IL-13 and IFN-γ mRNA production by CD3(+) cells. Increased expression of TLR9 mRNA was only observed in CD3(+) cells from AD dogs. No significantly increased gene expression was found in the CD11c(+) subset upon stimulation, for those genes evaluated. The results indicate that PBMC of healthy and atopic dogs are sensitive to stimulation with CpG ODN class C, with a resulting Th1 cytokine response in AD dogs and a mixed Th1/Th2/Treg cytokine response in healthy dogs. From this study, little evidence was found to support the use of CpG ODN class C for therapeutic purposes in dogs affected with AD.

Membrane-bound metallothionein 1 of murine dendritic cells promotes the expansion of regulatory T cells in vitro.

Exposure to environmental toxicants can alter a range of cellular functions involved in the immune response. Increased expression of the stress protein metallothionein 1 (MT1) is one example hereof. Previously, it has been reported that MT1 has several immunosuppressive properties. Furthermore, we earlier showed that functionally tolerogenic dendritic cells (DCs) expressed increased mRNA levels of MT1. Here, we demonstrate that dexamethasone-treated murine DCs are functionally tolerogenic and produce MT1. However, these DCs do not actively transport MT1 to the cell membrane and their regulatory function does not depend on MT1. Alternatively, ZnCl2-treated murine DCs transport MT1 to the cell surface are tolerogenic and promote the expansion of T cells with a regulatory phenotype. Moreover, the membrane-bound MT1 was shown to be essential for ZnCl2-treated DCs to exert their regulatory function. On the basis of this, MT1 can be used as a new marker for functionally tolerogenic DCs. Additionally, we have found a new mechanism for tolerogenic DCs to exert their immune regulatory function.

PLGA nanoparticles enhance the expression of retinaldehyde dehydrogenase enzymes in dendritic cells and induce FoxP3(+) T-cells in vitro.

Many autoimmune diseases and other chronic inflammatory disorders are characterized by defective FoxP3(+) regulatory T-cell (Treg) mediated suppression. A potential treatment option for these disorders is to increase the number and activity of Tregs locally. Both PLGA (poly-lactic-co-glycolic acid) and TMC-TPP (N-trimethyl chitosan tripolyphosphate) nanoparticles (NP) have been described to enhance T cell activation upon nasal application. Since, PLGA NP and TMC-TPP NP differentially affect CD4(+) T-cell differentiation, we investigated in vitro the capacity of both delivery systems to trigger retinoic acid (RA) production in dendritic cells (DCs) as a strategy to enhance the induction of FoxP3(+) T-cells. We generated ovalbumin (OVA)-encapsulated PLGA NP and TMC-TPP NP that were similar in size (400nm) but differed in their surface charge and other physico-chemical properties. We demonstrate that OVA-specific T-cells that are activated by cervical lymph node (CLN)-derived DCs treated with PLGA NP or TMC-TPP NP show more FoxP3 expression than T-cells that are activated by inguinal lymph node (ILN) cells. We demonstrate that only OVA-encapsulated PLGA NP enhance the induction of FoxP3 in activated T-cells via a TGF-ß and RA dependent mechanism by enhancing retinaldehyde dehydrogenase enzyme (RALDH) expression in CLN-derived DCs that is required for RA production. Additionally, detailed analysis of the CD4(+) T-cell response reveals that PLGA NP induce both IL-10 and IFN-γ production, while TMC-TPP NP induce mainly Th17 production. Underlining that both APC origin and NP characteristics determine the expression level of FoxP3 in activated T-cells. In conclusion, our data suggest that PLGA NP enhance the induction of FoxP3(+) T-cells in the CLN through modulation of DC function and we suggest that they might be a suitable nasal delivery system to treat a wide variety of autoimmune diseases and other chronic inflammatory disorders.

Tolerogenic dendritic cells that inhibit autoimmune arthritis can be induced by a combination of carvacrol and thermal stress.

Tolerogenic dendritic cells (DCs) can induce regulatory T cells and dampen pathogenic T cell responses. Therefore, they are possible therapeutic targets in autoimmune diseases. In this study we investigated whether mouse tolerogenic DCs are induced by the phytonutrient carvacrol, a molecule with known anti-inflammatory properties, in combination with a physiological stress. We show that treatment of DCs with carvacrol and thermal stress led to the mRNA expression of both pro- and anti-inflammatory mediators. Interestingly, treated DCs with this mixed gene expression profile had a reduced ability to activate pro-inflammatory T cells. Furthermore, these DCs increased the proportion of FoxP3(+) regulatory T cells. In vivo, prophylactic injection of carvacrol-thermal stress treated DCs pulsed with the disease inducing antigen was able to suppress disease in a mouse model of arthritis. These findings suggest that treatment of mouse bone marrow derived DCs with carvacrol and thermal stress induce a functionally tolerogenic DC that can suppress autoimmune arthritis. Herewith carvacrol seems to offer novel opportunities for the development of a dietary based intervention in chronic inflammatory diseases.

