Sl Field

Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia

We previously demonstrated prolonged, profound CD4+ T-lymphopenia in rheumatoid arthritis (RA) patients following lymphocyte-depleting therapy. Poor reconstitution could result either from reduced de novo T-cell production through the thymus or from poor peripheral expansion of residual T-cells. Interleukin-7 (IL-7) is known to stimulate the thymus to produce new T-cells and to allow circulating mature T-cells to expand, thereby playing a critical role in T-cell homeostasis. In the present study we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also compromised in RA. To investigate whether such an IL-7 deficiency could account for the prolonged lymphopenia observed in RA following therapeutic lymphodepletion, we compared RA patients and patients with solid cancers treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but this was sustained in RA patients at 12 months, as compared with the reconstitution that occurred in cancer patients by 3–4 months. Both cohorts produced naïve T-cells containing T-cell receptor excision circles. The main distinguishing feature between the groups was a failure to expand peripheral T-cells in RA, particularly memory cells during the first 3 months after treatment. Most importantly, there was no increase in serum IL-7 levels in RA, as compared with a fourfold rise in non-RA control individuals at the time of lymphopenia. Our data therefore suggest that RA patients are relatively IL-7 deficient and that this deficiency is likely to be an important contributing factor to poor early T-cell reconstitution in RA following therapeutic lymphodepletion. Furthermore, in RA patients with stable, well controlled disease, IL-7 levels were positively correlated with the T-cell receptor excision circle content of CD4+ T-cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort.

NSAIDS inhibit in vitro MSC chondrogenesis but not osteogenesis: implications for mechanism of bone formation inhibition in man

The non‐steroidal anti‐inflammatory drugs (NSAIDs) are widely used for analgesia but may inhibit bone formation. We investigated whether the reported NSAID effect on bone is related to inhibition of bone marrow mesenchymal stem cell (MSC) proliferation and osteogenic and chondrogenic differentiation and evaluated both cyclooxygenase (COX)‐1 and COX‐2 specific drugs. The effects of seven COX‐1 and COX‐2 inhibitors on MSC proliferation and osteogenic and chondrogenic differentiation were tested using Vybrant, sodium 3′‐[1‐(phenylaminocarbonyl)‐ 3,4‐tetrazolium]‐bis (4‐methoxy‐6‐nitro) benzene sulfonic acid hydrate (XTT), functional and quantitative assays of MSC differentiation. The MSC expression of COX‐1 and COX‐2 and prostaglandin E2 (PGE‐2) levels were evaluated serially during lineage differentiation by quantitative PCR and ELISA. None of the NSAIDs at broad range of concentration (range 10−3 to 100 μg/ml) significantly affected MSC proliferation. Surprisingly, MSC osteogenic differentiation inhibition was not evident. However, NSAIDs affected chondrogenic potential with a reduction in sulphated glycosaminoglycans (sGAG) content by 45% and 55% with diclofenac and ketorolac, respectively (P < 0.05 compared to controls). Parecoxib and meloxicam, more COX‐2 specific reagents inhibited sGAG to a lesser degree, 22% and 27% respectively (P < 0.05 compared to controls). Cartilage pellet immunohistochemistry confirmed the above results. Pellet chondrogenesis was associated with increased COX‐1 expression levels but not COX‐2, and COX‐1 specific drugs suppressed MSC PGE‐2 more than COX‐2 specific inhibitors. These findings suggest that NSAIDs may inhibit bone formation via blockage of MSC chondrogenic differentiation which is an important intermediate phase in normal endochondral bone formation.

