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Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by excessive immune responses resulting in inflammation of the joints. Although current therapies can be successful in dampening inflammation, a long-lived state of tolerance is seldom achieved. Therefore, novel therapies are needed that restore and maintain tolerance in patients with RA. Targeting regulatory T cells (Tregs) is a successful strategy to achieve tolerance, as was shown in studies performed in animal models and in human clinical trials. The antigen-specificity of Tregs is crucial for their effectiveness and allows for very specific targeting of these cells. However, which antigen is suitable for autoimmune diseases such as RA, for which the autoantigens are largely unknown? Heat shock proteins (HSPs) are ubiquitously expressed and can be up-regulated during inflammation. Additionally, HSPs, or HSP-derived peptides are immunogenic and can be recognised by a variety of immune cells, including Tregs. Therefore, this review highlights the potential of HSP-specific Tregs to control inflammatory immune responses. Targeting HSP-specific Tregs in RA can be achieved via the administration of HSPs (derived peptides), thereby controlling inflammatory responses. This makes HSPs attractive candidates for therapeutic intervention in chronic autoimmune diseases, with the ultimate goal of inducing long-lasting tolerance.

Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 induces B29-specific regulatory T cells, which suppressed experimental arthritis in mice by cross-recognition of their mammalian HSP70 homologs (1). The aim of this study is to characterize the B29 binding and specific CD4(+) T cell responses in the context of human MHC molecules (HLA). Binding of B29 peptide and its mammalian homologs to HLA molecules was examined with competitive binding assays. The effect of B29 immunization was assessed in proteoglycan-induced arthritis in HLA-DQ8 transgenic mice, followed by ex vivo restimulation with B29 to examine the T cell response. Human PBMC were used to investigate the presence of B29-specific T cells with immunoregulatory potential. We found a high to moderate binding affinity for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was functional, as B29 immunization resulted in suppression of arthritis and T cell responses in HLA-DQ8 transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4(+) T cells, which were cross-reactive with the mammalian homologs. With HLA-DR4(+) tetramers specific for B29 or mB29b we showed expansion of cross-reactive T cells, especially the human CD4(+) CD25(+) FoxP3(+) T cell population after in vitro stimulation with B29. These results demonstrated a conserved fine-specificity and functionality of the B29-induced regulatory T cell responses in the context of the human MHC. Based on these findings, a translational path of the B29 experimental findings into a clinical immunomodulatory therapeutic approach comes within reach. This article is protected by copyright. All rights reserved.

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.

Objectives: To explore the immunosuppressive effect and mechanism of action of intraperitoneal (ip) and intra-articular (ia) mesenchymal stem cell (MSC) injection in proteoglycan induced arthritis (PGIA). Methods: MSC were administered ip or ia after establishment of arthritis. We used serial bioluminescence imaging (BLI) to trace luciferase-transfected MSC. Mice were sacrificed at different time points to examine immunomodulatory changes in blood and secondary lymphoid organs. Results: Both ip and local ia MSC injection resulted in a beneficial clinical and histological effect on established PGIA. BLI showed that MSC ip and ia in arthritic mice are largely retained for several weeks in the peritoneal cavity or injected joint respectively, without signs of migration. Following MSC treatment pathogenic PG-specific IgG2a antibodies in serum decreased. The Th2 cytokine IL-4 was only upregulated in PG-stimulated lymphocytes from spleens in ip treated mice and in lymphocytes from draining lymph nodes in ia treated mice. An increase in production of IL-10 was seen with equal distribution. Although IFN-? was also elevated, the IFN-?/IL-4 ratio in MSC treated mice was opposite to the ratio in (untreated) active PGIA. Conclusions: MSC treatment, both ip and ia, suppresses PGIA, a non-collagen induced arthritis model. MSC are largely retained for weeks in the injection region. MSC treatment induced at the region of injection a deviation of PG-specific immune responses, suggesting a more regulatory phenotype with production of IL-4 and IL-10, but also of IFN-γ, and a systemic decrease of pathogenic PG-specific IgG2a antibodies. These findings underpin the potential of MSC treatment in resistant arthritis.

