Intra-articular bioactivity of a p38 MAPK inhibitor and development of an extended-release system.

In the treatment of arthritic diseases, oral or systemic administration of anti-inflammatory substances, such as p38 MAPK inhibitors, is hampered by numerous side effects. To overcome them, formulations of rapid and extended drug delivery systems were studied in intra-articular administration. For the first time, VX-745, a highly selective p38 MAPK inhibitor, demonstrated in vivo bioactivity, similar to dexamethasone activity, following intra-articular administration in an antigen-induced arthritic (AIA) mouse model. The in vitro bioactivity of VX-745 was also shown on synoviocytes, reducing the IL-6 concentration. Process and formulation parameters (i.e., polymer concentration, aqueous/organic phase ratio, emulsification speed and process, and evaporation pressure) and particle characterisation (i.e., drug loading, size of particle, and surface aspect) were extensively examined to produce optimised formulations. Indeed, a burst release provides a rapid saturation of intracellular p38 MAPK to relieve patients from pain and inflammation. Then, drug diffusion would be sufficient to maintain an effective dose over 2-3 months. This study confirms the effectiveness of encapsulated p38 MAPK inhibitors in extended drug delivery systems and seems to be a promising strategy for intra-articular treatment.

Ag-presenting CpG-activated pDCs prime Th17 cells that induce tumor regression.

Plasmacytoid dendritic cells (pDC) rapidly and massively produce type I IFN and other inflammatory cytokines in response to foreign nucleic acids, thereby indirectly influencing T-cell responses. Moreover, antigen (Ag)-presenting pDCs directly regulate T-cell differentiation. Depending on the immune environment, pDCs exhibit either tolerogenic or immunogenic properties. Here, we show that CpG-activated pDCs promote efficient Th17 differentiation. Indeed, Th17 responses are defective in mice selectively lacking MHCII on pDCs upon antigenic challenge. Importantly, in those mice, the frequency of Th17 cells infiltrating solid tumors is impaired. As a result, the recruitment of infiltrating leukocytes in tumors, including tumor-specific cytotoxic T lymphocytes (CTL), is altered and results in increased tumor growth. Importantly, following immunization with tumor Ag and CpG-B, MHCII-restricted Ag presentation by pDCs promotes the differentiation of antitumor Th17 cells that induce intratumor CTL recruitment and subsequent regression of established tumors. Our results highlight a new role for Ag presenting activated pDCs in promoting the development of Th17 cells and impacting on antitumor immunity.

STIM1 juxtaposes ER to phagosomes, generating Ca²âº hotspots that boost phagocytosis.

BACKGROUND: Endoplasmic reticulum (ER) membranes are recruited to phagosomes, but the mechanism and functional significance of this ER recruitment is not known. Here, we show that the ER Ca(2+) sensor stromal interaction molecule 1 (STIM1) sustains high-efficiency phagocytosis by recruiting thin ER cisternae that interact productively but do not fuse with phagosomes. RESULTS: Endogenous STIM1 was recruited to phagosomes upon ER Ca(2+) depletion in mouse neutrophils, and exogenous YFP-STIM1 puncta coincided with localized Ca(2+) elevations around phagosomes in fibroblasts expressing phagocytic receptors. STIM1 ablation decreased phagocytosis, ER-phagosome contacts, and periphagosomal Ca(2+) elevations in both neutrophils and fibroblasts, whereas STIM1 re-expression in Stim1(-/-) fibroblasts rescued these defects, promoted the formation and elongation of tight ER-phagosome contacts upon ER Ca(2+) depletion and increased the shedding of periphagosomal actin rings. Re-expression of a signaling-deficient STIM1 mutant unable to open Ca(2+) channels recruited ER cisternae to the vicinity of phagosomes but failed to rescue phagocytosis, actin shedding, and periphagosomal Ca(2+) elevations. The periphagosomal Ca(2+) hotspots were decreased by extracellular Ca(2+) chelation and by Ca(2+) channels inhibitors, revealing that the Ca(2+) ions originate at least in part from phagosomes. CONCLUSIONS: Our findings indicate that STIM1 recruits ER cisternae near phagosomes for signaling purposes and that the opening of phagosomal Ca(2+) channels generates localized Ca(2+) elevations that promote high-efficiency phagocytosis.

Long-term amelioration of established collagen-induced arthritis achieved with short-term therapy combining anti-CD3 and anti-tumor necrosis factor treatments.

