Post-injury immunosuppression and secondary infections are caused by an AIM2 inflammasome-driven signaling cascade.

Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1ß (IL-1ß) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.

Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death.

Small molecules that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biology research and as clinical anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, we introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biology, and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.

FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins.

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. KEY POINTS: • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells.

New View on Endothelial Cell Migration: Switching Modes of Migration Based on Matrix Composition.

OBJECTIVE: Cell-matrix interactions are crucial for regulating cellular activities, such as migration. This is of special importance for morphogenic processes, such as angiogenesis (the development of new blood vessels). Most of our understanding of cell migration relies on 2-dimensional (2D) experiments. However, the awareness that 3D settings might elicit different results has increased. Knowledge about endothelial cell (EC) behavior in 3D environments and the influence of matrix composition on EC migration, in particular, is still limited. APPROACH AND RESULTS: We characterize the migration of single ECs through 2 structurally different hydrogels: spongy Matrigel and fibrillar collagen I. Our observations reveal an elongated migration phenotype in Matrigel and a rounded phenotype with pronounced cell blebs (blebs >2 µm) in collagen I, which have not previously been described in ECs. Directed migration seems to depend on Rac1 and Cdc42 in collagen, but not in Matrigel (shown using appropriate pharmacological inhibitors). By applying anti-integrin antibodies and supplementing laminin in collagen gels, we identify laminin as the main determinant of the elongated phenotype. Laminin seems to induce a morphological switch between modes of migration. As an in situ proof of principle, we performed live imaging of EC migration during vascular growth in a murine retina in the absence and presence of anti-integrin antibodies. CONCLUSIONS: We show that, surprisingly, ECs can evade the pharmacological inhibition of central signaling pathways involved in migration (contractility, small GTPases, and proteolysis) by shifting gears between modes of migration. This finding indicates an unexpected contextual plasticity of EC behavior.

Endothelial Dysfunction, and A Prothrombotic, Proinflammatory Phenotype Is Caused by Loss of Mitochondrial Thioredoxin Reductase in Endothelium.

OBJECTIVE: Although the investigation on the importance of mitochondria-derived reactive oxygen species (ROS) in endothelial function has been gaining momentum, little is known on the precise role of the individual components involved in the maintenance of a delicate ROS balance. Here we studied the impact of an ongoing dysregulated redox homeostasis by examining the effects of endothelial cell-specific deletion of murine thioredoxin reductase 2 (Txnrd2), a key enzyme of mitochondrial redox control. APPROACH AND RESULTS: We analyzed the impact of an inducible, endothelial cell-specific deletion of Txnrd2 on vascular remodeling in the adult mouse after femoral artery ligation. Laser Doppler analysis and histology revealed impaired angiogenesis and arteriogenesis. In addition, endothelial loss of Txnrd2 resulted in a prothrombotic, proinflammatory vascular phenotype, manifested as intravascular cellular deposits, as well as microthrombi. This phenotype was confirmed by an increased leukocyte response toward interleukin-1 in the mouse cremaster model. In vitro, we could confirm the attenuated angiogenesis measured in vivo, which was accompanied by increased ROS and an impaired mitochondrial membrane potential. Ex vivo analysis of femoral arteries revealed reduced flow-dependent vasodilation in endothelial cell Txnrd2-deficient mice. This endothelial dysfunction could be, at least partly, ascribed to inadequate nitric oxide signaling. CONCLUSIONS: We conclude that the maintenance of mitochondrial ROS via Txnrd2 in endothelial cells is necessary for an intact vascular homeostasis and remodeling and that Txnrd2 plays a vitally important role in balancing mitochondrial ROS production in the endothelium.

Intercellular Transport of Nanomaterials is Mediated by Membrane Nanotubes In Vivo.