Previous studies have suggested that murine peritoneal cavity-derived B-1a cells possess similarities with described regulatory B cell subsets. The aim of the current study was to examine the potential immunoregulatory function of peritoneal cavity-derived B(-1a) cells. In vitro activation of peritoneal cavity-derived B- and B-1a cells shows that activation of these B cells with anti-CD40 and LPS induces these cells to secrete more IL-10, IL-6 and IgM as compared to splenic B cells. In a suppression assay, CD40/TLR4-activated peritoneal cavity B cells possess regulatory B cell functions as they inhibit the capacity of CD4+ T cells to produce both tumor necrosis factor-α and interferon-γ. Splenic B cells did not show this, whereas non-activated peritoneal cavity B cells augmented the capacity of CD4+ T cells to produce tumor necrosis factor-α, while the ability to produce interferon-γ was not altered. The current paper compares splenic B cells to peritoneal cavity B(-1a) cells in an in vitro activation- and an suppression-assay and concludes that peritoneal cavity B(-1a) cells possess properties that appear similar to splenic autoimmune-suppressive regulatory B cell subsets described in the literature.

Many existing therapies in autoimmune diseases are based on systemic suppression of inflammation and the observed side effects of these therapies illustrate the pressing need for more specific interventions. Regulatory T-cells (Treg) are pivotal controllers of (auto-aggressive) immune responses and inflammation, and decreased Treg numbers and/or functioning have been associated with autoimmune disease. Therefore, Treg became frequently studied targets for more specific immunotherapy. Especially antigen-specific targeting of Treg would enable local and tailor made interventions, while obviating the negative side effect of general immuno-suppression. Self-antigens that participate in inflammation, irrespective of the etiology of the different autoimmune diseases, are held to be candidate antigens for antigen-specific interventions. Rather than tolerance induction to disease inciting self-antigens, which are frequently unknown, general self-antigens expressed at sites of inflammation would allow targeting of disease independent, but inflammatory-site specific, regulatory mechanisms. Preferably, such self-antigens should be abundantly expressed and up-regulated at the inflammatory-site. In this perspective heat shock proteins (Hsp) have several characteristics that make them highly attractive targets for antigen-specific Treg inducing therapy. The development of an antigen-specific Treg inducing vaccine is a major novel goal in the field of immunotherapy in autoimmune diseases. However, progress is hampered not only by the lack of effective antigens, but also by the fact that other factors such as dose, route, and the presence or absence of an adjuvant, turned out to be critical unknowns, with respect to the effective induction of Treg. In addition, the use of a Treg inducing adjuvant might be required to achieve an effective regulatory response, in the case of ongoing inflammation. Future goals in clinical trials will be the optimization of natural Treg expansion (or the induction of adaptive Treg) without loss of their suppressive function or the concomitant induction of non-regulatory T-cells. Here, we will discuss the potential use of protein/peptide-based vaccines combined with Treg inducing adjuvants for the development of therapeutic vaccines against chronic inflammatory conditions.

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 (400 nm) 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.

Innate-like murine B-1a cells are well known for their ability to secrete natural IgM. Their non-Ab mediated functions, including Ag presentation to CD4+ T cells, are less well explored. Using combined adoptive transfer experiments with peptide-pulsed peritoneal cavity (PerC)-derived B-1a cells and CFSE-labeled T cells, we show that B-1a cells present Ag to CD4+ T cells from the periphery in vivo. In vitro characterization, using co-cultures in which B-1a or splenic B cells presented whole OVA protein to OVA-specific Tg T cells, shows that B-1a cells differentially promote intracellular cytokine-expressing T cells. PerC-derived B-1a cells increase the percentage of IL-10-producing T cells along with IL-4- and IFN-γ-producing CD4+ T cells. These data suggest that B cells in the PerC have the potential to influence peripheral immune responses without the necessity to migrate out of this location. This, to our knowledge previously undescribed, immuno-logical pathway potentially plays a role in the presentation of gut microbiota-derived Ags to peripheral T cells.

