Immunogenicity of third dose COVID-19 vaccine strategies in patients who are immunocompromised with suboptimal immunity following two doses (OCTAVE-DUO): an open-label, multicentre, randomised, controlled, phase 3 trial.

BACKGROUND: The humoral and T-cell responses to booster COVID-19 vaccine types in multidisease immunocompromised individuals who do not generate adequate antibody responses to two COVID-19 vaccine doses, is not fully understood. The OCTAVE DUO trial aimed to determine the value of third vaccinations in a wide range of patients with primary and secondary immunodeficiencies. METHODS: OCTAVE-DUO was a prospective, open-label, multicentre, randomised, controlled, phase 3 trial investigating humoral and T-cell responses in patients who are immunocompromised following a third vaccine dose with BNT162b2 or mRNA-1273, and of NVX-CoV2373 for those with lymphoid malignancies. We recruited patients who were immunocompromised from 11 UK hospitals, aged at least 18 years, with previous sub-optimal responses to two doses of SARS-CoV-2 vaccine. Participants were randomly assigned 1:1 (1:1:1 for those with lymphoid malignancies), stratified by disease, previous vaccination type, and anti-spike antibody response following two doses. Individuals with lived experience of immune susceptibility were involved in the study design and implementation. The primary outcome was vaccine-specific immunity defined by anti-SARS-CoV-2 spike antibodies (Roche Diagnostics UK and Ireland, Burgess Hill, UK) and T-cell responses (Oxford Immunotec, Abingdon, UK) before and 21 days after the third vaccine dose analysed by a modified intention-to-treat analysis. The trial is registered with the ISRCTN registry, ISRCTN 15354495, and the EU Clinical Trials Register, EudraCT 2021-003632-87, and is complete. FINDINGS: Between Aug 4, 2021 and Mar 31, 2022, 804 participants across nine disease cohorts were randomly assigned to receive BNT162b2 (n=377), mRNA-1273 (n=374), or NVX-CoV2373 (n=53). 356 (45%) of 789 participants were women, 433 (55%) were men, and 659 (85%) of 775 were White. Anti-SARS-CoV-2 spike antibodies measured 21 days after the third vaccine dose were significantly higher than baseline pre-third dose titres in the modified intention-to-treat analysis (median 1384 arbitrary units [AU]/mL [IQR 4·3-7990·0] compared with median 11·5 AU/mL [0·4-63·1]; p<0·001). Of participants who were baseline low responders, 380 (90%) of 423 increased their antibody concentrations to more than 400 AU/mL. Conversely, 166 (54%) of 308 baseline non-responders had no response after the third dose. Detectable T-cell responses following the third vaccine dose were seen in 494 (80%) of 616 participants. There were 24 serious adverse events (BNT612b2 eight [33%] of 24, mRNA-1273 12 [50%], NVX-CoV2373 four [17%]), two (8%) of which were categorised as vaccine-related. There were seven deaths (1%) during the trial, none of which were vaccine-related. INTERPRETATION: A third vaccine dose improved the serological and T-cell response in the majority of patients who are immunocompromised. Individuals with chronic renal disease, lymphoid malignancy, on B-cell targeted therapies, or with no serological response after two vaccine doses are at higher risk of poor response to a third vaccine dose. FUNDING: Medical Research Council, Blood Cancer UK.

Application of clinical and molecular profiling data to improve patient outcomes in psoriatic arthritis.

Achieving a good outcome for a person with Psoriatic Arthritis (PsA) is made difficult by late diagnosis, heterogenous clinical disease expression and in many cases, failure to adequately suppress inflammatory disease features. Single-centre studies have certainly contributed to our understanding of disease pathogenesis, but to adequately address the major areas of unmet need, multi-partner, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient-representative organizations. In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for better short-term and long-term outcomes.

Improving outcomes in Psoriatic Arthritis Psoriatic Arthritis (PsA) is a form of arthritis which is found in approximately 30% of people who have the skin condition, Psoriasis. Frequently debilitating and progressive, achieving a good outcome for a person with PsA is made difficult by late diagnosis, disease clinical features and in many cases, failure to adequately control features of inflammation. Research studies from individual centres have certainly contributed to our understanding of why people develop PsA but to adequately address the major areas of unmet need, multi-centre, collaborative research programmes are now required. HIPPOCRATES is a 5-year, Innovative Medicines Initiative (IMI) programme which includes 17 European academic centres experienced in PsA research, 5 pharmaceutical industry partners, 3 small-/medium-sized industry partners and 2 patient representative organisations (see appendix). In this review, the ambitious programme of work to be undertaken by HIPPOCRATES is outlined and common approaches and challenges are identified. The participation of patient research partners in all stages of the work of HIPPOCRATES is highlighted. It is expected that, when completed, the results will ultimately allow for changes in the approaches to diagnosing, managing and treating PsA allowing for improvements in short-term and long-term outcomes.