A case of mistaken identity: HSPs are no DAMPs but DAMPERs.

Until recently, the immune system was seen solely as a defense system with its primary task being the elimination of unwanted microbial invaders. Currently, however, the functional significance of the immune system has obtained a much wider perspective, to include among others the maintenance and restoration of homeostasis following tissue damage. In this latter aspect, there is a growing interest in the identification of molecules involved, such as the so-called danger or damage-associated molecular patterns (DAMPs), also called alarmins. Since heat shock proteins are archetypical molecules produced under stressful conditions, such as tissue damage or inflammation, they are frequently mentioned as prime examples of DAMPs (Bianchi, J Leukoc Biol 81:1-5, 2007; Kono and Rock, Nat Rev Immunol 8:279-289, 2008; Martin-Murphy et al., Toxicol Lett 192:387-394, 2010). See for instance also a recent review (Chen and Nunez, Science 298:1395-1401, 2010). Contrary to this description, we recently presented some of the arguments against a role of heat shock protein as DAMPs (Broere et al., Nat Rev Immunol 11:565-c1, 2011). With this perspective and reflection article, we hope to elaborate on this debate and provide additional thoughts to further ignite this discussion on this critical and evolving issue.

A novel heat-shock protein coinducer boosts stress protein Hsp70 to activate T cell regulation of inflammation in autoimmune arthritis.

OBJECTIVE: Stress proteins, such as members of the heat-shock protein (HSP) family, are up-regulated by cells in inflamed tissue and can be viewed functionally as "biomarkers" for the immune system to monitor inflammation. Exogenous administration of stress proteins has induced immunoregulation in various models of inflammation and has also been shown to be effective in clinical trials in humans. This study was undertaken to test the hypothesis that boosting of endogenous HSP expression can restore effective immunoregulation through T cells specific for stress proteins. METHODS: Stress protein expression was manipulated in vivo and in vitro with a food component (carvacrol), and immune recognition of stress proteins was studied. RESULTS: Carvacrol, a major compound in the oil of many Origanum species, had a notable capacity to coinduce cellular Hsp70 expression in vitro and, upon intragastric administration, in Peyer's patches of mice in vivo. As a consequence, carvacrol specifically promoted T cell recognition of endogenous Hsp70, as demonstrated in vitro by the activation of an Hsp70-specific T cell hybridoma and in vivo by amplified T cell responses to Hsp70. Carvacrol administration also increased the number of CD4+CD25+FoxP3+ T cells, systemically in the spleen and locally in the joint, and almost completely suppressed proteoglycan-induced experimental arthritis. Furthermore, protection against arthritis could be transferred with T cells isolated from carvacrol-fed mice. CONCLUSION: These findings illustrate that a food component can boost protective T cell responses to a self stress protein and down-regulate inflammatory disease, i.e., that the immune system can respond to diet.

Highly diverse TCR delta chain repertoire in bovine tissues due to the use of up to four D segments per delta chain.

Tissue-specific distribution of gammadelta TCRs with limited TCR diversity is a common phenomenon in species with a low percentage of gammadelta T cells like humans and mice. We set out to investigate whether this is also the case in cattle (Bos taurus), a species with high percentages of gammadelta T cells. Using a method that was independent of variable (V) segment-specific primers, we generated 65 unique TCR delta chain sequences. We found no evidence for preferential use of certain Vdelta segments in lymph node, skin, spleen, small intestine, large intestine, and blood. The delta chain CDR3 length distribution was very wide in each tissue, which was confirmed by spectratyping. The highly variable CDR3 length was due to the use of up to four diversity (D) segments by one bovine delta chain. Human and murine delta chains contain only one or two D segments. The five functional Ddelta segments that we describe here were identified at cDNA and genomic level, and are the first ruminant D segments described. Fourteen TCR delta chain sequences used novel Vdelta1 segments, and one expressed a novel member of the Vdelta3 family. The number of known functional Vdelta segments in cattle including these new ones is 42 now, but the total number may be much higher. A high number of Vdelta segments in combination with the use of up to four out of five D segments, and the possibility of using non-template encoded (N) nucleotides on either side of these, makes the potential bovine delta chain repertoire much bigger than any known TCR chain. This situation is quite different from the situation in humans and mice, and suggests that the differences between gammadelta high and gammadelta low species in distribution, diversity, and function of gammadelta T cells may be substantial.