John Edward Connolly
Email Address
miccje@nus.edu.sg
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Publication Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis(OXFORD UNIV PRESS, 2023-06-21) Tay, Sen Hee; Zharkova, Olga; Lee, Hui Yin; Toh, Michelle Min Xuan; Libau, Eshele Anak; Celhar, Teja; Narayanan, Sriram; Ahl, Patricia Jennifer; Ong, Wei Yee; Joseph, Craig; Lim, Jeffrey Chun Tatt; Wang, Lingzhi; Larbi, Anis; Liang, Shen; Lateef, Aisha; Akira, Shizuo; Ling, Lieng Hsi; Thamboo, Thomas Paulraj; Yeong, Joe Poh Seng; Lee, Bernett Teck Kwong; Edwards, Steven W; Wright, Helen L; MacAry, Paul Anthony; Connolly, John E; Fairhurst, Anna-Marie; Assoc Prof Paul A MacAry; MEDICINE; MICROBIOLOGY AND IMMUNOLOGYOBJECTIVES: Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of systemic lupus erythematosus (SLE). Despite evidence showing the importance of platelet neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and TLR7 in clinical disease. METHODS: Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets, and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet: HDN mixing studies using TLR7 deficient mice and Klinefelter syndrome patients. RESULTS: SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the PBMC layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7, and that the association results in increased NETosis. The neutrophil-to-platelet ratio (NPR), is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of lupus nephritis. CONCLUSIONS: LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils, which may be an important therapeutic opportunity for lupus nephritis.Publication Brief report: Decreased expression of CD244 (SLAMF4) on monocytes and platelets in patients with systemic lupus erythematosus(Springer London, 2018) Mak, A; Thornhill, S.I; Lee, H.Y; Lee, B; Poidinger, M; Connolly, J.E; Fairhurst, A.-M; BIOLOGY; MEDICINE; MICROBIOLOGY AND IMMUNOLOGYThe signalling lymphocyte activation molecule (SLAM) family receptors play important roles in modulating immune responses. Previous studies in murine models and patients have suggested an association of the SLAM family (SLAMF) members with the development of autoimmunity, particularly systemic lupus erythematosus (SLE). Since previous investigations on CD244 expression have focussed on NK and T cells, the aim of this study was to evaluate the surface expression of major SLAMF members across monocytes and polymorphonuclear cells in an Asian SLE cohort and explore their potential associations with SLE-related disease activity and autoantibodies. Thirty-nine SLE patients and twenty-nine healthy controls (HC) were evaluated for the expression of CD150, CD84, CD229, CD48, CD244, CD352 and CD319. We determined a significantly lower expression of CD244 on monocytes in SLE patients compared to HC. Furthermore, monocyte CD244 expression was negatively associated with several serum autoantibody titres. Our findings suggest that this molecule plays an important role in immune tolerance mechanisms and should be investigated further. © 2017, The Author(s).Publication Autophagy governs protumorigenic effects of mitotic slippage-induced senescence(2018) Jakhar, R; Luijten, M.N.H; Wong, A.X.F; Cheng, B; Guo, K; Neo, S.P; Au, B; Kulkarni, M; Lim, K.J; Maimaiti, J; Chong, H.C; Lim, E.H; Tan, T.B.K; Ong, K.W; Sim, Y; Wong, J.S.L; Khoo, J.B.K; Ho, J.T.S; Chua, B.T; Sinha, I; Wang, X; Connolly, J.E; Gunaratne, J; Crasta, K.C; ANATOMY; MICROBIOLOGY AND IMMUNOLOGY; DUKE-NUS MEDICAL SCHOOLThe most commonly utilized class of chemotherapeutic agents administered as a first-line therapy are antimitotic drugs; however, their clinical success is often impeded by chemoresistance and disease relapse. Hence, a better understanding of the cellular pathways underlying escape from cell death is critical. Mitotic slippage describes the cellular process where cells exit antimitotic drug-enforced mitotic arrest and "slip" into interphase without proper chromosome segregation and cytokinesis. The current report explores the cell fate consequence following mitotic slippage and assesses a major outcome following treatment with many chemotherapies, therapy-induced senescence. It was found that cells postslippage entered senescence and could impart the senescenceassociated secretory phenotype (SASP). SASP factor production elicited paracrine protumorigenic effects, such as migration, invasion, and vascularization. Both senescence and SASP factor development were found to be dependent on autophagy. Autophagy induction during mitotic slippage involved the autophagy activator AMPK and endoplasmic reticulum stress response protein PERK. Pharmacologic inhibition of autophagy or silencing of autophagy-related ATG5 led to a bypass of G1 arrest