Double-negative-2 B cells are the major synovial plasma cell precursor in rheumatoid arthritis.

B cells are key pathogenic drivers of chronic inflammation in rheumatoid arthritis (RA). There is limited understanding of the relationship between synovial B cell subsets and pathogenic antibody secreting cells (ASCs). This knowledge is crucial for the development of more targeted B-cell depleting therapies. While CD11c+ double-negative 2 (DN2) B cells have been suggested as an ASC precursor in lupus, to date there is no proven link between the two subsets in RA. We have used both single-cell gene expression and BCR sequencing to study synovial B cells from patients with established RA, in addition to flow cytometry of circulating B cells. To better understand the differentiation patterns within the diseased tissue, a combination of RNA-based trajectory inference and clonal lineage analysis of BCR relationships were used. Both forms of analysis indicated that DN2 B cells serve as a major precursors to synovial ASCs. This study advances our understanding of B cells in RA and reveals the origin of pathogenic ASCs in the RA synovium. Given the significant role of DN2 B cells as a progenitor to pathogenic B cells in RA, it is important to conduct additional research to investigate the origins of DN2 B cells in RA and explore their potential as therapeutic targets in place of the less specific pan-B cells depletion therapies currently in use.

IĸB Protein BCL3 as a Controller of Osteogenesis and Bone Health.

OBJECTIVE: IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. METHODS: To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6-14) lacking BCL3 (Bcl3-/- ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3-/- mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3-7) were assessed. Osteoclast differentiation and function in Bcl3-/- mice (n = 3-5) was assessed. Adult 20-week Bcl3-/- and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3-/- mice (n = 11-13). RESULTS: Evaluation of Bcl3-/- mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3-/- cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3-/- mice exhibited decreased pathological osteophyte formation (P < 0.05). CONCLUSION: Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.

SARS-CoV-2-specific immune responses and clinical outcomes after COVID-19 vaccination in patients with immune-suppressive disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune responses and infection outcomes were evaluated in 2,686 patients with varying immune-suppressive disease states after administration of two Coronavirus Disease 2019 (COVID-19) vaccines. Overall, 255 of 2,204 (12%) patients failed to develop anti-spike antibodies, with an additional 600 of 2,204 (27%) patients generating low levels (<380 AU ml-1). Vaccine failure rates were highest in ANCA-associated vasculitis on rituximab (21/29, 72%), hemodialysis on immunosuppressive therapy (6/30, 20%) and solid organ transplant recipients (20/81, 25% and 141/458, 31%). SARS-CoV-2-specific T cell responses were detected in 513 of 580 (88%) patients, with lower T cell magnitude or proportion in hemodialysis, allogeneic hematopoietic stem cell transplantation and liver transplant recipients (versus healthy controls). Humoral responses against Omicron (BA.1) were reduced, although cross-reactive T cell responses were sustained in all participants for whom these data were available. BNT162b2 was associated with higher antibody but lower cellular responses compared to ChAdOx1 nCoV-19 vaccination. We report 474 SARS-CoV-2 infection episodes, including 48 individuals with hospitalization or death from COVID-19. Decreased magnitude of both the serological and the T cell response was associated with severe COVID-19. Overall, we identified clinical phenotypes that may benefit from targeted COVID-19 therapeutic strategies.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14- mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

JAK inhibitors disrupt T cell-induced proinflammatory macrophage activation.