Anim Genet. 2012 Sep 19. doi: 10.1111/age.12002. [Epub ahead of print] Tandem repeats modify the structure of the canine CD1D gene. Looringh van Beeck FA, Leegwater PA, Herrmann T, Broere F, Rutten VP, Willemse T, Van Rhijn I. Source Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands. Abstract Among the CD1 proteins that present lipid antigens to T cells, CD1d is the only one that stimulates a population of T cells with an invariant T-cell receptor known as NKT cells. Sequencing of a 722 nucleotide gap in the dog (Canis lupus familiaris) genome revealed that the canine CD1D gene lacks a sequence homologous to exon 2 of human CD1D, coding for the start codon and signal peptide. Also, the canine CD1D gene contains three different short tandem repeats that disrupt the expected gene structure. Because canine CD1D cDNA lacks sequences homologous to human exon 2 and 3, the functionality of canine CD1d protein may be affected, and this could have consequences for the development and activation of canine NKT cells. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics. PMID: 22988997 [PubMed - as supplied by publisher]

Anim Genet. 2012 Sep 19. doi: 10.1111/age.12002. [Epub ahead of print] Tandem repeats modify the structure of the canine CD1D gene. Looringh van Beeck FA, Leegwater PA, Herrmann T, Broere F, Rutten VP, Willemse T, Van Rhijn I. Source Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands. Abstract Among the CD1 proteins that present lipid antigens to T cells, CD1d is the only one that stimulates a population of T cells with an invariant T-cell receptor known as NKT cells. Sequencing of a 722 nucleotide gap in the dog (Canis lupus familiaris) genome revealed that the canine CD1D gene lacks a sequence homologous to exon 2 of human CD1D, coding for the start codon and signal peptide. Also, the canine CD1D gene contains three different short tandem repeats that disrupt the expected gene structure. Because canine CD1D cDNA lacks sequences homologous to human exon 2 and 3, the functionality of canine CD1d protein may be affected, and this could have consequences for the development and activation of canine NKT cells. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics. PMID: 22988997 [PubMed - as supplied by publisher]

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4 + CD25 + Foxp3 + T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen–specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after transfer. Given that ( i ) B29 administration by itself suppressed disease, ( ii ) our findings were made with wild-type (T-cell receptor nontransgenic) Tregs, and ( iii ) the B29 human homolog is presented by HLA class II, we are nearing translation of antigen-specific Treg activation as a promising intervention for chronic inflammatory diseases.

Natural bioactive compounds have been studied for a long time for their chemopreventive and therapeutic potential in several chronic inflammatory diseases, including cancer. However, their physicochemical properties generally result in poor chemical stability and lack of in vivo bioavailability. Very few human clinical trials have addressed absorption, distribution, metabolism, and excretion of these compounds in relation to efficacy. This limits the use of these valuable natural compounds in the clinic. In this study, we examined caffeic acid (derivatives), carvacrol (derivatives), thymol, pterostilbene (derivatives), and N-(3-oxo-dodecanoyl)-l-homoserine lactone. These are natural compounds with strong anti-inflammatory properties derived from plants and bacteria. However, these compounds have poor water solubility or are chemically unstable. To overcome these limitations we have prepared liposomal formulations. Our results show that lipophilic 3-oxo-C(12)-homoserine lactone and stilbene derivatives can be loaded into liposomal lipid bilayer with efficiencies of 50-70%. Thereby, the liposomes solubilize these compounds, allowing intravenous administration without use of solvents. When compounds could not be loaded into the lipid bilayer (carvacrol and thymol) or are rapidly extracted from the liposomes in the presence of serum albumin (3-oxo-C(12)-homoserine lactone and pterostilbene derivatives), derivatization of the compound into a water-soluble prodrug was shown to improve loading efficiency and encapsulation stability. The phosphate forms of carvacrol and pterostilbene were loaded into the aqueous interior of the liposomes and encapsulation was unaffected by the presence of serum albumin. Chemical instability of resveratrol was improved by liposome-encapsulation, preventing inactivating cis-trans isomerization. For caffeic acid, liposomal encapsulation did not prevent oxidation into a variety of products. Still, by derivatization into a phenyl ester, the compound could be stably encapsulated without chemical degradation. Despite the instability of liposome-association of 3-oxo-C(12)-homoserine lactone and resveratrol, intravenous administration of these compounds inhibited tumor growth for approximately 70% in a murine tumor model, showing that simple solubilization can have important therapeutic benefits.