So-called membrane nanotubes are cellular protrusions between cells whose functions include cell communication, environmental sampling, and protein transfer. It has been previously reported that systemically administered carboxyl-modified quantum dots (cQDs) are rapidly taken up by perivascular macrophages in skeletal muscle of healthy mice. Expanding these studies, it is found, by means of in vivo fluorescence microscopy on the mouse cremaster muscle, rapid uptake of cQDs not only by perivascular macrophages but also by tissue-resident cells, which are localized more than 100 µm distant from the closest vessel. Confocal microscopy on muscle tissue, immunostained for the membrane dye DiI, reveals the presence of continuous membranous structures between MHC-II-positive, F4/80-positive cells. These structures contain microtubules, components of the cytoskeleton, which clearly colocalize with cQDs. The cQDs are exclusively found inside endosomal vesicles. Most importantly, by using in vivo fluorescence microscopy, this study detected fast (0.8 µm s(-1) , mean velocity), bidirectional movement of cQDs in such structures, indicating transport of cQD-containing vesicles along microtubule tracks by the action of molecular motors. The findings are the first to demonstrate membrane nanotube function in vivo and they suggest a previously unknown route for the distribution of nanomaterials in tissue.

Influence of Surface Modifications on the Spatiotemporal Microdistribution of Quantum Dots In Vivo.

For biomedical applications of nanoconstructs, it is a general prerequisite to efficiently reach the desired target site. In this regard, it is crucial to determine the spatiotemporal distribution of nanomaterials at the microscopic tissue level. Therefore, the effect of different surface modifications on the distribution of microinjected quantum dots (QDs) in mouse skeletal muscle tissue has been investigated. In vivo real-time fluorescence microscopy and particle tracking reveal that carboxyl QDs preferentially attach to components of the extracellular matrix (ECM), whereas QDs coated with polyethylene glycol (PEG) show little interaction with tissue constituents. Transmission electron microscopy elucidates that carboxyl QDs adhere to collagen fibers as well as basement membranes, a type of ECM located on the basolateral side of blood vessel walls. Moreover, carboxyl QDs have been found in endothelial junctions as well as in caveolae of endothelial cells, enabling them to translocate into the vessel lumen. The in vivo QD distribution is confirmed by in vitro experiments. The data suggest that ECM components act as a selective barrier depending on QD surface modification. For future biomedical applications, such as targeting of blood vessel walls, the findings of this study offer design criteria for nanoconstructs that meet the requirements of the respective application.

Multiphoton Microscopy of Nonfluorescent Nanoparticles In Vitro and In Vivo.

Nanotechnology holds great promise for a plethora of potential applications. The interaction of engineered nanomaterials with living cells, tissues, and organisms is, however, only partly understood. Microscopic investigations of nano-bio interactions are mostly performed with a few model nanoparticles (NPs) which are easy to visualize, such as fluorescent quantum dots. Here the possibility to visualize nonfluorescent NPs with multiphoton excitation is investigated. Signals from silver (Ag), titanium dioxide (TiO2 ), and silica (SiO2 ) NPs in nonbiological environments are characterized to determine signal dependency on excitation wavelength and intensity as well as their signal stability over time. Ag NPs generate plasmon-induced luminescence decaying over time. TiO2 NPs induce photoluminescent signals of variable intensities and in addition strong third harmonic generation (THG). Optimal settings for microscopic detection are determined and then applied for visualization of these two particle types in living cells, in murine muscle tissue, and in the murine blood stream. Silica NPs produce a THG signal, but in living cells it cannot be discriminated sufficiently from endogenous cellular structures. It is concluded that multiphoton excitation is a viable option for studies of nano-bio interactions not only for fluorescent but also for some types of nonfluorescent NPs.

Spatiotemporal expression dynamics of selectins govern the sequential extravasation of neutrophils and monocytes in the acute inflammatory response.