OBJECTIVES: Macrophage subsets, activated by T cells, are increasingly recognised to play a central role in rheumatoid arthritis (RA) pathogenesis. Janus kinase (JAK) inhibitors have proven beneficial clinical effects in RA. In this study, we investigated the effect of JAK inhibitors on the generation of cytokine-activated T (Tck) cells and the production of cytokines and chemokines induced by Tck cell/macrophage interactions. METHODS: CD14+ monocytes and CD4+ T cells were purified from peripheral blood mononuclear cells from buffy coats of healthy donors. As representative JAK inhibitors, tofacitinib or ruxolitinib were added during Tck cell differentiation. Previously validated protocols were used to generate macrophages and Tck cells from monocytes and CD4+ T cells, respectively. Cytokine and chemokine including TNF, IL-6, IL-15, IL-RA, IL-10, MIP1α, MIP1ß and IP10 were measured by ELISA. RESULTS: JAK inhibitors prevented cytokine-induced maturation of Tck cells and decreased the production of proinflammatory cytokines TNF, IL-6, IL-15, IL-1RA and the chemokines IL-10, MIP1α, MIP1ß, IP10 by Tck cell-activated macrophages in vitro (p<0.05). CONCLUSIONS: Our findings show that JAK inhibition disrupts T cell-induced macrophage activation and reduces downstream proinflammatory cytokine and chemokine responses, suggesting that suppressing the T cell-macrophage interaction contributes to the therapeutic effect of JAK inhibitors.

Phenotypic heterogeneity in psoriatic arthritis: towards tissue pathology-based therapy.

Psoriatic arthritis (PsA) is a heterogeneous disease involving multiple potential tissue domains. Most outcome measures used so far in randomized clinical trials do not sufficiently reflect this domain heterogeneity. The concept that pathogenetic mechanisms might vary across tissues within a single disease, underpinning such phenotype diversity, could explain tissue-distinct levels of response to different therapies. In this Review, we discuss the tissue, cellular and molecular mechanisms that drive clinical heterogeneity in PsA phenotypes, and detail existing tissue-based research, including data generated using sophisticated interrogative technologies with single-cell precision. Finally, we discuss how these elements support the need for tissue-based therapy in PsA in the context of existing and new therapeutic modes of action, and the implications for future PsA trial outcomes and design.

Targeting 3D chromosomal architecture at the RANK loci to suppress myeloma-driven osteoclastogenesis.

Bone disease represents a major cause of morbidity and mortality in Multiple Myeloma (MM); primarily driven by osteoclasts whose differentiation is dependent on expression of RANKL by MM cells. Notably, costimulation by ITAM containing receptors (i.e., FcγR) can also play a crucial role in osteoclast differentiation. Modeling the pathology of the bone marrow microenvironment with an ex vivo culture system of primary human multiple myeloma cells, we herein demonstrate that FcγR-mediated signaling, via staphylococcal protein A (SpA) IgG immune-complexes, can act as a critical negative regulator of MM-driven osteoclast differentiation. Interrogation of the mode-of-action revealed that FcγR-mediated signaling causes epigenetic modulation of chromosomal 3D architecture at the RANK promoter; with altered spatial orientation of a proximal super enhancer. Combined this leads to substantial down-regulation of RANK at a transcript, protein, and functional level. These observations shed light on a novel mechanism regulating RANK expression and provide a rationale for targeting FcγR-signaling for the amelioration of osteolytic bone pathology in disease.

A Specific Calprotectin Neo-epitope [CPa9-HNE] in Serum from Inflammatory Bowel Disease Patients Is Associated with Neutrophil Activity and Endoscopic Severity.

BACKGROUND AND AIMS: Endoscopy and the use of faecal calprotectin [faecal CP] are among the least-favoured methods for assessing disease activity by inflammatory bowel disease [IBD] patients; the handling/processing of faecal samples is also impractical. Therefore, we sought to develop a novel neo-epitope serum calprotectin enzyme-linked immunosorbent assay [ELISA], CPa9-HNE, with the aim of quantifying neutrophil activity and neutrophil extracellular trap [NET]-osis and proposing a non-invasive method for monitoring disease activity in IBD patients. METHODS: In vitro cleavage was performed by mixing calprotectin [S100A9/S100A8] with human neutrophil elastase [HNE], and a novel HNE-derived calprotectin neo-epitope [CPa9-HNE] was identified by mass spectrometry for ELISA development. The CPa9-HNE ELISA was quantified in supernatants from ex vivo activated neutrophils and serum samples from patients with ulcerative colitis [UC, n = 43], Crohn's disease [CD, n = 93], and healthy subjects [HS, n = 23]. For comparison, faecal CP and MRP8/14 biomarkers were also measured. RESULTS: CPa9-HNE was specific for activated neutrophils ex vivo. Serum CPa9-HNE levels were 4-fold higher in CD [p <0.0001] and UC [p <0.0001] patients than in HS. CPa9-HNE correlated well with the Simple Endoscopic Score [SES]-CD score [r = 0.61, p <0.0001], MES [r = 0.46, p = 0.0141], and the full Mayo score [r = 0.52, p = 0.0013]. CPa9-HNE was able to differentiate between CD and UC patients in endoscopic remission and moderate/severe disease activity (CD: area under the curve [AUC] = 0.82 [p = 0.0003], UC: AUC = 0.87 [p = 0.0004]). The performance of CPa9-HNE was equipotent or slightly better than that of faecal CP. CONCLUSIONS: Serum CPa9-HNE levels were highly associated with CD and UC patients. CPa9-HNE correlated with the SES-CD score and the full Mayo score, indicating a strong association with disease activity.

Annexin-A1: The culprit or the solution?

Annexin-A1 has a well-defined anti-inflammatory role in the innate immune system, but its function in adaptive immunity remains controversial. This glucocorticoid-induced protein has been implicated in a range of inflammatory conditions and cancers, as well as being found to be overexpressed on the T cells of patients with autoimmune disease. Moreover, the formyl peptide family of receptors, through which annexin-A1 primarily signals, has also been implicated in these diseases. In contrast, treatment with recombinant annexin-A1 peptides resulted in suppression of inflammatory processes in murine models of inflammation. This review will focus on what is currently known about annexin-A1 in health and disease and discuss the potential of this protein as a biomarker and therapeutic target.

Human olfactory mesenchymal stromal cell transplantation ameliorates experimental autoimmune encephalomyelitis revealing an inhibitory role for IL16 on myelination.

One of the therapeutic approaches for the treatment of the autoimmune demyelinating disease, multiple sclerosis (MS) is bone marrow mesenchymal stromal cell (hBM-MSCs) transplantation. However, given their capacity to enhance myelination in vitro, we hypothesised that human olfactory mucosa-derived MSCs (hOM-MSCs) may possess additional properties suitable for CNS repair. Herein, we have examined the efficacy of hOM-MSCs versus hBM-MSCs using the experimental autoimmune encephalomyelitis (EAE) model. Both MSC types ameliorated disease, if delivered during the initial onset of symptomatic disease. Yet, only hOM-MSCs improved disease outcome if administered during established disease when animals had severe neurological deficits. Histological analysis of spinal cord lesions revealed hOM-MSC transplantation reduced blood-brain barrier disruption and inflammatory cell recruitment and enhanced axonal survival. At early time points post-hOM-MSC treatment, animals had reduced levels of circulating IL-16, which was reflected in both the ability of immune cells to secrete IL-16 and the level of IL-16 in spinal cord inflammatory lesions. Further in vitro investigation revealed an inhibitory role for IL-16 on oligodendrocyte differentiation and myelination. Moreover, the availability of bioactive IL-16 after demyelination was reduced in the presence of hOM-MSCs. Combined, our data suggests that human hOM-MSCs may have therapeutic benefit in the treatment of MS via an IL-16-mediated pathway, especially if administered during active demyelination and inflammation.

Nanovibrational stimulation inhibits osteoclastogenesis and enhances osteogenesis in co-cultures.

Models of bone remodelling could be useful in drug discovery, particularly if the model is one that replicates bone regeneration with reduction in osteoclast activity. Here we use nanovibrational stimulation to achieve this in a 3D co-culture of primary human osteoprogenitor and osteoclast progenitor cells. We show that 1000 Hz frequency, 40 nm amplitude vibration reduces osteoclast formation and activity in human mononuclear CD14+ blood cells. Additionally, this nanoscale vibration both enhances osteogenesis and reduces osteoclastogenesis in a co-culture of primary human bone marrow stromal cells and bone marrow hematopoietic cells. Further, we use metabolomics to identify Akt (protein kinase C) as a potential mediator. Akt is known to be involved in bone differentiation via transforming growth factor beta 1 (TGFß1) and bone morphogenetic protein 2 (BMP2) and it has been implicated in reduced osteoclast activity via Guanine nucleotide-binding protein subunit α13 (Gα13). With further validation, our nanovibrational bioreactor could be used to help provide humanised 3D models for drug screening.

Analysis of Prostate Cancer Tumor Microenvironment Identifies Reduced Stromal CD4 Effector T-cell Infiltration in Tumors with Pelvic Nodal Metastasis.

