E. Taher

Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway

Objectives To establish if changes in Th1/Th17 cell populations previously reported in experimental arthritis occur in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor α (TNFα) agents, and whether the therapeutic response to anti-TNFα is compromised in patients and mice because of elevated Th17/IL-17 levels. Finally, to assess the efficacy of combined blockade of anti-TNFα and anti-IL-17 in experimental arthritis. Methods A longitudinal study of two independent cohorts (cohort 1, n=24; cohort 2, n=19) of patients with RA treated with anti-TNFα biological agents was carried out to assess their Th17/IL-17 levels before and after the start of anti-TNFα therapy. IL-12/23p40 production was assessed in plasma Peripheral blood lymphocytes (PBLs) and monocytes. Mice with collagen-induced arthritis (CIA) were treated with anti-TNFα alone, anti-IL17 alone or a combination of the two. Efficacy of treatment and response was assessed from changes in Disease Activity Score 28–erythrocyte sedimentation rate scores in patients, and in clinical scores and histological analysis in CIA. Results Significant increases in circulating Th17 cells were observed in patients after anti-TNFα therapy and this was accompanied by increased production of IL-12/23p40. There was an inverse relationship between baseline Th17 levels and the subsequent response of patients with RA to anti-TNFα therapy. In addition, PBLs from non-responder patients showed evidence of increased IL-17 production. Similarly, in anti-TNFα-treated mice, there was a strong correlation between IL-17 production and clinical score. Finally combined blockade of TNFα and IL-17 in CIA was more effective than monotherapy, particularly with respect to the duration of the therapeutic effect. Conclusions These findings, which need to be confirmed in a larger cohort, suggest that a Th17-targeted therapeutic approach may be useful for anti-TNFα non-responder patients or as an adjunct to anti-TNFα therapy, provided that safety concerns can be addressed.

B Lymphocyte Cytokines and Rheumatic Autoimmune Disease

There is some debate as to which type of lym-phocyte prevails in the pathogenesis of autoimmunity.This controversy is borne out by the fact that T cellshave, for years, been viewed as competent in their ownright to initiate rheumatoid arthritis (RA), primarySjo¨gren’s syndrome (SS), and systemic lupus erythema-tosus (SLE), while B cells were confined to a subservientrole as producers of antibodies to self antigens. Doubtshave, however, been cast when T cell–directed treat-ments proved to be disappointing.B cells have attracted significant interest follow-ing new information on the key roles they play in RA,SS, and SLE. Encouraged by the promising results ofsmall-scale clinical trials of the anti-CD20 antibodyrituximab in patients with RA (1), several groups haveembarked on similar treatment of patients with RA, SS,or SLE (2). The clinical benefits were found to becorrelated with a decline in B cell numbers, but not witha reduction in the levels of most autoantibodies, exceptfor rheumatoid factor and anti–cyclic citrullinated pep-tide (3). This finding implies that B cell depletion mayameliorate these disorders through mechanisms otherthan suppressing autoantibody production. Given the Bcell dependence of T cell activation in the rheumatoidjoint, it makes sense that heavy infiltration of B cells intothe synovium accompanies a modest response to ritux-imab, that patients in whom depletion of circulating Bcells is incomplete have worse outcomes than those inwhom it is complete (4), and that removal of B cellssignificantly reduces the severity of experimental arthri-tis in mice (5).Furthermore, consistent with this view are theresults of experiments in SLE-prone mice geneticallyengineered to be deficient in B cells. While mice with noB lymphocytes do not suffer from end-organ damage,those with B cells incapable of antibody secretion retainmany features of the syndrome (6). Such observationsindicate that B cells promote autoimmunity by interact-ing with any one of several pathways that impact theregulation of immunity to the self. Although a role of Blymphocytes in SLE, RA, and SS has been suspected formany years, recent discoveries have unveiled new in-sights into B cell–derived cytokines, includinginterferon- (IFN ) and interleukin-4 (IL-4), that mod-ulate the response. They are likely to serve as effectorsof some biologic functions of B cells.Early studies of the functions of B lymphocytesfocused on splitting the B cell population into subpopu-lations. For example, B lymphocytes are classified basedon the expression of CD5 as B-1 cells, which express thisT cell marker, and B-2 cells, which do not. The ensuingmodel of the maturation of B lymphocytes asserts thatB-2 cells leave the bone marrow as immature B cells,populate secondary lymphoid organs as transitional type1 (T1) cells, and evolve into type 2 (T2) cells, whichdifferentiate either into marginal zone or follicular Bcells. Upon antigen encounter, these latter B cellsinitiate the development of a germinal center (GC).