Development of vaccines in autoimmune diseases has received wide attention over the last decade. However, many vaccines showed limited clinical efficacy. To enhance vaccine efficacy in infectious diseases, biocompatible and biodegradable polymeric nanoparticles have gained interest as antigen delivery systems. We investigated in mice whether antigen-encapsulated PLGA (poly-lactic-co-glycolic acid), PLGA-TMC (N-trimethyl chitosan) or TMC-TPP (tri-polyphosphate) nanoparticles can also be used to modulate the immunological outcome after nasal vaccination. These three nanoparticles enhanced the antigen presentation by dendritic cells, as shown by increased in vitro and in vivo CD4(+) T-cell proliferation. However, only nasal PLGA nanoparticles were found to induce an immunoregulatory response as shown by enhanced Foxp3 expression in the nasopharynx associated lymphoid tissue and cervical lymph nodes. Nasal administration of OVA-containing PLGA particle resulted in functional suppression of an OVA-specific Th-1 mediated delayed-type hypersensitivity reaction, while TMC-TPP nanoparticles induced humoral immunity, which coincided with the enhanced generation of OVA-specific B-cells in the cervical lymph nodes. Intranasal treatment with Hsp70-mB29a peptide-loaded PLGA nanoparticles suppressed proteoglycan-induced arthritis, leading to a significant reduction of disease. We have uncovered a role for PLGA nanoparticles to enhance CD4(+) T-cell mediated immunomodulation after nasal application. The exploitation of this differential regulation of nanoparticles to modulate nasal immune responses can lead to innovative vaccine development for prophylactic or therapeutic vaccination in infectious or autoimmune diseases.

Nasal vaccination is a promising, needle-free alternative to classical vaccination. Nanoparticulate delivery systems have been reported to overcome the poor immunogenicity of nasally administered soluble antigens, but the characteristics of the ideal particle are unknown. This study correlates differences in physicochemical characteristics of nanoparticles to their adjuvant effect, using ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA NP), N-trimethyl chitosan (TMC) based NP (TMC NP) and TMC-coated PLGA NP (PLGA/TMC NP). PLGA NP and PLGA/TMC NP were prepared by emulsification/solvent extraction and TMC NP by ionic complexation. The NP were characterized physicochemically. Their toxicity and interaction with and stimulation of monocyte derived dendritic cells (DC) were tested in vitro. Furthermore, the residence time and the immunogenicity (serum IgG titers and secretory IgA levels in nasal washes) of the nasally applied OVA formulations were assessed in Balb/c mice. All NP were similar in size, whereas only PLGA NP carried a negative zeta potential. The NP were nontoxic to isolated nasal epithelium. Only TMC NP increased the nasal residence time of OVA compared to OVA administered in PBS and induced DC maturation. After i.m. administration all NP systems induced higher IgG titers than OVA alone, PLGA NP and TMC NP being superior to PLGA/TMC NP. Nasal immunization with the slow antigen releasing particles, PLGA NP and PLGA/TMC NP, did not induce detectable antibody titers. In contrast, nasal immunization with the positively charged, fast antigen releasing TMC NP led to high serum antibody titers and sIgA levels. In conclusion, particle charge and antigen release pattern of OVA-loaded NP has to be adapted to the intended route of administration. For nasal vaccination, TMC NP, releasing their content within several hours, being mucoadhesive and stimulating the maturation of DC, were superior to PLGA NP and PLGA/TMC NP which lacked some or all of these characteristics. (C) 2010 Elsevier Ltd. All rights reserved.