BACKGROUND: Pelvic nodal metastasis in prostate cancer impacts patient outcome negatively. OBJECTIVE: To explore tumor-infiltrating immune cells as a potential predictive tool for regional lymph node (LN) metastasis. DESIGN SETTING AND PARTICIPANTS: We applied multiplex immunofluorescence and targeted transcriptomic analysis on 94 radical prostatectomy specimens in patients with (LN+) or without (LN-) pelvic nodal metastases. Both intraepithelial and stromal infiltrations of immune cells and differentially expressed genes (mRNA and protein levels) were correlated with the nodal status. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The identified CD4 effector cell signature of nodal metastasis was validated in a comparable independent patient cohort of 184 informative cases. Patient outcome analysis and decision curve analysis were performed with the CD4 effector cell density-based signature. RESULTS AND LIMITATIONS: In the discovery cohort, both tumor epithelium and stroma from patients with nodal metastasis had significantly lower infiltration of multiple immune cell types, with stromal CD4 effector cells highlighted as the top candidate marker. Targeted gene expression analysis and confirmatory protein analysis revealed key alteration of extracellular matrix components in tumors with nodal metastasis. Of note, stromal CD4 immune cell density was a significant independent predictor of LN metastasis (odds ratio [OR] = 0.15, p = 0.004), and was further validated as a significant predictor of nodal metastasis in the validation cohort (OR = 0.26, p < 0.001). CONCLUSIONS: Decreased T-cell infiltrates in the primary tumor (particularly CD4 effector cells) are associated with a higher risk of LN metastasis. Future evaluation of CD4-based assays on prostate cancer diagnostic biopsy materials may improve selection of at-risk patients for the treatment of LN metastasis. PATIENT SUMMARY: In this report, we found that cancer showing evidence of cancer metastasis to the lymph nodes tends to have less immune cells present within the tumor. We conclude that the extent of immune cells present within a prostate tumor can help doctors determine the most appropriate treatment plan for individual patients.

BIOlogical Factors that Limit sustAined Remission in rhEumatoid arthritis (the BIO-FLARE study): protocol for a non-randomised longitudinal cohort study.

BACKGROUND: Our knowledge of immune-mediated inflammatory disease (IMID) aetiology and pathogenesis has improved greatly over recent years, however, very little is known of the factors that trigger disease relapses (flares), converting diseases from inactive to active states. Focussing on rheumatoid arthritis (RA), the challenge that we will address is why IMIDs remit and relapse. Extrapolating from pathogenetic factors involved in disease initiation, new episodes of inflammation could be triggered by recurrent systemic immune dysregulation or locally by factors within the joint, either of which could be endorsed by overarching epigenetic factors or changes in systemic or localised metabolism. METHODS: The BIO-FLARE study is a non-randomised longitudinal cohort study that aims to enrol 150 patients with RA in remission on a stable dose of non-biologic disease-modifying anti-rheumatic drugs (DMARDs), who consent to discontinue treatment. Participants stop their DMARDs at time 0 and are offered an optional ultrasound-guided synovial biopsy. They are studied intensively, with blood sampling and clinical evaluation at weeks 0, 2, 5, 8, 12 and 24. It is anticipated that 50% of participants will have a disease flare, whilst 50% remain in drug-free remission for the study duration (24 weeks). Flaring participants undergo an ultrasound-guided synovial biopsy before reinstatement of previous treatment. Blood samples will be used to investigate immune cell subsets, their activation status and their cytokine profile, autoantibody profiles and epigenetic profiles. Synovial biopsies will be examined to profile cell lineages and subtypes present at flare. Blood, urine and synovium will be examined to determine metabolic profiles. Taking into account all generated data, multivariate statistical techniques will be employed to develop a model to predict impending flare in RA, highlighting therapeutic pathways and informative biomarkers. Despite initial recruitment to time and target, the SARS-CoV-2 pandemic has impacted significantly, and a decision was taken to close recruitment at 118 participants with complete data. DISCUSSION: This study aims to investigate the pathogenesis of flare in rheumatoid arthritis, which is a significant knowledge gap in our understanding, addressing a major unmet patient need. TRIAL REGISTRATION: The study was retrospectively registered on 27/06/2019 in the ISRCTN registry 16371380 .

Clonal haematopoiesis of indeterminate potential: intersections between inflammation, vascular disease and heart failure.