senescence, reduced SASP-associated paracrine tumorigenic effects, and increased DNA damage after Sphase entry with a concomitant increase in apoptosis. Consistent with this, the autophagy inhibitor chloroquine and microtubule-stabilizing drug paclitaxel synergistically inhibited tumor growth in mice. Sensitivity to this combinatorial treatment was dependent on p53 status, an important factor to consider before treatment. © 2018 American Association for Cancer Research.Publication Bayesian analysis of cytokines and chemokine identifies immune pathways of HBsAg loss during chronic hepatitis B treatment(Springer Science and Business Media LLC, 2021-12-01) Narayanan, S; Au, VB; Khakpoor, A; Yan, C; Ahl, PJ; Kaliaperumal, N; Lee, B; Xiang, WW; Wang, J; Lee, C; Tay, A; Lim, SG; Connolly, JE; Dr Atefeh Khakpoor; MEDICINE; MICROBIOLOGY AND IMMUNOLOGY; DUKE-NUS MEDICAL SCHOOLOur objective was to examine differences in cytokine/chemokine response in chronic hepatitis B(CHB) patients to understand the immune mechanism of HBsAg loss (functional cure) during antiviral therapy. We used an unbiased machine learning strategy to unravel the immune pathways in CHB nucleo(t)side analogue-treated patients who achieved HBsAg loss with peg-interferon-α(peg-IFN-α) add-on or switch treatment in a randomised clinical trial. Cytokines/chemokines from plasma were compared between those with/without HBsAg loss, at baseline, before and after HBsAg loss. Peg-IFN-α treatment resulted in higher levels of IL-27, IL-12p70, IL-18, IL-13, IL-4, IL-22 and GM-CSF prior to HBsAg loss. Probabilistic network analysis of cytokines, chemokines and soluble factors suggested a dynamic dendritic cell driven NK and T cell immune response associated with HBsAg loss. Bayesian network analysis showed a dominant myeloid-driven type 1 inflammatory response with a MIG and I-TAC central module contributing to HBsAg loss in the add-on arm. In the switch arm, HBsAg loss was associated with a T cell activation module exemplified by high levels of CD40L suggesting T cell activation. Our findings show that more than one immune pathway to HBsAg loss was found with peg-IFN-α therapy; by myeloid-driven Type 1 response in one instance, and T cell activation in the other.Publication A Flow Cytometry-Based Assay for High-Throughput Detection and Quantification of Neutrophil Extracellular Traps in Mixed Cell Populations(WILEY, 2019-03-01) Zharkova, Olga; Tay, Sen Hee; Lee, Hui Yin; Shubhita, Tripathi; Ong, Wei Yee; Lateef, Aisha; MacAry, Paul Anthony; Lim, Lina Hsiu Kim; Connolly, John Edward; Fairhurst, Anna-Marie; Assoc Prof Paul A MacAry; MEDICINE; PHYSIOLOGY; SURGERY; MICROBIOLOGY AND IMMUNOLOGYNeutrophil extracellular traps (NETs) are web-like structures composed of decondensed chromatin and antimicrobial proteins that are released into the extracellular space during microbial infections. This active cell death program is known as NETosis. To date, florescence microscopy is the widely accepted method for visualization and quantification of NETs. However, this method is subjective, time consuming and yields low numbers of analyzed polymorphonuclear cells (PMNs) per sample. Increasing interest has emerged on the identification of NETs using flow cytometry techniques. However, flow cytometry analysis of NETs requires particular precautions for sample preparation to obtain reproducible data. Herein, we describe a flow cytometry-based assay for high-throughput detection and quantification of NETosis in mixed cell populations. We used fluorescent-labeled antibodies against cell markers on PMNs together with a combination of nucleic acid stains to measure NETosis in whole blood (WB) and purified PMNs. Using plasma membrane-impermeable DNA-binding dye, SYTOX Orange (SO), we found that cell-appendant DNA of NETting PMNs were positive for SO and DAPI. The combination of optimally diluted antibody and nucleic acid dyes required no washing and yielded low background fluorescence. Significant correlations were found for NETosis from WB and purified PMNs. We then validated the assay by comparing with time-lapse live cell fluorescence microscopy and determined very good intraassay and interassay variances. The assay was then applied to a disease associated with NETosis, systemic lupus erythematosus (SLE). We examined PMA-induced NETosis in peripheral PMNs from SLE patients and controls and in bone marrow PMNs from multiple murine models. In summary, this assay is observer-independent and allows for rapid assessment of a large number of PMNs per sample. Use of this assay does not require sophisticated microscopic equipment like imaging flow cytometers and may be a starting point to analyze extracellular trap formation from immune cells other than PMNs. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.Publication Chromosomal instability-induced senescence potentiates cell non-autonomous tumourigenic effects(Nature Publishing Group, 2018) He, Q.; Au, B.; Kulkarni, M.; Shen, Y.; Lim, K.J.; Maimaiti, J.; Wong, C.K.; Luijten, M.N.H.; Chong, H.C.; Lim, E.H.; Rancati, G.; Sinha, I.; Fu, Z.; Wang, X.; Connolly, J.E.; Crasta, K.C.; MICROBIOLOGY AND IMMUNOLOGYChromosomal instability (CIN), a high rate of chromosome loss or gain, is often associated with poor prognosis and drug resistance in cancers. Aneuploid, including near-polyploid, cells contain an abnormal number of chromosomes and exhibit CIN. The post-mitotic cell fates following generation of different degrees of chromosome mis-segregation and aneuploidy are unclear. Here we used aneuploidy inducers, nocodazole and reversine, to create different levels of aneuploidy. A higher extent of aneuploid and near-polyploid cells in a given population led to senescence. This was in contrast to cells with relatively lower levels of abnormal ploidy that continued to proliferate. Our findings revealed that senescence was accompanied by DNA damage and robust p53 activation. These senescent cells acquired the senescence-associated secretory phenotype (SASP). Depletion of p53 reduced the number of senescent cells with concomitant increase in cells undergoing DNA replication. Characterisation of these SASP factors demonstrated that they conferred paracrine pro-tumourigenic effects such as invasion, migration and angiogenesis both in vitro and in vivo. Finally, a correlation between increased aneuploidy and senescence was observed at the invasive front in breast carcinomas. Our findings demonstrate functional non-equivalence of discernable aneuploidies on tumourigenesis and suggest a cell non-autonomous mechanism by which aneuploidy-induced senescent cells and SASP can affect the tumour microenvironment to promote tumour progression. © 2018, The Author(s).Publication A multicenter phase II randomized trial of durvalumab (MEDI-4736) versus physician's choice chemotherapy in recurrent ovarian clear cell adenocarcinoma (MOCCA)(BMJ PUBLISHING GROUP, 2020-08-01) Ngoi, Natalie YL; Heong, Valerie; Ow, Samuel; Chay, Wen Yee; Kim, Hee Seung; Choi, Chel Hun; Goss, Geraldine; Goh, Jeffrey C; Tai, Bee Choo; Lim, Diana G; Kaliaperumal, Nivashini; Au, Veonice B; Connolly, John E; Kim, Jae-Weon; Friedlander, Michael; Kim, Kidong; Tan, David SP; Dr Gkeok Stzuan Diana Lim; MEDICINE; MICROBIOLOGY AND IMMUNOLOGY; SAW SWEE HOCK SCHOOL OF PUBLIC HEALTH; PATHOLOGY© IGCS and ESGO 2020. Re-use permitted under CC BY-NC. No commercial re-use. Published by BMJ. BACKGROUND: The optimal treatment of recurrent ovarian clear cell carcinoma remains unknown. There is increasing rationale to support the role of immune checkpoint inhibitors targeting the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis in ovarian clear cell carcinoma. PRIMARY OBJECTIVE: To evaluate the efficacy of durvalumab (MEDI-4736) compared with standard chemotherapy in patients with recurrent ovarian clear cell carcinoma. STUDY HYPOTHESIS: Patients with recurrent ovarian clear cell carcinoma treated with durvalumab will have improved progression-free survival compared with those treated with chemotherapy of physician's choice. TRIAL DESIGN: The MOCCA study is a multicenter, open-label, randomized phase II trial in patients with recurrent ovarian clear cell carcinoma, which recruited from eight sites across Gynecologic Cancer Group Singapore (GCGS), Korean Gynecologic-Oncology Group (KGOG), and Australia New Zealand Gynecological Oncology Group (ANZGOG). Enrolled patients were randomized in a 2:1 ratio to receive durvalumab or physician's choice of chemotherapy until disease progression, intolerable toxicity, or withdrawal of patient consent. MAJOR INCLUSION/EXCLUSION CRITERIA: Eligible patients required histologically documented diagnosis of recurrent ovarian clear cell carcinoma, as evidenced by WT1 negativity. All patients must have been of Eastern Cooperative Oncology Group (ECOG) performance status 2 or better, and have had previous treatment with, and progressed or recurred after prior platinum-based chemotherapy. No more than four prior lines of treatment were allowed and prior immune checkpoint inhibitor treatment was not permitted. PRIMARY ENDPOINTS: The primary endpoint was the median progression-free survival following treatment with durvalumab, compared with physician's choice of chemotherapy. Progression-free survival was defined as the time from the first day of treatment to the first observation of disease progression, or death due to any cause, or last follow-up. SAMPLE SIZE: The target sample size was 46 patients. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: Accrual has been completed and results are expected to be presented by mid-2021. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03405454.Publication Upon viral exposure, myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors(American Society of Hematology, 2006) Piqueras, B; Connolly, J; Freitas, H; Palucka, A.K; Banchereau, J; MICROBIOLOGY AND IMMUNOLOGYHost response to viral infection involves distinct effectors of innate and adaptive immunity, whose mobilization needs to be coordinated to ensure protection. Here we show that influenza virus triggers, in human blood dendritic-cell (DC) subsets (ie, plasmacytoid and myeloid DCs), a coordinated chemokine (CK) secretion program with 3 successive waves. The first one, occurring at early time points (2 to 4 hours), includes CKs potentially attracting effector cells such as neutrophils, cytotoxic T cells, and natural killer (NK) cells (CXCL16, CXCL1, CXCL2, and CXCL3). The second one occurs within 8 to 12 hours and includes CKs attracting effector memory T cells (CXCL8, CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11). The third wave, which occurs after 24 to 48 hours, when DCs have reached the lymphoid organs, includes CCL19, CCL22, and CXCL13, which attract naive T and B lymphocytes. Thus, human blood DC subsets carry a common program of CK production, which allows for a coordinated attraction of the different immune effectors in response to viral infection. © 2006 by The American Society of Hematology.Publication Singapore Ocular Tuberculosis Immunity Study (SPOTIS): Role of T-lymphocyte profiling in patients with presumed ocular tuberculosis(Taylor & Francis, 2020-07-14) Paul Hutchinson; Aera Kee; Rupesh Agrawal; Nobuyo Iwata; Mayjane Tumulak; John Connolly; Soon Phaik Chee; Jay Siak; MICROBIOLOGY AND IMMUNOLOGY; LIFE SCIENCES INSTITUTE; DUKE-NUS MEDICAL SCHOOLObjective: A prospective clinical study to assess the utility of CD4 + T cell lymphocyte profiling from peripheral blood in patients with ocular tuberculosis (TB). Methods: Thirty-six Asian patients with presumed diagnosis of ocular TB were recruited for T-lymphocyte profiling. MTB antigen specific CD4 assay was set up, and flow cytometric data were analyzed using FlowJo software. Results: There was no significant difference between treatment responders and non-responders for the proportion of CD4 + T cells specific for PPD or ESAT-6+ CFP-10, but treatment responders did have significantly higher frequency of CD38+ (p = .0357) and CD38+ HLA-DR+ (p = .0357) on the PPD-specific CD4 + T cells. Conclusion: This study is one of the first of its kind to look into MTB specific T cell activation marker profiling of peripheral blood in patients with ocular TB. Further studies need to be undertaken to assess the utility of CD4 + T cell phenotypes as a biomarker for ocular TB.Publication Dysfunctional bronchial cilia are a feature of Chronic Obstructive Pulmonary Disease (COPD)(Taylor & Francis, 2021-09-01) Biju Thomas; Mariko Siyue Koh; Chris O'Callaghan; JOHN ALLEN; Andrew Rutman; Rob Hirst; John Connolly; Su Ying Low; Ong Thun How; Loo Chian Min; Darren Wan Teck Lim; Oon Lin Ean Lynette; Qixian He; Oon Hoe Teoh; Therese Lapperre; MEDICINE; MICROBIOLOGY AND IMMUNOLOGY; DEAN'S OFFICE (DUKE-NUS MEDICAL SCHOOL); DUKE-NUS MEDICAL SCHOOLImpaired mucociliary clearance may increase COPD exacerbation risk. We aimed to compare bronchial ciliary function and epithelial ultrastructure of COPD patients to healthy controls and explore its relationship to exacerbator phenotypes (frequent [FE] and infrequent [IFE] exacerbator). In this cross-sectional study, 16 COPD patients and 12 controls underwent bronchial brushings. Ciliary beat frequency (CBF) and dyskinesia index (DI; % of dyskinetic cilia) were assessed using digital high-speed video microscopy, and epithelial ultrastructure using transmission electron microscopy (TEM). Bronchial epithelium in COPD showed lower CBF and higher DI, compared to controls (median [IQR] CBF: 6.8 (6.1–7.2) Hz vs 8.5 (7.7–8.9) Hz, p<0.001 and DI: 73.8 (60.7–89.8) % vs 14.5 (11.2–16.9) %, p<0.001, respectively). This was true for FE and IFE phenotypes of COPD, which were similar in terms of bronchial CBF or DI. Subgroup analyses demonstrated lower CBF and higher DI in FE and IFE COPD phenotypes compared to controls, irrespective of smoking status. TEM showed more loss of cilia, extrusion of cells, cytoplasmic blebs and dead cells in COPD patients versus controls. Profound dysfunction of bronchial cilia is a feature of COPD irrespective of exacerbation phenotype and smoking status, which is likely to contribute to poor mucus clearance in COPD.
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