Negative impact of Interleukin-9 on synovial regulatory T cells in rheumatoid arthritis.

In Rheumatoid Arthritis (RA), regulatory T cells (Tregs) have been found to be enriched in the synovial fluid. Despite their accumulation, they are unable to suppress synovial inflammation. Recently, we showed the synovial enrichment of interleukin-9 (IL-9) producing helper T cells and its positive correlation with disease activity. Therefore, we investigated the impact of IL-9 on synovial Tregs in RA. Here, we confirmed high synovial Tregs in RA patients, however these cells were functionally impaired in terms of suppressive cytokine production (IL-10 and TGF-ß). Abrogating IL-9/ IL-9 receptor interaction could restore the suppressive cytokine production of synovial Tregs and reduce the synovial inflammatory T cells producing IFN-γ, TNF-α, IL-17. However, blocking these inflammatory cytokines failed to show any effect on IL-9 producing T cells, highlighting IL-9's hierarchy in the inflammatory network. Thus, we propose that blocking IL-9 might dampen synovial inflammation by restoring Tregs function and inhibiting inflammatory T cells.

Staphylococci planktonic and biofilm environments differentially affect osteoclast formation.

INTRODUCTION: The pathophysiology of chronic implant-related bone infections is characterized by an increase in osteoclast numbers and enhanced bone resorption. Biofilms are a major reason for chronicity of such infections as the biofilm matrix protects bacteria against antibiotics and impairs the function of immune cells. Macrophages are osteoclast precursor cells and therefore linked to inflammation and bone destruction. OBJECTIVE AND METHOD: Investigations on the impact of biofilms on the ability of macrophages to form osteoclasts are yet missing and we, therefore, analyzed the effect of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) planktonic and biofilm environments on osteoclastogenesis using RAW 264.7 cells and conditioned media (CM). RESULTS: Priming with the osteoclastogenic cytokine RANKL before CM addition enabled the cells to differentiate into osteoclasts. This effect was highest in SE planktonic or SA biofilm CM. Simultaneous stimulation with CM and RANKL, however, suppressed osteoclast formation and resulted in formation of inflammation-associated multinucleated giant cells (MGCs) which was most pronounced in SE planktonic CM. CONCLUSION: Our data indicate that the biofilm environment and its high lactate levels are not actively promoting osteoclastogenesis. Hence, the inflammatory immune response against planktonic bacterial factors through Toll-like receptors seems to be the central cause for the pathological osteoclast formation. Therefore, immune stimulation or approaches that aim at biofilm disruption need to consider that this might result in enhanced inflammation-mediated bone destruction.

Distinct immune-effector and metabolic profile of CD8+ T cells in patients with autoimmune polyarthritis induced by therapy with immune checkpoint inhibitors.

OBJECTIVES: Rheumatic immune-related adverse events (irAE) such as (poly)arthritis in patients undergoing immune checkpoint inhibitor (ICI) treatment pose a major clinical challenge. ICI therapy improves CD8+ T cell (CD8) function, but CD8 contributes to chronic inflammation in autoimmune arthritis (AA). Thus, we investigated whether immune functional and metabolic changes in CD8 explain the development of musculoskeletal irAE in ICI-treated patients. METHODS: Peripheral CD8 obtained from ICI-treated patients with and without arthritis irAEs and from AA patients with and without a history of malignancy were stimulated in media containing 13C-labelled glucose with and without tofacitinib or infliximab. Changes in metabolism, immune-mediator release, expression of effector cell-surface molecules and inhibition of tumour cell growth were quantified. RESULTS: CD8 from patients with irAE showed significantly lower frequency and expression of cell-surface molecule characteristic for activation, effector-functions, homing, exhaustion and apoptosis and reduced release of cytotoxic and proinflammatory immune mediators compared with CD8 from ICI patients who did not develop irAE. This was accompanied by a higher glycolytic rate and ATP production. Gene-expression analysis of pre-ICI-treated CD8 revealed several differentially expressed transcripts in patients who later developed arthritis irAEs. In vitro tofacitinib or infliximab treatment did not significantly change the immune-metabolic profile nor the capacity to release cytolytic mediators that inhibit the growth of the human lung cancer cell line H838. CONCLUSIONS: Our study shows that CD8 from ICI-treated patients who develop a musculoskeletal irAE has a distinct immune-effector and metabolic profile from those that remain irAE free. This specific irAE profile overlaps with the one observed in CD8 from AA patients and may prove useful for novel therapeutic strategies to manage ICI-induced irAEs.

Influence of bag volume on reproducibility of inert gas rebreathing pulmonary blood flow measurements in patients with pulmonary diseases.

PURPOSE: Non-invasive inert gas rebreathing (IGR) has shown promising results in the determination of pulmonary blood flow. The volume of the rebreathing bag (V bag) is proposed by the system. However, elderly patients or those with severe pulmonary disease may be unable to rebreathe this volume entirely. We evaluated the effect of adapting V bag on the reproducibility of IGR. METHODS: A total of 270 valid measurements were obtained from 45 patients with obstruction (group A), restriction (group B), and in healthy controls (group C). Two measurements for each of three different V bag of 1,200, 1,700, and 2,200 ml were conducted in the supine position. RESULTS: We found no statistically significant difference of the repeated measurements neither between the different V bag in groups A to C nor between the three groups for identical V bag. There was a weak yet significantly worse coefficient of variation between a V bag of 2,200 ml in group A compared with group C with 2,200 and 1,200 ml, respectively. Intraclass correlation coefficient and repeatability coefficient yielded significantly worse values in group A for a V bag of 2,200 ml compared with healthy controls and lower bag volumes. No difference could be found intraclass nor interclass in groups B and C. CONCLUSIONS: V bag can be altered between 1,200 and 2,200 ml in most situations without affecting the reproducibility. Attention has to be paid to extreme volumes in obstructive patients. Nevertheless, V bag should be chosen as large as possible and therefore has to be carefully adapted, particularly in patients with obstruction or restriction.