Protein phosphorylation and kinome profiling reveal altered regulation of multiple signaling pathways in B lymphocytes from patients with systemic lupus erythematosus

OBJECTIVE The cause of B lymphocyte hyperactivity and autoantibody production in systemic lupus erythematosus (SLE) remains unclear. Previously, we identified abnormalities in the level and translocation of signaling molecules in B cells in SLE patients. The present study was undertaken to examine the extent of signaling abnormalities that relate to altered B cell responses in SLE. METHODS B lymphocytes from 88 SLE patients and 72 healthy controls were isolated from blood by negative selection. Protein tyrosine phosphorylation and cellular kinase levels were analyzed by Western blotting, flow cytometry, and a kinome array protocol. Changes in protein phosphorylation were determined in ex vivo B cells and following B cell receptor engagement. RESULTS Differences in tyrosine phosphorylation in B cells from patients with SLE, compared with matched controls, were demonstrated. Further, the kinome array analysis identified changes in the activation of key kinases, i.e., the activity of phosphatidylinositol 3-kinase, which regulates survival and differentiation, was up-regulated and the activity of Rac and Rho kinases, which regulate the cytoskeleton and migration, was increased. In contrast, the activity of ATR, which regulates the cell cycle, was down-regulated in SLE patients compared with controls. Differences in signaling pathways were seen in all SLE B lymphocyte subsets that manifested phenotypic features of immature, mature, and memory cells. CONCLUSION This study revealed dysregulation in multiple signaling pathways that control key responses in B cells of SLE patients. Data generated in this study provide a molecular basis for further analysis of the altered B lymphocyte responses in SLE.

CD5 expression promotes multiple intracellular signaling pathways in B lymphocyte.

CD5(+) B lymphocytes have distinct functional properties compared with B lymphocytes that lack CD5. However, it remains unclear if and how the CD5 molecule modulates B lymphocyte biology and responses. Our recent studies have revealed that CD5 promotes constitutive activation of multiple signaling pathways including extracellular signal-regulated kinases (ERK1/2), phosphatidylinositol 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) and calcineurin-NFAT signaling pathways. Further, changes in cytokine production including the production of IL-10 are related to the activation of the transcription factors NFAT2 and STAT3. All in all, these studies provide a framework for understanding how CD5 impacts B lymphocyte biology and responses.

Targeted delivery of cytokine therapy to rheumatoid tissue by a synovial targeting peptide

Objectives The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. Methods Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. Results In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1β in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. Conclusions The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.

Harnessing the Therapeutic Potential of Th17 Cells.

Th17 cells provide protective immunity to infections by fungi and extracellular bacteria as well as cancer but are also involved in chronic inflammation. The cells were first identified by their ability to produce interleukin 17A (IL-17A) and, subsequently, associated with chronic inflammation and autoimmunity. Th17 cells have some gene profile similarity with stem cells and can remain dormant in mucosal tissues for long periods. Indeed, recent studies suggest that functionally distinct subsets of pro- and anti-inflammatory Th17 cells can interchange phenotype and functions. For development, Th17 cells require activation of the transcription factors STAT3 and RORγt while RUNX1, c-Maf, and Aiolos are involved in changes of phenotype/functions. Attempts to harness Th17 cells against pathogens and cancer using vaccination strategies are being explored. The cells gain protective abilities when induced to produce interferon γ (IFNγ). In addition, treatment with antibodies to IL-17 is effective in treating patients with psoriasis, psoriatic arthritis, and refectory rheumatoid arthritis. Moreover, since RORγt is a nuclear receptor, it is likely to be a potential future drug target for modulating Th17 functions. This review explores pathways through which Th17 subsets are induced, the molecular basis of their plasticity, and potential therapeutic strategies for their modulation in diseases.

Enhanced propensity of T lymphocytes in patients with systemic lupus erythematosus to apoptosis in the presence of tumour necrosis factor alpha

Objective: To determine the effect of inflammation through exposure to tumour necrosis factor (TNF)α on T lymphocytes in patients with systemic lupus erythematosus (SLE). Methods: We studied the effect of TNFα on T‐lymphocyte apoptosis in patients with SLE, rheumatoid arthritis (RA), and in healthy controls. Apoptosis of CD4 and CD8 T lymphocytes and naive and memory subpopulations was determined by flow cytometry using 7‐amino‐actinomycin D (7AAD) and propidium iodide (PI). In SLE, apoptosis was studied in patients with active and inactive disease and in patients on different medications. Results: TNFα enhanced apoptosis of anti‐CD3‐activated T lymphocytes. The percentage of apoptotic cells was significantly higher in T lymphocytes from patients with SLE than RA patients and healthy controls. After 3 days of culture, 38% of CD4+ and 37% of CD8+ cells from SLE patients underwent apoptosis in the presence of TNFα compared with 25% CD4+ and 26% CD8+ T cells from the controls (p<0.001). In healthy controls, more memory than naive T lymphocytes underwent apoptosis. By contrast, in patients with SLE, more naive T cells underwent apoptosis with TNFα (p<0.01). Enhanced apoptosis of T cells in SLE was independent of disease activity or medication. Finally, inhibition experiments showed that apoptosis in the presence of TNFα was only partly blocked with anti‐Fas ligand (FasL) antibody. Conclusions: This study demonstrates that T lymphocytes in patients with SLE are more prone to apoptosis in the presence of TNFα than T lymphocytes from healthy controls. Defects in TNFα signalling pathways rather than distribution of TNF receptors (TNFRs) probably explain the enhanced apoptosis in SLE.