Abnormal T cell differentiation persists in patients with rheumatoid arthritis in clinical remission and predicts relapse

Objectives: An abnormal CD4+ T cell subset related to inflammation exposure (inflammation-related cells, IRC) has been identified in rheumatoid arthritis (RA). Patients with inflammatory and non-inflammatory diseases were used to examine the relationship between inflammation and this T cell subset in vivo. Methods: Blood was collected from healthy controls and patients with RA (active disease or in clinical remission), Crohn’s disease and osteoarthritis. IRC and chemokine receptors were quantified by flow cytometry. Thymic activity and apoptotic factors were measured by real-time polymerase chain reaction. Circulating cytokines were measured by enzyme-linked immunosorbent assay. CXCR4 and SDF1 in synovial biopsies were measured using immunohistochemistry. Results: IRC were identified in patients with RA (p<0.0001) and Crohn’s disease (p = 0.005), but not in those with osteoarthritis. In RA in remission, IRC persisted (p<0.001). In remission, hyperproliferation of IRC was lost, chemokine receptor expression was significantly lowered (p<0.007), Bax expression dropped significantly (p<0.001) and was inversely correlated with IRC (rho = −0.755, p = 0.03). High IRC frequency in remission was associated with relapse within 18 months (OR = 6.4, p<0.001) and a regression model predicted 72% of relapse. Conclusions: These results suggest a model in which, despite the lack of systemic inflammation, IRC persist in remission, indicating that IRC are an acquired feature of RA. They have, however, lost their hyper-responsiveness, acquired a potential for survival, and no longer express chemokine receptors. IRC persistence in remission confirms their important role in chronic inflammation as circulating precursors of pathogenic cells. This was further demonstrated by much higher incidence of relapse in patients with high IRC frequency in remission.

Predicting oocyte fertilisability in intracytoplasmic sperm injection cycles: a retrospective observational study

Abstract Background Conception assisted by intracytoplasmic sperm injection (ICSI) requires oocyte stripping for morphological evaluation of maturity status. However, this approach prevents further maturation and poorly predicts fertilisability, so more robust assessment strategies are needed. Given that cytokines orchestrate oocyte development, we aimed to assess the association of follicular fluid cytokine profiles with maturation stage and develop predictive machine learning-based methods to identify those with the greatest fertilisation potential. Methods In this retrospective study, follicular fluid was collected at oocyte retrieval from 64 women and linked to oocyte maturity status or fate—namely, germinal vesicle (n=26), metaphase I (51), metaphase II not fertilised (51), and metaphase II fertilised (84). 51 follicular fluid cytokines were profiled by multiplex immunoassay. Machine learning-based classifiers to predict oocyte fertilisability were subjected to iterative feature reduction to a threshold suitable for developing a clinically viable assessment of oocyte maturity. Women gave written, informed consent. Findings Cytokine profiles varied dynamically throughout maturation. When applied to naive samples with known outcome, classifiers developed using tumour necrosis factor-related apoptosis-inducing ligand and interleukin 18 profiles alone correctly discriminated 89% of metaphase II not fertilised oocytes (ie, those with the highest fertilisability) with high confidence from all other maturation stages. Interpretation These classifiers offer the prospect of cost-effective, point-of-care testing, and streamlined ICSI-based workflows. This assessment circumvents stripping such that immature or low fertilisability oocytes could benefit from in-vitro maturation and increase the pool of usable oocytes. Further studies will confirm the robustness of these classifiers in women with a broader morbidity spectrum and their translational value across a range of clinical settings. Funding Infertility Research Trust.

A Bayesian view of murine seminal cytokine networks

It has long been established that active agents in seminal fluid are key to initiating and coordinating mating-induced immunomodulation. This is in part governed by the actions of a network of cytokine interactions which, to date, remain largely undefined, and whose interspecific evolutionary conservation is unknown. This study applied Bayesian methods to illustrate the interrelationships between seminal profiles of interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-gamma, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1) alpha, MIP-1beta, regulated on activation normal T cell expressed and secreted (RANTES), tumour necrosis factor (TNF)-alpha, leptin, inducible protein (IP)-10 and vascular endothelial growth factor (VEGF) in a rat model. IL-2, IL-9, IL-12 (p70), IL-13, IL-18, eotaxin, IFN-gamma, IP-10, KC, leptin, MCP-1, MIP-1alpha and TNF-alpha were significantly higher in serum, whilst IL-1beta, IL-5, IL-6, IL-10, IL-17, G-CSF and GM-CSF were significantly higher in seminal fluid. When compared to mouse profiles, only G-CSF was present at significantly higher levels in the seminal fluid in both species. Bayesian modelling highlighted key shared features across mouse and rat networks, namely TNF-alpha as the terminal node in both serum and seminal plasma, and MCP-1 as a central coordinator of seminal cytokine networks through the intermediary of KC and RANTES. These findings reveal a marked interspecific conservation of seminal cytokine networks.