Bacterial and Metabolic Factors of Staphylococcal Planktonic and Biofilm Environments Differentially Regulate Macrophage Immune Activation.

Biofilm formation is a leading cause for chronic implant-related bone infections as biofilms shield bacteria against the immune system and antibiotics. Additionally, biofilms generate a metabolic microenvironment that shifts the immune response towards tolerance. Here, we compared the impact of the metabolite profile of bacterial environments on macrophage immune activation using Staphylococcus aureus (SA) and epidermidis (SE) conditioned media (CM) of planktonic and biofilm cultures. The biofilm environment had reduced glucose and increased lactate concentrations. Moreover, the expression of typical immune activation markers on macrophages was reduced in the biofilm environment compared to the respective planktonic CM. However, all CM caused a predominantly pro-inflammatory macrophage cytokine response with a comparable induction of Tnfa expression. In biofilm CM, this was accompanied by higher levels of anti-inflammatory Il10. Planktonic CM, on the other hand, induced an IRF7 mediated Ifnb gene expression which was absent in the biofilm environments. For SA but not for SE planktonic CM, this was accompanied by IRF3 activation. Stimulation of macrophages with TLR-2/-9 ligands under varying metabolic conditions revealed that, like in the biofilm setting, low glucose concentration reduced the Tnfa to Il10 mRNA ratio. However, the addition of extracellular L-lactate but not D-lactate increased the Tnfa to Il10 mRNA ratio upon TLR-2/-9 stimulation. In summary, our data indicate that the mechanisms behind the activation of macrophages differ between planktonic and biofilm environments. These differences are independent of the metabolite profiles, suggesting that the production of different bacterial factors is ultimately more important than the concentrations of glucose and lactate in the environment.

Reduction of Proinflammatory Effector Functions Through Remodeling of Fatty Acid Metabolism in CD8+ T Cells From Rheumatoid Arthritis Patients.

OBJECTIVE: Rheumatoid arthritis (RA) CD8+ T cells maintain their effector proinflammatory phenotype by changing their metabolism toward aerobic glycolysis. However, their massive energy and biosynthesis needs may require additional substrates other than glucose. Since systemic alterations in lipid metabolism have been reported in RA patients, we explored the role of fatty acid (FA) metabolism in CD8+ T cells to identify potential targets to curb their proinflammatory potential. METHODS: The expression of FA metabolism-related genes was analyzed for total CD8+ T cells and CD8+ T cell subsets in the data of RA patients and healthy controls retrieved from the GEO database. Functional assays were performed using peripheral blood CD8+ T cells isolated from RA (n = 31), psoriatic arthritis (n = 26), and spondyloarthritis (n = 21) patients receiving different therapies (disease-modifying antirheumatic drugs, biologics, and JAK inhibitors) and from healthy controls (n = 14). We quantified the expression of FA transporters, lipid uptake, intracellular FA content, cytokine production, activation, proliferation, and capacity to inhibit tumor cell growth, either with or without FA metabolism inhibitors. RESULTS: The CD8+ T cell gene expression profile of FA metabolism-related genes was significantly different between untreated RA patients and healthy controls. RA patients who had a good clinical response after 6 months of methotrexate therapy had significantly increased expression of FA metabolism-related genes. Cell surface expression of the FA transporters FA binding protein 4 (FABP4) and G protein-coupled receptor 84 (GPR84) and FA uptake were higher in effector and memory CD8+ T cells from RA patients compared to those from healthy controls. In vitro blockade of FA metabolism significantly impaired CD8+ T cell effector functions. CONCLUSION: RA CD8+ T cells present an altered FA metabolism, which could provide potential therapeutic targets to control their proinflammatory profile, particularly therapies directed against the transport and oxidation of free FA.

Multi-Physical Tissue Modeling of a Human Urinary Bladder.

A multi-physical model of a human urinary bladder is an essential element for the potential application of electrical impedance spectroscopy during transurethral resection surgery, where measurements are taken at different fill levels inside the bladder. This work derives a multi-physical bladder tissue model that incorporates the electrical impedance properties with dependence on mechanical deformation due to filling of the bladder. The volume and ratio of the intracellular to extracellular tissue fluid heavily influence the electrical impedance characteristics and thus provide the connection between the mechanical and electrical domains. Modeling the fluid within the tissue links both the physical and histological processes and enables useful inferences of the properties from empiric observations. This is demonstrated by taking impedance measurements at different fill volumes. The resulting model provides a tool to analyze impedance measurements during surgery at different stress levels. In addition, this model can be used to determine patient-specific tissue parameters.

Effect of Increased Lactate Dehydrogenase A Activity and Aerobic Glycolysis on the Proinflammatory Profile of Autoimmune CD8+ T Cells in Rheumatoid Arthritis.

OBJECTIVE: CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA. METHODS: Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing 13 C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by 1 H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed. RESULTS: RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly 13 C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane. CONCLUSION: Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.