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Objectives:\ud \ud Fatty acid oxidation (FAO) and glycolysis have been implicated in immune regulation and activation of macrophages. However, investigation of human monocyte intracellular metabolism in the context of the hypoxic and inflammatory rheumatoid arthritis (RA) synovium is lacking. We hypothesized that exposure of monocytes to the hypoxic and inflammatory RA environment would have a profound impact on their metabolic state, and potential to contribute to disease pathology.\ud Methods:\ud \ud Human monocytes were isolated from buffy coats and exposed to hypoxia. Metabolic profiling of monocytes was carried out by LC-MS metabolomics. Inflammatory mediator release after LPS or RA-synovial fluid (RA-SF) stimulation was analysed by ELISA. FAO was inhibited by etomoxir or enhanced with exogenous carnitine supplementation. Transcriptomics of RA blood monocytes and RA-SF macrophages was carried out by microarray.\ud Results:\ud \ud Hypoxia exacerbated monocyte-derived CCL20 and IL-1β release in response to LPS, and increased glycolytic intermediates at the expense of carnitines. Modulation of carnitine identified a novel role for FAO in the production of CCL20 in response to LPS. Transcriptional analysis of RA blood monocytes and RA-SF macrophages revealed that fatty acid metabolism was altered and CCL20 increased when monocytes enter the synovial environment. In vitro analysis of monocytes showed that RA-SF increases carnitine abundance and CCL20 production in hypoxia, which was exacerbated by exogenous carnitine.\ud Conclusion:\ud \ud This work has revealed a novel inflammatory mechanism in RA that links FAO to CCL20 production in human monocytes, which could subsequently contribute to RA disease pathogenesis by promoting the recruitment of Th17 cells and osteoclastogenesis.

Nisha Kurian,1 Taylor S Cohen,2 Lisa Öberg,3 Erica De Zan,3 Gabriel Skogberg,4 Stefan Vollmer,3 Engin Baturcam,3 Petter Svanberg,5 Britta Bonn,5 Paul D Smith,6 Outi Vaarala,3 Danen M Cunoosamy3 1Respiratory Inflammation and Autoimmune (RIA) Precision Medicine Unit, Precision Medicine, Oncology R&D, AstraZeneca, Gothenburg, Sweden; 2Microbial Sciences, Medimmune, Gaithersburg, MD, USA; 3Translational Science and Experimental Medicine, Research and Early Development, RIA, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 4Bioscience, Research and Early Development, RIA, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 5Drug Metabolism and Pharmacokinetics, Research and Early Development, RIA, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 6Bioscience, Oncology R&D, AstraZeneca, Cambridge, UKCorrespondence: Nisha KurianRespiratory Inflammation and Autoimmune (RIA) Precision Medicine Unit, Precision Medicine, Oncology R&D, AstraZeneca, Gothenburg, SwedenTel +46 72 197 9662Email Nisha.e.kurian@astrazeneca.comBackground: Unlike p38 mitogen-activated protein Kinases (MAPK) that has been extensively studied in the context of lung-associated pathologies in COPD, the role of the dual-specificity mitogen-activated protein kinase kinase (MEK1/2) or its downstream signaling molecule extracellular signal-regulated kinases 1/2 (ERK1/2) in COPD is poorly understood.Objectives: The aim of this study was to address whether MEK1/2 pathway activation is linked to COPD and that targeting this pathway can improve lung inflammation through decreased immune-mediated inflammatory responses without compromising bacterial clearance.Methods: Association of MEK1/2 pathway activation to COPD was investigated by immunohistochemistry using lung tissue biopsies from COPD and healthy individuals and through analysis of sputum gene expression data from COPD patients. The anti-inflammatory effect of MEK1/2 inhibition was assessed on cytokine release from lipopolysaccharide-stimulated alveolar macrophages. The effect of MEK1/2 inhibition on bacterial clearance was assessed using Staphylococcus aureus killing assays with RAW 264.7 macrophage cell line and human neutrophils.Results: We report here MEK1/2 pathway activation demonstrated by increased pERK1/2 staining in bronchial epithelium and by the presence of MEK gene activation signature in sputum samples from COPD patients. Inhibition of MEK1/2 resulted in a superior anti-inflammatory effect in human alveolar macrophages in comparison to a p38 inhibitor. Furthermore, MEK1/2 inhibition led to an increase in bacterial killing in human neutrophils and RAW 264.7 cells that was not observed with the p38 inhibitor.Conclusion: Our data demonstrate the activation of MEK1/2 pathway in COPD and highlight a dual function of MEK1/2 inhibition in improving host defense responses whilst also controlling inflammation.Keywords: exacerbation, infection, alveolar macrophage, p38 MAPK, steroid, transcriptomics, sputum

Figure S1. MEK inhibitor works to reduce pERK1/2 and enhances IFN-β release. Figure S2. Challenge with poly(I:C) is similar but not identical to challenge with RV2. Figure S3. PI3Ki does not cause cytotoxicity. Figure S4. Enhanced IFN-β response due to MEKi is not associated with increased IRF7 protein expression. Figure S5. Challenge with poly(I:C) is similar but not identical to challenge with RV2 or RSVA2. Figure S6. Induction of ISG15 mRNA by poly(I:C) is similar to that of RV2. Figure S7. MEKi reduces phosphorylation of STAT3 at S727. Table S1. NHBE donors. Table S2. Structure of small molecules and PROTACs. Table S3. List of antibodies used for immunoblotting. Table S4. Target sequence of short interfering RNA (siRNA). Table S5. List of Taqman primer/probe. (DOCX 1110 kb)

Human rhinovirus is frequently seen as an upper respiratory tract infection but growing evidence proves the virus can cause lower respiratory tract infections in patients with chronic inflammatory lung diseases including chronic obstructive pulmonary disease (COPD). In addition to airway epithelial cells, macrophages are crucial for regulating inflammatory responses to viral infections. However, the response of macrophages to HRV has not been analyzed in detail. We used in vitro monocyte-derived human macrophages to study the cytokine secretion of macrophages in response to the virus. Our results showed that macrophages were competent at responding to HRV, as a robust cytokine response was detected. However, after subsequent exposure to non-typeable Haemophilus influenzae (NTHi) or to LPS, HRV-treated macrophages secreted reduced levels of pro-inflammatory or regulatory cytokines. This “paralyzed” phenotype was not mimicked if the macrophages were pre-treated with LPS or CpG instead of the virus. These results begin to deepen our understanding into why patients with COPD show HRV-induced exacerbations and why they mount a defective response toward NTHi.