OBJECTIVE: Leukocyte recruitment to the site of inflammation is a key event in a variety of cardiovascular pathologies. Infiltrating neutrophils constitute the first line of defense that precedes a second wave of emigrating monocytes reinforcing the inflammatory reaction. The mechanisms initiating this sequential process remained largely obscure. APPROACH AND RESULTS: Using advanced in vivo microscopy and in vitro/ex vivo techniques, we identified individual spatiotemporal expression patterns of selectins and their principal interaction partners on neutrophils, resident/inflammatory monocytes, and endothelial cells. Coordinating the intraluminal trafficking of neutrophils and inflammatory monocytes to common sites of extravasation, selectins assign different sites to these immune cells for their initial interactions with the microvascular endothelium. Whereas constitutively expressed leukocyte L-selectin/CD62L and endothelial P-selectin/CD62P together with CD44 and P-selectin glycoprotein ligand-1/CD162 initiate the emigration of neutrophils, de novo synthesis of endothelial E-selectin/CD62E launches the delayed secondary recruitment of inflammatory monocytes. In this context, P-selectin/CD62P and L-selectin/CD62L together with P-selectin glycoprotein ligand-1/CD162 and CD44 were found to regulate the flux of rolling neutrophils and inflammatory monocytes, whereas E-selectin/CD62E selectively adjusts the rolling velocity of inflammatory monocytes. Moreover, selectins and their interaction partners P-selectin glycoprotein ligand-1/CD162 and CD44 differentially control the intraluminal crawling behavior of neutrophils and inflammatory monocytes collectively enabling the sequential extravasation of these immune cells to inflamed tissue. CONCLUSIONS: Our findings provide novel insights into the mechanisms initiating the sequential infiltration of the perivascular tissue by neutrophils and monocytes in the acute inflammatory response and might thereby contribute to the development of targeted therapeutic strategies for prevention and treatment of cardiovascular diseases.

Matrix metalloproteinases modulate ameboid-like migration of neutrophils through inflamed interstitial tissue.

In vitro studies suggest that leukocytes locomote in an ameboid fashion independently of pericellular proteolysis. Whether this motility pattern applies for leukocyte migration in inflamed tissue is still unknown. In vivo microscopy on the inflamed mouse cremaster muscle revealed that blockade of serine proteases or of matrix metalloproteinases (MMPs) significantly reduces intravascular accumulation and transmigration of neutrophils. Using a novel in vivo chemotaxis assay, perivenular microinjection of inflammatory mediators induced directional interstitial migration of neutrophils. Blockade of actin polymerization, but not of actomyosin contraction abolished neutrophil interstitial locomotion. Multiphoton laser scanning in vivo microscopy showed that the density of the interstitial collagen network increases in inflamed tissue, thereby providing physical guidance to infiltrating neutrophils. Although neutrophils locomote through the interstitium without pericellular collagen degradation, inhibition of MMPs, but not of serine proteases, diminished their polarization and interstitial locomotion. In this context, blockade of MMPs was found to modulate expression of adhesion/signaling molecules on neutrophils. Collectively, our data indicate that serine proteases are critical for neutrophil extravasation, whereas these enzymes are dispensable for neutrophil extravascular locomotion. By contrast, neutrophil interstitial migration strictly relies on actin polymerization and does not require the pericellular degradation of collagen fibers but is modulated by MMPs.

Glycolysis is governed by growth regime and simple enzyme regulation in adherent MDCK cells.

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model's predictive power supports the design of more efficient bioprocesses.

Tissue plasminogen activator promotes postischemic neutrophil recruitment via its proteolytic and nonproteolytic properties.

OBJECTIVE: Neutrophil infiltration of the postischemic tissue considerably contributes to organ dysfunction on ischemia/reperfusion injury. Beyond its established role in fibrinolysis, tissue-type plasminogen activator (tPA) has recently been implicated in nonfibrinolytic processes. The role of this serine protease in the recruitment process of neutrophils remains largely obscure. APPROACH AND RESULTS: Using in vivo microscopy on the postischemic cremaster muscle, neutrophil recruitment and microvascular leakage, but not fibrinogen deposition at the vessel wall, were significantly diminished in tPA(-/-) mice. Using cell transfer techniques, leukocyte and nonleukocyte tPA were found to mediate ischemia/reperfusion-elicited neutrophil responses. Intrascrotal but not intra-arterial application of recombinant tPA induced a dose-dependent increase in the recruitment of neutrophils, which was significantly higher compared with stimulation with a tPA mutant lacking catalytic activity. Whereas tPA-dependent transmigration of neutrophils was selectively reduced on the inhibition of plasmin or gelatinases, neutrophil intravascular adherence was significantly diminished on the blockade of mast cell activation or lipid mediator synthesis. Moreover, stimulation with tPA caused a significant elevation in the leakage of fluorescein isothiocyanate dextran to the perivascular tissue, which was completely abolished on neutrophil depletion. In vitro, tPA-elicited macromolecular leakage of endothelial cell layers was abrogated on the inhibition of its proteolytic activity. CONCLUSIONS: Endogenously released tPA promotes neutrophil transmigration to reperfused tissue via proteolytic activation of plasmin and gelatinases. As a consequence, tPA on transmigrating neutrophils disrupts endothelial junctions allowing circulating tPA to extravasate to the perivascular tissue, which, in turn, amplifies neutrophil recruitment through the activation of mast cells and release of lipid mediators.