OBJECTIVE: The goal of rheumatoid arthritis (RA) treatment is to achieve clinical remission in order to limit structural damage and physical disability. To this end, recent emphasis has been placed on aggressive treatment early in the course of disease with drugs such as anti-tumor necrosis factor (anti-TNF) agents. As T cells are also thought to play an important role in the initiation of RA, we hypothesized that targeting both TNF and T cells would result in better outcomes. The aim of this study was to examine the efficacy of combined therapy with anti-CD3 and anti-TNF in experimental RA. METHODS: Two anti-mouse antibodies were developed as surrogate reagents for anti-TNF and anti-CD3 therapies. Collagen-induced arthritis (CIA) was induced in DBA/1 mice, and antibodies were injected intraperitoneally, either alone on in combination, at predetermined subtherapeutic doses. The frequency and number of pathogenic and regulatory CD4+ T cell subsets in the draining lymph nodes were determined in order to investigate the mechanisms of action. RESULTS: Strikingly, the combination of the two antibodies demonstrated a potent synergy in established CIA, with long-term inhibition of disease progression and protection from joint destruction. The results did not demonstrate any enhancement of CD25+FoxP3+ regulatory T cells, but a profound depletion of pathogenic T cells from the draining lymph nodes was associated with reduced numbers of T cells in the joints. CONCLUSION: A short course of combination therapy with anti-CD3 and anti-TNF efficiently depletes pathogenic T cells from the draining lymph nodes, reducing the numbers of T cells in the joints and affording long-lasting inhibition of established CIA.

Autoimmunity and inflammation are independent of class II transactivator type PIV-dependent class II major histocompatibility complex expression in peripheral tissues during collagen-induced arthritis.

OBJECTIVE: To determine the regulation of class II major histocompatibility complex (MHC) expression in fibroblast-like synoviocytes (FLS) in order to investigate their role as nonprofessional antigen-presenting cells in collagen-induced arthritis (CIA). METHODS: Expression of class II MHC, class II MHC transactivator (CIITA), and Ciita isoforms PI, PIII, and PIV was examined by real-time quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry in human synovial tissues, arthritic mouse joints, and human and murine FLS. CIA was induced in mice in which isoform PIV of Ciita was knocked out (PIV(-/-) ), in PIV(-/-) mice transgenic for CIITA in the thymus (K14 CIITA), and in their control littermates. RESULTS: HLA-DRA, total CIITA, and CIITA PIII messenger RNA levels were significantly increased in synovial tissue samples from patients with rheumatoid arthritis compared with the levels in tissue from patients with osteoarthritis. Human FLS expressed surface class II MHC via CIITA PIII and PIV, while class II MHC expression in murine FLS was entirely mediated by PIV. Mice with a targeted deletion of CIITA PIV lack CD4+ T cells and were protected against CIA. The expression of CIITA was restored in the thymus of PIV(-/-) K14 CIITA-transgenic mice, which had a normal CD4+ T cell repertoire and normal surface levels of class II MHC on professional antigen-presenting cells, but did not induce class II MHC on FLS. Synovial inflammation and immune responses against type II collagen were similar in PIV(-/-) K14 CIITA-transgenic mice and control mice with CIA, but bone erosion was significantly reduced in the absence of PIV. CONCLUSION: Overexpression of class II MHC is tightly correlated with CIITA expression in arthritic synovium and in FLS. Selective targeting of Ciita PIV in peripheral tissues abrogates class II MHC expression by murine FLS but does not protect against inflammation and autoimmune responses in CIA.

Regulation of peripheral inflammation by the central nervous system.

In inflammatory disorders such as rheumatoid arthritis, cytokines and danger signals are sensed by the central nervous system, which adapts behavior and physiologic responses during systemic stress. The central nervous system can also signal the periphery to modulate inflammation through efferent hormonal and neuronal pathways. The brain and spinal cord are involved in this bidirectional interaction. A variety of neuronal pathways that modulate synovial inflammation have been implicated, including the sympathetic and the parasympathetic branches of the autonomic system. Another mechanism, the dorsal root reflex, involves antidromic signaling along somatic afferent fibers that influences joint inflammation by releasing neuropeptides and other neuromediators in the periphery. Some of the neurotransmitters and neuroreceptors involved have been identified in preclinical models and represent novel targets for the treatment of rheumatic diseases.

Acetylcholine regulation of synoviocyte cytokine expression by the alpha7 nicotinic receptor.