TNFα in the regulation of Treg and Th17 cells in rheumatoid arthritis and other autoimmune inflammatory diseases.

HighlightsTNF&agr; mediates cell responses and inflammation by binding to TNF receptors 1 and 2.In T cells, TNF&agr; can promote cell death or proliferation depending on context.TNF&agr; enhances T cell responses but sustained exposure renders them hypo‐responsive.TNF&agr; signalling molecule polymorphisms are autoimmune disease susceptibility factors.Tregs and Th17 cells are modulated by TNF&agr; and its blockade alters their numbers. ABSTRACT TNF&agr; is a principal pro‐inflammatory cytokine vital for immunity to infections. However, its excessive production is involved in chronic inflammation and disease pathology in autoimmune diseases. Evidence for its pathogenic role is validated by the fact that its neutralisation by therapeutic agents in vivo is beneficial in ameliorating disease and controlling symptoms. Paradoxically, however, treatment with TNF&agr; inhibitors can either have no clinical effects, or even exacerbate disease in some patients. The explanation for such contradictory outcomes may lay in how and which downstream signalling pathways are activated and drive disease. TNF&agr; causes its effects by binding to either or both of two membrane‐bound receptors, TNFR1 and TNFR2. Engagement of the receptors can induce cell death or cell proliferation. T cells both produce and respond to TNF&agr; and depending on whether the cytokine is membrane‐bound or soluble and the level of expression of its two receptors, the biological outcome can be distinct. In addition, polymorphisms in genes encoding TNF&agr; and T cell signalling proteins can significantly impact the outcome of TNF&agr; receptor engagement. Early studies revealed that effector T cells in patients with rheumatoid arthritis (RA) are hyporesponsive due to chronic exposure to TNF&agr;. However, recent evidence indicates that the relationship between TNF&agr; and T cell responses is complex and, at times, can be paradoxical. In addition, there is controversy as to the specific effects of TNF&agr; on different T cell subsets. This review will summarise knowledge on how TNF&agr; modulates T cell responses and the effect of engaging either of its two receptors. Furthermore, we discuss how such interactions can dictate the outcome of treatment with TNF&agr; inhibitors.

Repopulation of B-lymphocytes with restricted gene expression using haematopoietic stem cells engineered with lentiviral vectors.

B-lymphocytes play a key role in the pathogenesis of many immune-mediated diseases, such as autoimmune and atopic diseases. Therefore, targeting B-lymphocytes provides a rationale for refining strategies to treat such diseases for long-term clinical benefits and minimal side effects. In this study we describe a protocol for repopulating irradiated mice with B-lymphocytes engineered for restricted expression of transgenes using haematopoietic stem cells. A self-inactivating lentiviral vector, which encodes enhanced green fluorescence protein (EGFP) from the spleen focus-forming virus (SFFV) promoter, was used to generate new vectors that permit restricted EGFP expression in B-lymphocytes. To achieve this, the SFFV promoter was replaced with the B-lymphocyte-restricted CD19 promoter. Further, an immunoglobulin heavy chain enhancer (Eμ) flanked by the associated matrix attachment regions (MARs) was inserted upstream of the CD19 promoter. Incorporation of the Eμ-MAR elements upstream of the CD19 promoter resulted in enhanced, stable and selective transgene expression in human and murine B-cell lines. In addition, this modification permitted enhanced selective EGFP expression in B-lymphocytes in vivo in irradiated mice repopulated with transduced bone marrow haematopoietic stem cells (BMHSCs). The study provides evidence for the feasibility of targeting B-lymphocytes for therapeutic restoration of normal B-lymphocyte functions in patients with B-cell-related diseases.

B-Lymphocyte Signalling Abnormalities and Lupus Immunopathology

Lupus is a complex autoimmune rheumatic disease of unknown aetiology. The disease is associated with diverse features of immunological abnormality in which B-lymphocytes play a central role. However, the cause of atypical B-lymphocyte responses remains unclear. In this article, we provide a synopsis of current knowledge on intracellular signalling abnormalities in B-lymphocytes in lupus and their potential effects on the response of these cells in mouse models and in patients. There are numerous reported defects in the regulation of intracellular signalling proteins and pathways in B-lymphocytes in lupus that, potentially, affect critical biological responses. Most of the evidence for these defects comes from studies of disease models and genetically engineered mice. However, there is also increasing evidence from studying B-lymphocytes from patients and from genome-wide linkage analyses for parallel defects to those observed in mice. These studies provide molecular and genetic explanations for the key immunological abnormalities associated with lupus. Most of the new information appears to relate to defects in intracellular signalling that impact B-lymphocyte tolerance, cytokine production and responses to infections. Some of these abnormalities will be discussed within the context of disease pathogenesis.