Transient targeting of phosphoinositide 3-kinase acts as a roadblock in mast cells' route to allergy.

BACKGROUND: Tissue mast cell numbers are dynamically regulated by recruitment of progenitors from the vasculature. It is unclear whether progenitors are recruited during allergic sensitization and whether recruitment promotes allergic responses. OBJECTIVE: We sought to (1) determine the effect of mast cell recruitment on acute allergic responses and (2) to define the role of phosphoinositide 3-kinase (PI3K) isoforms in sequential steps to allergic responses. METHODS: Gene-targeted mice for PI3Kγ or PI3Kδ or mice treated with isoform-specific PI3K inhibitors (a novel PI3Kγ-specific inhibitor [NVS-PI3-4] and the PI3Kδ inhibitor IC87114) were used to monitor IgE-mediated mast cell recruitment, migration, adhesion by means of intravital microscopy, degranulation, TNF-α release, and subsequent endothelial cell activation in vivo or in bone marrow-derived mast cells. RESULTS: Functional PI3Kγ, but not PI3Kδ, was crucial for mast cell accumulation in IgE-challenged skin, TNF-α release from IgE/antigen-stimulated mast cells, and mast cell/endothelial interactions and chemotaxis. PI3Kγ-deficient bone marrow-derived mast cells did not adhere to the endothelium in TNF-α-treated cremaster muscle, whereas PI3Kδ was not required. Depletion of TNF-α blocked IgE-induced mast cell recruitment, which links tissue mast cell-derived cytokine release to endothelial activation and mast cell recruitment. Interference with mast cell recruitment protected against anaphylaxis and was superior to blockage of tissue mast cell degranulation. CONCLUSIONS: Interference with mast cell recruitment to exacerbated tissues provides a novel strategy to alleviate allergic reactions and surpassed attenuation of tissue mast cell degranulation. This results in prolonged drug action and allows for reduction of drug doses required to block anaphylaxis, an important feature for drugs targeting inflammatory disease in general.

Bridging Smart Nanosystems with Clinically Relevant Models and Advanced Imaging for Precision Drug Delivery.

Intracellular delivery of nano-drug-carriers (NDC) to specific cells, diseased regions, or solid tumors has entered the era of precision medicine that requires systematic knowledge of nano-biological interactions from multidisciplinary perspectives. To this end, this review first provides an overview of membrane-disruption methods such as electroporation, sonoporation, photoporation, microfluidic delivery, and microinjection with the merits of high-throughput and enhanced efficiency for in vitro NDC delivery. The impact of NDC characteristics including particle size, shape, charge, hydrophobicity, and elasticity on cellular uptake are elaborated and several types of NDC systems aiming for hierarchical targeting and delivery in vivo are reviewed. Emerging in vitro or ex vivo human/animal-derived pathophysiological models are further explored and highly recommended for use in NDC studies since they might mimic in vivo delivery features and fill the translational gaps from animals to humans. The exploration of modern microscopy techniques for precise nanoparticle (NP) tracking at the cellular, organ, and organismal levels informs the tailored development of NDCs for in vivo application and clinical translation. Overall, the review integrates the latest insights into smart nanosystem engineering, physiological models, imaging-based validation tools, all directed towards enhancing the precise and efficient intracellular delivery of NDCs.