Ageing is a major risk factor for the development of cardiovascular disease (CVD) and cancer. Whilst the cumulative effect of exposure to conventional cardiovascular risk factors is important, recent evidence highlights clonal haematopoiesis of indeterminant potential (CHIP) as a further key risk factor. CHIP reflects the accumulation of somatic, potentially pro-leukaemic gene mutations within haematopoietic stem cells over time. The most common mutations associated with CHIP and CVD occur in genes that also play central roles in the regulation of inflammation. While CHIP carriers have a low risk of haematological malignant transformation (<1% per year), their relative risk of mortality is increased by 40% and this reflects an excess of cardiovascular events. Evidence linking CHIP, inflammation and atherosclerotic disease has recently become better defined. However, there is a paucity of information about the role of CHIP in the development and progression of heart failure, particularly heart failure with preserved ejection fraction (HFpEF). While systemic inflammation plays a role in the pathophysiology of both heart failure with reduced and preserved ejection fraction (EF), it may be of greater relevance in the pathophysiology of HFpEF, which is also strongly associated with ageing. This review describes CHIP and its pathogenetic links with ageing, inflammation and CVD, while providing insight into its putative role in HFpEF.

TNF is a homoeostatic regulator of distinct epigenetically primed human osteoclast precursors.

OBJECTIVES: Circulating myeloid precursors are responsible for post-natal osteoclast (OC) differentiation and skeletal health, although the exact human precursors have not been defined. Enhanced osteoclastogenesis contributes to joint destruction in rheumatoid arthritis (RA) and tumour necrosis factor (TNF) is a well-known pro-osteoclastogenic factor. Herein, we investigated the interplay between receptor activator of nuclear factor kappa-Β ligand (RANK-L), indispensable for fusion of myeloid precursors and the normal development of OCs, and TNF in directing the differentiation of diverse pre-OC populations derived from human peripheral blood. METHODS: Flow cytometric cell sorting and analysis was used to assess the potential of myeloid populations to differentiate into OCs. Transcriptomic, epigenetic analysis, receptor expression and inhibitor experiments were used to unravel RANK-L and TNF signalling hierarchy. RESULTS: TNF can act as a critical homoeostatic regulator of CD14+ monocyte (MO) differentiation into OCs by inhibiting osteoclastogenesis to favour macrophage development. In contrast, a distinct previously unidentified CD14-CD16-CD11c+ myeloid pre-OC population was exempt from this negative regulation. In healthy CD14+ MOs, TNF drove epigenetic modification of the RANK promoter via a TNFR1-IKKß-dependent pathway and halted osteoclastogenesis. In a subset of patients with RA, CD14+ MOs exhibited an altered epigenetic state that resulted in dysregulated TNF-mediated OC homoeostasis. CONCLUSIONS: These findings fundamentally re-define the relationship between RANK-L and TNF. Moreover, they have identified a novel pool of human circulating non-MO OC precursors that unlike MOs are epigenetically preconditioned to ignore TNF-mediated signalling. In RA, this epigenetic preconditioning occurs in the MO compartment providing a pathological consequence of failure of this pathway.

Apremilast inhibits inflammatory osteoclastogenesis.

OBJECTIVES: Psoriatic arthritis (PsA) is associated with bone erosion and inflammation-induced bone loss, which are mediated by osteoclasts (OC) and modulated by inflammatory cytokines. Apremilast (APR) (a selective phosphodiesterase 4 inhibitor) is efficacious in PsA and acts by inhibiting cytokine production. However, there are no direct data informing whether and how APR affects osteoclast formation in humans. METHODS: Osteoclastogenic cytokine production by activated human peripheral blood mononuclear cells (PBMCs) was measured in the presence and absence of APR. Effects of APR on osteoclast differentiation were tested (i) in co-cultures of activated PBMCs and human CD14+ blood monocytes as well as (ii) in CD14+ blood monocytes stimulated with activated-PBMCs supernatant, TNF or IL-17A. Bone resorption was measured on OsteoAssay plates. Effects of APR on ex vivo osteoclast differentiation were compared in PsA, pre-PsA and psoriasis patients, as well as in healthy controls. RESULTS: APR significantly impaired the expression of key osteoclastogenic cytokines in activated PBMCs. Furthermore, APR dose-dependently and significantly inhibited activated PBMC-driven osteoclast differentiation and ex vivo osteoclast differentiation of PBMCs derived from PsA and pre-PsA patients, but not from psoriasis patients or healthy controls. TNF and IL-17A-enhanced osteoclastogenesis and osteolytic activity of CD14+ blood monocytes from PsA patients was also significantly inhibited by APR. Finally, APR inhibited expression of the key osteoclast fusion protein dendritic cell-specific transmembrane protein. CONCLUSION: Phosphodiesterase 4 targeting by APR not only inhibits osteoclastogenic cytokine production, but also directly suppresses inflammation-driven osteoclastogenesis. These data provide initial evidence that APR has the potential to provide a direct bone protective effect in PsA.