Circulating levels of IL-7 in rheumatoid arthritis

We have previously demonstrated reduced circulating levels of IL-7 in active rheumatoid arthritis (RA) compared with health [1]. Controversy exist as to whether IL-7 correlates or not with C-reactive protein (CRP) in active disease or with other disease activity markers. Normal IL-7 levels were, however, found in 50% of patients in clinical remission and correlated with the recovery of thymic activity [1]. Patients in clinical remission may represent a heterogeneous group, and the aim of this work was to identify predictors of IL-7 recovery in demographic, clinical, imaging and functional data and to compare them with IL-7 in active disease. One hundred and six patients deemed to be in clinical remission were recruited: stable disease for the preceding 6 months, previous disease duration of at least 12 months, no clinically significant synovitis, CRP below 15 mg/l for the preceding 6 months. Blood and serum were collected from these patients (n = 106). Clinical data and imaging data were gathered at the time of sampling. High-sensitivity ELISA was used for cytokine analysis, proliferation assays for T-cell function (mitogen, T-cell receptor stimulation, IL-2, recall antigens); real-time PCR to quantify T-bet and GATA3 expression, and DNA sequencing was carried out to investigate two genetic polymorphisms in the IL-7 gene. Several studies in healthy controls indicate that normal levels of circulating IL-7 are between 10 and 25 pg/ml. In remission, circulating levels of IL-7 vary between 2.47 and 23.85 pg/ml. Recovery of T-cell function was directly related to levels of circulating IL-7 in vivo (P < 0.001, R = 0.873 for phytohaemagglutinin, R = 0.786 for T-cell receptor stimulation and R = 0.821 for recall antigen). Age and sex had no effect on IL-7. Combining active (n = 35) and remission patients (n = 106) showed no indication of a relationship between IL-7 and routine measures of disease activity (CRP, DAS28, erythrocyte sedimentation rate, plasma viscosity and joint counts), suggesting that recovery of IL-7 is not an indicator of remission. To confirm this observation, we used imaging data (MRI and US assessment of hand and wrist) to address whether subclinical disease could predict lack of IL-7 recovery. Evidence of subclinical synovitis was found in 96% of patients in remission [2] despite no evidence of clinically significant synovitis and there was no relationship between IL-7 and imaging scores. However, we found that levels of IL-7 in remission and the age of the patient at disease onset correlate (P < 0.001, R = 0.498, n = 86). A family history of RA and smoking at the time of onset (both self-reported) were also strongly associated with lower levels of IL-7 (both P < 0.001, n = 66). We analysed two polymorphisms in the promoter and enhancer regions of the IL-7 gene. We found no allele frequency difference between RA patients and healthy controls, remission patients with or without family history, or in relation to IL-7 levels. Finally we analysed circulating cytokines known to regulate IL-7 expression, and found a strong correlation between IL-7 and IFNγ (P < 0.03, R = 0.650, n = 10). Since IL-7 is a co-activator of Th1 polarisation, we analysed the expression of T-bet and found a direct relationship between the two (P < 0.01, R = 0.601, n = 15) but not with GATA3, which is a regulator of Th2 polarisation. Recent evidence suggests an important role for IL-7 in the pathogenesis of RA [3]. Our data demonstrate that circulating IL-7 is not an indicator of disease activity. Despite the relationship with age at onset (genetic anticipation) and the family history association, levels of IL-7 in the circulation are apparently not driven by these two polymorphisms. Our results suggest that Th1 polarisation and IL-7 are related and that the mechanism by which IL-7 is recovered in remission may be associated with the recovery of Th1 polarisation.