Real-World Sarilumab Use and Rule Testing to Predict Treatment Response in Patients with Rheumatoid Arthritis: Findings from the RISE Registry.

INTRODUCTION: Clinical trial findings may not be generalizable to routine practice. This study evaluated sarilumab effectiveness in patients with rheumatoid arthritis (RA) and tested the real-world applicability of a response prediction rule, derived from trial data using machine learning (based on C-reactive protein [CRP] > 12.3 mg/l and seropositivity [anticyclic citrullinated peptide antibodies, ACPA +]). METHODS: Sarilumab initiators from the ACR-RISE Registry, with ≥ 1 prescription on/after its FDA approval (2017-2020), were divided into three cohorts based on progressively restrictive criteria: Cohort A (had active disease), Cohort B (met eligibility criteria of a phase 3 trial in RA patients with inadequate response/intolerance to tumor necrosis factor inhibitors [TNFi]), and Cohort C (characteristics matched to the phase 3 trial baseline). Mean changes in Clinical Disease Activity Index (CDAI) and Routine Assessment of Patient Index Data 3 (RAPID3) were evaluated at 6 and 12 months. In a separate cohort, predictive rule was tested based on CRP levels and seropositive status (ACPA and/or rheumatoid factor); patients were categorized into rule-positive (seropositive with CRP > 12.3 mg/l) and rule-negative groups to compare the odds of achieving CDAI low disease activity (LDA)/remission and minimal clinically important difference (MCID) over 24 weeks. RESULTS: Among sarilumab initiators (N = 2949), treatment effectiveness was noted across cohorts, with greater improvement noted for Cohort C at 6 and 12 months. Among the predictive rule cohort (N = 205), rule-positive (vs. rule-negative) patients were more likely to reach LDA (odds ratio: 1.5 [0.7, 3.2]) and MCID (1.1 [0.5, 2.4]). Sensitivity analyses (CRP > 5 mg/l) showed better response to sarilumab in rule-positive patients. CONCLUSIONS: In real-world setting, sarilumab demonstrated treatment effectiveness, with greater improvements in the most selective population, mirroring phase 3 TNFi-refractory and rule-positive RA patients. Seropositivity appeared a stronger driver for treatment response than CRP, although optimization of the rule in routine practice requires further data.

Rheumatoid arthritis (RA) is a condition that may cause joint damage, if untreated. Sarilumab is an advanced medication, approved for treating moderate-to-severe RA in patients not responding to initial standard medicines. Clinical trials have shown that sarilumab improves RA symptoms; however, some people do not respond. This is a common problem in RA treatment. Physicians measure proteins in people's blood (called biomarkers; e.g., anticyclic citrullinated peptide antibodies [ACPA], C-reactive protein [CRP], and rheumatoid factor [RF]) to predict a medicine's response. A previous study showed that people with positive blood tests for ACPA and CRP (> 12.3 mg/l) responded well to sarilumab; this study was based on machine learning (a branch of science using computers) and identified factors that could be linked to treatment benefits. The present study analyzed routine data of 2949 people from the ACR-RISE Registry and showed an improvement in RA symptoms after 6 and 12 months of sarilumab, with a greater improvement noted in patients previously treated with other medicines. Biomarkers were tested in 205 people to check whether they could predict treatment response in day-to-day life. People were called rule-positive if they tested positive for RF and/or ACPA with CRP > 12.3 mg/l, and otherwise rule-negative. After 24 weeks of treatment, rule-positive people had a greater chance of disease improvement than rule-negative people. These results showed the benefits of sarilumab in RA in routine care and suggested the usefulness of machine learning in identifying biomarkers that physicians can use to make treatment decisions.

