Rachel Spiering

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.

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.

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.

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-β 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.

Mycobacterial and mouse HSP70 have immuno-modulatory effects on dendritic cells

Previously, it has been shown that heat shock protein 70 (HSP70) can prevent inflammatory damage in experimental autoimmune disease models. Various possible underlying working mechanisms have been proposed. One possibility is that HSP70 induces a tolerogenic phenotype in dendritic cells (DCs) as a result of the direct interaction of the antigen with the DC. Tolerogenic DCs can induce antigen-specific regulatory T cells and dampen pathogenic T cell responses. We show that treatment of murine DCs with either mycobacterial (Mt) or mouse HSP70 and pulsed with the disease-inducing antigen induced suppression of proteoglycan-induced arthritis (PGIA), although mouse HSP70-treated DCs could ameliorate PGIA to a greater extent. In addition, while murine DCs treated with Mt- or mouse HSP70 had no significantly altered phenotype as compared to untreated DCs, HSP70-treated DCs pulsed with pOVA (ovalbumin peptide 323–339) induced a significantly increased production of IL-10 in pOVA-specific T cells. IL-10-producing T cells were earlier shown to be involved in Mt HSP70-induced suppression of PGIA. In conclusion, this study indicates that Mt- and mouse HSP70-treated BMDC can suppress PGIA via an IL-10-producing T cell-dependent manner.

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.

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.

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.

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.