ß2 Integrins differentially regulate γδ T cell subset thymic development and peripheral maintenance.

The γδ T cells reside predominantly at barrier sites and play essential roles in immune protection against infection and cancer. Despite recent advances in the development of γδ T cell immunotherapy, our understanding of the basic biology of these cells, including how their numbers are regulated in vivo, remains poor. This is particularly true for tissue-resident γδ T cells. We have identified the ß2 family of integrins as regulators of γδ T cells. ß2-integrin-deficient mice displayed a striking increase in numbers of IL-17-producing Vγ6Vδ1+ γδ T cells in the lungs, uterus, and circulation. Thymic development of this population was normal. However, single-cell RNA sequencing revealed the enrichment of genes associated with T cell survival and proliferation specifically in ß2-integrin-deficient IL-17+ cells compared to their wild-type counterparts. Indeed, ß2-integrin-deficient Vγ6+ cells from the lungs showed reduced apoptosis ex vivo, suggesting that increased survival contributes to the accumulation of these cells in ß2-integrin-deficient tissues. Furthermore, our data revealed an unexpected role for ß2 integrins in promoting the thymic development of the IFNγ-producing CD27+ Vγ4+ γδ T cell subset. Together, our data reveal that ß2 integrins are important regulators of γδ T cell homeostasis, inhibiting the survival of IL-17-producing Vγ6Vδ1+ cells and promoting the thymic development of the IFNγ-producing Vγ4+ subset. Our study introduces unprecedented mechanisms of control for γδ T cell subsets.

MicroRNA-17-5p Reduces Inflammation and Bone Erosions in Mice With Collagen-Induced Arthritis and Directly Targets the JAK/STAT Pathway in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

OBJECTIVE: We undertook this study to examine microRNA (miRNA) expression across rheumatoid arthritis (RA) phenotypes, along with the effects and mechanisms of action of miRNA-17-5p (miR-17). METHODS: A miRNA array was performed in synovial tissue biopsied from patients with naive erosive RA (n = 3) and patients with nonerosive RA (n = 3). MicroRNA-17 lipoplex was delivered intraarticularly in the murine collagen-induced arthritis model. Clinical, histologic, and structural effects were studied over the course of arthritis. In-depth studies of the mechanisms of action of miR-17 were performed in primary RA fibroblast-like synoviocytes (FLS) isolated from synovial tissue. RESULTS: Fifty-five miRNAs including miR-17 were reduced in erosive RA. The miR-17 transfection into arthritic paws reduced the clinical inflammation score between day 2 and day 7 (2.8 versus 1.9; P = 0.03). Synovial B cell, T cell, macrophage, and polynuclear neutrophil infiltration was significantly reduced. Structural damage was also decreased, as shown by a reduction in the number of osteoclasts detected using tartrate-resistant acid phosphatase staining (osteoclast surface/bone surface 32% versus 18%; P = 0.005) and erosion score by computed tomography analysis (2.9 versus 1.7; P = 0.023). Proinflammatory cytokines from the interleukin-6 (IL-6) family and IL-1ß expression were also significantly reduced, but tumor necrosis factor was not. MicroRNA-17 directly targeted the 3'-untranslated regions of STAT3 and JAK1. STAT3 and JAK1 messenger RNA (mRNA) and protein expression were reduced in RA FLS following miR-17 transfection. STAT3 and JAK1 mRNA and activation of STAT3, as assessed by immunohistochemistry, were also reduced in injected paws (% stained area 93% versus 62%; P = 0.035). CONCLUSION: We demonstrate an antiinflammatory and antierosive role of miR-17 in vivo. This effect involves the suppression of the IL-6 family autocrine-amplifying loop through the direct targeting of JAK1 and STAT3.