Additive efficacy of a bispecific anti-TNF/IL-6 nanobody compound in translational models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases affecting primarily the joints. Despite successful therapies including antibodies against tumor necrosis factor (TNF) and interleukin-6 (IL-6) receptor, only 20 to 30% of patients experience remission. We studied whether inhibiting both TNF and IL-6 would result in improved efficacy. Using backtranslation from single-cell RNA sequencing (scRNA-seq) data from individuals with RA, we hypothesized that TNF and IL-6 act synergistically on fibroblast-like synoviocytes (FLS) and T cells. Coculture of FLS from individuals with RA and T cells supported this hypothesis, revealing effects on both disease-driving pathways and biomarkers. Combining anti-TNF and anti-IL-6 antibodies in collagen-induced arthritis (CIA) mouse models resulted in sustained long-term remission, improved histology, and effects on bone remodeling pathways. These promising data initiated the development of an anti-TNF/IL-6 bispecific nanobody compound 1, with similar potencies against TNF and IL-6. We observed additive efficacy of compound 1 in a FLS/T cell coculture affecting arthritis and T helper 17 (TH17) pathways. This nanobody compound transcript signature inversely overlapped with described RA endotypes, indicating a potential efficacy in a broader patient population. In summary, we showed superiority of a bispecific anti-TNF/IL-6 nanobody compound or combination treatment over monospecific treatments in both in vitro and in vivo models. We anticipate improved efficacy in upcoming clinical studies.

Urokinase-type plasminogen activator promotes paracellular transmigration of neutrophils via Mac-1, but independently of urokinase-type plasminogen activator receptor.

BACKGROUND: Urokinase-type plasminogen activator (uPA) has recently been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury. The underlying mechanisms remain largely unclear. METHODS AND RESULTS: Using in vivo microscopy on the mouse cremaster muscle, I/R-elicited firm adherence and transmigration of neutrophils were found to be significantly diminished in uPA-deficient mice and in mice treated with the uPA inhibitor WX-340, but not in uPA receptor (uPAR)-deficient mice. Interestingly, postischemic leukocyte responses were significantly reduced on blockade of the integrin CD11b/Mac-1, which also serves as uPAR receptor. Using a cell transfer technique, postischemic adherence and transmigration of wild-type leukocytes were significantly decreased in uPA-deficient animals, whereas uPA-deficient leukocytes exhibited a selectively reduced transmigration in wild-type animals. On I/R or stimulation with recombinant uPA, >90% of firmly adherent leukocytes colocalized with CD31-immunoreactive endothelial junctions as detected by in vivo fluorescence microscopy. In a model of hepatic I/R, treatment with WX-340 significantly attenuated postischemic neutrophil infiltration and tissue injury. CONCLUSIONS: Our data suggest that endothelial uPA promotes intravascular adherence, whereas leukocyte uPA facilitates the subsequent paracellular transmigration of neutrophils during I/R. This process is regulated via CD11b/Mac-1, and does not require uPAR. Pharmacological blockade of uPA interferes with these events and effectively attenuates postischemic tissue injury.

Inhibitor of apoptosis proteins as novel targets in inflammatory processes.

OBJECTIVE: Inhibitor of apoptosis proteins (IAPs), such as X-linked or cellular IAP 1/2 (XIAP, cIAP1/2), are important regulators of apoptosis. IAP antagonists are currently under clinical investigation as anticancer agents. Interestingly, IAPs participate in the inflammation-associated TNF receptor signaling complex and regulate NFκB signaling. This raises the question about the role of IAPs in inflammation. Here, we investigated the anti-inflammatory potential of IAP inhibitors and the role of IAPs in inflammatory processes of endothelial cells. METHODS AND RESULTS: In mice, the small molecule IAP antagonist A-4.10099.1 (ABT) suppressed antigen-induced arthritis, leukocyte infiltration in concanavalin A-evoked liver injury, and leukocyte transmigration in the TNFα-activated cremaster muscle. In vitro, we observed an attenuation of leukocyte-endothelial cell interaction by downregulation of the intercellular adhesion molecule-1. ABT did not impair NFκB signaling but decreased the TNFα-induced activation of the TGF-ß-activated kinase 1, p38, and c-Jun N-terminal kinase. These effects are based on the proteasomal degradation of cIAP1/2 accompanied by an altered ratio of the levels of membrane-localized TNF receptor-associated factors 2 and 5. CONCLUSIONS: Our results reveal IAP antagonism as a profound anti-inflammatory principle in vivo and highlight IAPs as important regulators of inflammatory processes in endothelial cells.