OBJECTIVE: The central nervous system can regulate peripheral inflammation, but the efferent neuronal routes and the mediators remain poorly defined. One candidate is the cholinergic pathway, which releases acetylcholine (ACh). This neurotransmitter can bind to the alpha7 cholinergic receptor (alpha7R) expressed by nonneuronal cells and reduce inflammation. To test this possibility, we evaluated the expression of alpha7R and its potential role as a target in rheumatoid arthritis (RA). METHODS: The expression of alpha7R in human synovium and fibroblast-like synoviocytes (FLS) was determined using immunohistochemical, Western blot, and quantitative polymerase chain reaction (PCR) analyses. The effects of ACh in vitro were determined in interleukin-1 (IL-1)-stimulated FLS using immunoassays for protein, quantitative PCR for messenger RNA (mRNA), luciferase reporter constructs for IL-6 and NF-kappaB promoter activity, and electrophoretic mobility shift assays. Expression of alpha7R was knocked down with small interfering RNA (siRNA) or was inhibited with the selective alpha7R antagonist methyllycaconitine (MLA). RESULTS: Protein and mRNA for alpha7R were demonstrated in RA and osteoarthritis synovium and cultured synoviocytes. Expression in synovium was mainly in the intimal lining. ACh significantly reduced the production of IL-6, CXCL8, CCL2, CCL3, CCL5, and granulocyte colony-stimulating factor by IL-1-stimulated FLS. This effect was blocked by the alpha7R antagonist MLA or by using alpha7R siRNA to knock down receptor expression. The selective alpha7R agonist PNU-282,987 decreased the production of IL-6 by IL-1-stimulated FLS. ACh did not reduce IL-6 transcription, but it decreased IL-6 mRNA half-life and reduced IL-6 mRNA steady-state levels. CONCLUSION: The alpha7 receptor is expressed in the synovium and by synoviocytes. Receptor ligation inhibits cytokine expression in FLS through a posttranscriptional mechanism. Therefore, alpha7R is a potential therapeutic target for inflammatory diseases.

Regulation of MHC class II gene expression by the class II transactivator.

MHC class II molecules are pivotal for the adaptive immune system, because they guide the development and activation of CD4+ T helper cells. Fulfilling these functions requires that the genes encoding MHC class II molecules are transcribed according to a strict cell-type-specific and quantitatively modulated pattern. This complex gene-expression profile is controlled almost exclusively by a single master regulatory factor, which is known as the class II transactivator. As we discuss here, differential activation of the three independent promoters that drive expression of the gene encoding the class II transactivator ultimately determines the exquisitely regulated pattern of MHC class II gene expression.

MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells.

Major histocompatibility complex (MHC) class II-restricted antigen presentation is essential for the function of dendritic cells (DCs). We show here that plasmacytoid DCs (pDCs) differ from all other DC subsets with respect to expression of CIITA, the 'master regulator' of MHC class II genes. The gene encoding CIITA is controlled by three cell type-specific promoters: pI, pIII and pIV. With gene targeting in mice, we demonstrate that pDCs rely strictly on the B cell promoter pIII, whereas macrophages and all other DCs depend on pI. The molecular mechanisms driving MHC class II expression in pDCs are thus akin to those operating in lymphoid rather than myeloid cells.

Mini-review: Specificity and expression of CIITA, the master regulator of MHC class II genes.

The class II transactivator (CIITA) has been referred to as the "master control factor" for the expression of MHC class II (MHCII) genes. As our knowledge on the specificity and function of CIITA grows, it is becoming increasingly evident that this sobriquet is entirely justified. First, despite extensive investigations, the major target genes of CIITA remain those implicated in the presentation of antigenic peptides by MHCII molecules. Although other putative target genes have been reported, the contribution of CIITA to their expression remains indirect, controversial or comparatively minor relative to its decisive role as a regulator of MHCII and related genes. Second, the most important parameter dictating MHCII expression is by far the expression pattern of the gene encoding CIITA (MHC2TA). The vast majority of signals that activate or repress MHCII expression under physiological and pathological situations converge on one or more of the three alternative promoters that drive transcription of the MHC2TA gene. In short, with respect to its specificity and its exquisitely controlled pattern of expression, CIITA is by a long stretch the single most important transcription factor for the regulation of genes required for MHCII-restricted antigen-presentation.

Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells.

Major histocompatibility complex class II (MHCII) expression is regulated by the transcriptional coactivator CIITA. Positive selection of CD4(+) T cells is abrogated in mice lacking one of the promoters (pIV) of the Mhc2ta gene. This is entirely due to the absence of MHCII expression in thymic epithelia, as demonstrated by bone marrow transfer experiments between wild-type and pIV(-/-) mice. Medullary thymic epithelial cells (mTECs) are also MHCII(-) in pIV(-/-) mice. Bone marrow-derived, professional antigen-presenting cells (APCs) retain normal MHCII expression in pIV(-/-) mice, including those believed to mediate negative selection in the thymic medulla. Endogenous retroviruses thus retain their ability to sustain negative selection of the residual CD4(+) thymocytes in pIV(-/-) mice. Interestingly, the passive acquisition of MHCII molecules by thymocytes is abrogated in pIV(-/-) mice. This identifies thymic epithelial cells as the source of this passive transfer. In peripheral lymphoid organs, the CD4(+) T-cell population of pIV(-/-) mice is quantitatively and qualitatively comparable to that of MHCII-deficient mice. It comprises a high proportion of CD1-restricted natural killer T cells, which results in a bias of the V beta repertoire of the residual CD4(+) T-cell population. We have also addressed the identity of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common gamma chain (CD132) or common beta chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia.