Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune­related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Proteomic analysis of chicken bone marrow-derived dendritic cells in response to an inactivated IBV + NDV poultry vaccine.

Inactivated poultry vaccines are subject to routine potency testing for batch release, requiring large numbers of animals. The replacement of in vivo tests for cell-based alternatives can be facilitated by the identification of biomarkers for vaccine-induced immune responses. In this study, chicken bone marrow-derived dendritic cells were stimulated with an inactivated vaccine for infectious bronchitis virus and Newcastle disease virus, as well as inactivated infectious bronchitis virus only, and lipopolysaccharides as positive control, or left unstimulated for comparison with the stimulated samples. Next, the cells were lysed and subjected to proteomic analysis. Stimulation with the vaccine resulted in 66 differentially expressed proteins associated with mRNA translation, immune responses, lipid metabolism and the proteasome. For the eight most significantly upregulated proteins, mRNA expression levels were assessed. Markers that showed increased expression at both mRNA and protein levels included PLIN2 and PSMB1. Stimulation with infectious bronchitis virus only resulted in 25 differentially expressed proteins, which were mostly proteins containing Src homology 2 domains. Stimulation with lipopolysaccharides resulted in 118 differentially expressed proteins associated with dendritic cell maturation and antimicrobial activity. This study provides leads to a better understanding of the activation of dendritic cells by an inactivated poultry vaccine, and identified PLIN2 and PSMB1 as potential biomarkers for cell-based potency testing.

Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line.

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1ß and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.

In vitro Chicken Bone Marrow-Derived Dendritic Cells Comprise Subsets at Different States of Maturation.

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo. This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II)low and MHC-IIhigh subsets with different morphologies. Compared with MHC-IIlow chBMDCs, the MHC-IIhigh subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-IIhigh chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-IIhigh chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-IIhigh chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-IIhigh subset back into the immature phenotype of the MHC-IIlow subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types.

Leucinostatin acts as a co-inducer for heat shock protein 70 in cultured canine retinal pigment epithelial cells.

Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.

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.

Lipidoid-polymer hybrid nanoparticles loaded with TNF siRNA suppress inflammation after intra-articular administration in a murine experimental arthritis model.

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.

APL1, an altered peptide ligand derived from human heat-shock protein 60, increases the frequency of Tregs and its suppressive capacity against antigen responding effector CD4 + T cells from rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by a chronic relapsing-remitting joint inflammation. Perturbations in the balance between CD4 + T cells producing IL-17 and CD4 + CD25(high)FoxP3 + Tregs correlate with irreversible bone and cartilage destruction in RA. APL1 is an altered peptide ligand derived from a CD4+ T-cell epitope of human HSP60, an autoantigen expressed in the inflamed synovium, which increases the frequency of CD4 + CD25(high)FoxP3+ Tregs in peripheral blood mononuclear cells from RA patients. The aim of this study was to evaluate the suppressive capacity of Tregs induced by APL1 on proliferation of effector CD4+ T cells using co-culture experiments. Enhanced Treg-mediated suppression was observed in APL1-treated cultures compared with cells cultured only with media. Subsequent analyses using autologous cross-over experiments showed that the enhanced Treg suppression in APL1-treated cultures could reflect increased suppressive function of Tregs against APL1-responsive T cells. On the other hand, APL1-treatment had a significant effect reducing IL-17 levels produced by effector CD4+ T cells. Hence, this peptide has the ability to increase the frequency of Tregs and their suppressive properties whereas effector T cells produce less IL-17. Thus, we propose that APL1 therapy could help to ameliorate the pathogenic Th17/Treg balance in RA patients.

Reduced TCR-dependent activation through citrullination of a T-cell epitope enhances Th17 development by disruption of the STAT3/5 balance.

Citrullination is a post-translational modification of arginine that commonly occurs in inflammatory tissues. Because T-cell receptor (TCR) signal quantity and quality can regulate T-cell differentiation, citrullination within a T-cell epitope has potential implications for T-cell effector function. Here, we investigated how citrullination of an immunedominant T-cell epitope affected Th17 development. Murine naïve CD4(+) T cells with a transgenic TCR recognising p89-103 of the G1 domain of aggrecan (agg) were co-cultured with syngeneic bone marrow-derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro-Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL-2, expressed lower levels of the IL-2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL-2 blockade in native p89-103-primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post-translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th-cell development in inflammatory disorders.

Surface coating of siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers: enhanced gene silencing and reduced adverse effects in vitro.

Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/ß-peptoid peptidomimetics with a systematically varied number of repeating lysine and homoarginine residues was shown to self-assemble with small interfering RNA (siRNA). The resulting well-defined nanocomplexes were coated with anionic lipids giving rise to net anionic liposomes. These complexes and the corresponding liposomes were optimized towards efficient gene silencing and low adverse effects. The optimal anionic liposomes mediated a high silencing effect, which was comparable to that of the control (cationic Lipofectamine 2000), and did not display any noticeable cytotoxicity and immunogenicity in vitro. In contrast, the corresponding nanocomplexes mediated a reduced silencing effect with a more narrow safety window. The surface coating with anionic lipid bilayers led to partial decomplexation of the siRNA-peptidomimetic nanocomplex core of the liposomes, which facilitated siRNA release. Additionally, the optimal anionic liposomes showed efficient intracellular uptake and endosomal escape. Therefore, these findings suggest that a more efficacious and safe formulation can be achieved by surface coating of the siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers.

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide.

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4+CD25- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25+ cells prior to immunization, allowing the in vivo formation of Tregs from CD4+CD25- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4+CD25+ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4+CD25- T cells.

Activated peritoneal cavity B-1a cells possess regulatory B cell properties.

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.

T cell recognition of naturally presented epitopes of self-heat shock protein 70.

Self-reactive T cells have shown to have a potential role as regulators of the immune system preventing or even suppressing autoimmunity. One of the most abundant proteins that can be eluted from human HLA molecules is heat shock protein 70 (HSP70). The aims of the current study are to identify HSP70 epitopes based on published HLA elution studies and to investigate whether T cells from healthy individuals may respond to such self-epitopes. A literature search and subsequent in silico binding prediction based on theoretical MHC binding motifs resulted in the identification of seven HSP70 epitopes. PBMCs of healthy controls proliferated after incubation with two of the seven peptides (H167 and H290). Furthermore H161, H290, and H443 induced CD69 expression or production of cytokines IFNγ or TNFα in healthy controls. The identification of these naturally presented epitopes and the response they elicit in the normal immune system make them potential candidates to study during inflammatory conditions as well as in autoimmune diseases.

Antigen-specific B lymphocytes acquire proteoglycan aggrecan from cartilage extracellular matrix resulting in antigen presentation and CD4+ T-cell activation.

The majority of studies examining antigen-presenting cell (APC) function have focused on the capture and presentation of antigens released from pathogens or damaged cells. However, antigen-specific B cells are also capable of efficiently extracting antigens that are either tethered to, or integrally part of the plasma membrane of various target cells. In this study we show that B cells are also highly efficient at extracting integral components of the extracellular matrix (ECM) for subsequent presentation. In particular we demonstrate that B cells specific for aggrecan, an integral component of cartilage ECM, acquire this rheumatoid arthritis candidate autoantigen in both a B-cell-receptor-dependent and a contact-dependent manner. We also demonstrate that the subsequent presentation of aggregan from ECM leads to CD4(+) T-cell activation and effector cell formation. Recent studies have identified B-cell-mediated antigen presentation as essential for the development of autoimmunity, but a unique role for B cells compared with other APC has yet to be defined. Our findings lead us to propose that the acquisition of ECM-derived autoantigens represents a mechanism that defines the APC requirement for B cells in the development of autoimmunity.

APL-1, an altered peptide ligand derived from human heat-shock protein 60, selectively induces apoptosis in activated CD4+ CD25+ T cells from peripheral blood of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4+ T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis and regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4+ T-cell epitope of human heat-shock protein of 60kDa, an autoantigen involved in the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4+ CD25(high)Foxp3+ Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4+ CD25+ T cells but not resting CD4+ CD25- T cells were identified as targets of APL-1. Furthermore, CD4+ T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4+ T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4+ CD25(high)Foxp3+ Tregs and apoptosis in activated CD4+ T cells. These results support further investigation of this candidate drug for the treatment of RA.

Heat shock proteins can be targets of regulatory T cells for therapeutic intervention in rheumatoid arthritis.

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.

Heat shock protein expression analysis in canine osteosarcoma reveals HSP60 as a potentially relevant therapeutic target.

Heat shock proteins (HSP) are highly conserved across eukaryotic and prokaryotic species. These proteins play a role in response to cellular stressors, protecting cells from damage and facilitating recovery. In tumor cells, HSPs can have cytoprotective effects and interfere with apoptotic cascades. This study was performed to assess the prognostic and predictive values of the gene expression of HSP family members in canine osteosarcoma (OS) and their potential for targeted therapy. Gene expressions for HSP were assessed using quantitative PCR (qPCR) on 58 snap-frozen primary canine OS tumors and related to clinic-pathological parameters. A significant increased expression of HSP60 was found in relation to shorter overall survival and an osteoblastic phenotype. Therefore, the function of HSP60 was investigated in more detail. Immunohistochemical analysis revealed heterogeneous staining for HSP60 in tumors. The highest immunoreactivity was found in tumors of short surviving dogs. Next HSP expression was shown in a variety of canine and human OS cell lines by qPCR and Western blot. In two highly metastatic cell lines HSP60 expression was silenced using siRNA resulting in decreased cell proliferation and induction of apoptosis in both cell lines. It is concluded that overexpression of HSP60 is associated with a poor prognosis of OS and should be evaluated as a new target for therapy.

Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis.

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.

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4(+) T helper type 1 subset differentiation.

Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis.