Stromal cells in chronic inflammation and tertiary lymphoid organ formation.

Inflammation is an unstable state. It either resolves or persists. Why inflammation persists and the factors that define tissue tropism remain obscure. Increasing evidence suggests that tissue-resident stromal cells not only provide positional memory but also actively regulate the differential accumulation of inflammatory cells within inflamed tissues. Furthermore, at many sites of chronic inflammation, structures that mimic secondary lymphoid tissues are observed, suggesting that chronic inflammation and lymphoid tissue formation share common activation programs. Similarly, blood and lymphatic endothelial cells contribute to tissue homeostasis and disease persistence in chronic inflammation. This review highlights our increasing understanding of the role of stromal cells in inflammation and summarizes the novel immunological role that stromal cells exert in the persistence of inflammatory diseases.

A longitudinal single-cell atlas of anti-tumour necrosis factor treatment in inflammatory bowel disease.

Precision medicine in immune-mediated inflammatory diseases (IMIDs) requires a cellular understanding of treatment response. We describe a therapeutic atlas for Crohn's disease (CD) and ulcerative colitis (UC) following adalimumab, an anti-tumour necrosis factor (anti-TNF) treatment. We generated ~1 million single-cell transcriptomes, organised into 109 cell states, from 216 gut biopsies (41 subjects), revealing disease-specific differences. A systems biology-spatial analysis identified granuloma signatures in CD and interferon (IFN)-response signatures localising to T cell aggregates and epithelial damage in CD and UC. Pretreatment differences in epithelial and myeloid compartments were associated with remission outcomes in both diseases. Longitudinal comparisons demonstrated disease progression in nonremission: myeloid and T cell perturbations in CD and increased multi-cellular IFN signalling in UC. IFN signalling was also observed in rheumatoid arthritis (RA) synovium with a lymphoid pathotype. Our therapeutic atlas represents the largest cellular census of perturbation with the most common biologic treatment, anti-TNF, across multiple inflammatory diseases.

CD200+ fibroblasts form a pro-resolving mesenchymal network in arthritis.

Fibroblasts are important regulators of inflammation, but whether fibroblasts change phenotype during resolution of inflammation is not clear. Here we use positron emission tomography to detect fibroblast activation protein (FAP) as a means to visualize fibroblast activation in vivo during inflammation in humans. While tracer accumulation is high in active arthritis, it decreases after tumor necrosis factor and interleukin-17A inhibition. Biopsy-based single-cell RNA-sequencing analyses in experimental arthritis show that FAP signal reduction reflects a phenotypic switch from pro-inflammatory MMP3+/IL6+ fibroblasts (high FAP internalization) to pro-resolving CD200+DKK3+ fibroblasts (low FAP internalization). Spatial transcriptomics of human joints indicates that pro-resolving niches of CD200+DKK3+ fibroblasts cluster with type 2 innate lymphoid cells, whereas MMP3+/IL6+ fibroblasts colocalize with inflammatory immune cells. CD200+DKK3+ fibroblasts stabilized the type 2 innate lymphoid cell phenotype and induced resolution of arthritis via CD200-CD200R1 signaling. Taken together, these data suggest a dynamic molecular regulation of the mesenchymal compartment during resolution of inflammation.

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Why does understanding the biology of fibroblasts in immunity really matter?

Fibroblasts are known for their ability to make and modify the extracellular matrix. However, there is more to them than meets the eye. It is now clear that they help define tissue microenvironments and support immune responses in organs. As technology advances, we have started to uncover the secrets of fibroblasts. In this Essay, we present fibroblasts as not only the builders and renovators of tissue environments but also the rheostat cells for immune circuits. Although they perform location-specific functions, they do not have badges of fixed identity. Instead, they display a spectrum of functional states and can swing between these states depending on the needs of the organ. As fibroblasts participate in a range of activities both in health and disease, finding the key factors that alter their development and functional states will be an important goal to restore homeostasis in maladapted tissues.

Notch signalling drives synovial fibroblast identity and arthritis pathology.

The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.

Abatacept in individuals at high risk of rheumatoid arthritis (APIPPRA): a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial.

BACKGROUND: Individuals with serum antibodies to citrullinated protein antigens (ACPA), rheumatoid factor, and symptoms, such as inflammatory joint pain, are at high risk of developing rheumatoid arthritis. In the arthritis prevention in the pre-clinical phase of rheumatoid arthritis with abatacept (APIPPRA) trial, we aimed to evaluate the feasibility, efficacy, and acceptability of treating high risk individuals with the T-cell co-stimulation modulator abatacept. METHODS: The APIPPRA study was a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial done in 28 hospital-based early arthritis clinics in the UK and three in the Netherlands. Participants (aged ≥18 years) at risk of rheumatoid arthritis positive for ACPA and rheumatoid factor with inflammatory joint pain were recruited. Exclusion criteria included previous episodes of clinical synovitis and previous use of corticosteroids or disease-modifying antirheumatic drugs. Participants were randomly assigned (1:1) using a computer-generated permuted block randomisation (block sizes of 2 and 4) stratified by sex, smoking, and country, to 125 mg abatacept subcutaneous injections weekly or placebo for 12 months, and then followed up for 12 months. Masking was achieved by providing four kits (identical in appearance and packaging) with pre-filled syringes with coded labels of abatacept or placebo every 3 months. The primary endpoint was the time to development of clinical synovitis in three or more joints or rheumatoid arthritis according to American College of Rheumatology and European Alliance of Associations for Rheumatology 2010 criteria, whichever was met first. Synovitis was confirmed by ultrasonography. Follow-up was completed on Jan 13, 2021. All participants meeting the intention-to-treat principle were included in the analysis. This trial was registered with EudraCT (2013-003413-18). FINDINGS: Between Dec 22, 2014, and Jan 14, 2019, 280 individuals were evaluated for eligibility and, of 213 participants, 110 were randomly assigned to abatacept and 103 to placebo. During the treatment period, seven (6%) of 110 participants in the abatacept group and 30 (29%) of 103 participants in the placebo group met the primary endpoint. At 24 months, 27 (25%) of 110 participants in the abatacept group had progressed to rheumatoid arthritis, compared with 38 (37%) of 103 in the placebo group. The estimated proportion of participants remaining arthritis-free at 12 months was 92·8% (SE 2·6) in the abatacept group and 69·2% (4·7) in the placebo group. Kaplan-Meier arthritis-free survival plots over 24 months favoured abatacept (log-rank test p=0·044). The difference in restricted mean survival time between groups was 53 days (95% CI 28-78; p<0·0001) at 12 months and 99 days (95% CI 38-161; p=0·0016) at 24 months in favour of abatacept. During treatment, abatacept was associated with improvements in pain scores, functional wellbeing, and quality-of-life measurements, as well as low scores of subclinical synovitis by ultrasonography, compared with placebo. However, the effects were not sustained at 24 months. Seven serious adverse events occurred in the abatacept group and 11 in the placebo group, including one death in each group deemed unrelated to treatment. INTERPRETATION: Therapeutic intervention during the at-risk phase of rheumatoid arthritis is feasible, with acceptable safety profiles. T-cell co-stimulation modulation with abatacept for 12 months reduces progression to rheumatoid arthritis, with evidence of sustained efficacy beyond the treatment period, and with no new safety signals. FUNDING: Bristol Myers Squibb.

Distinct fibroblast subsets drive inflammation and damage in arthritis.

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Single-cell RNA sequencing analysis of vestibular schwannoma reveals functionally distinct macrophage subsets.

BACKGROUND: Vestibular schwannomas (VSs) remain a challenge due to their anatomical location and propensity to growth. Macrophages are present in VS but their roles in VS pathogenesis remains unknown. OBJECTIVES: The objective was to assess phenotypic and functional profile of macrophages in VS with single-cell RNA sequencing (scRNAseq). METHODS: scRNAseq was carried out in three VS samples to examine characteristics of macrophages in the tumour. RT-qPCR was carried out on 10 VS samples for CD14, CD68 and CD163 and a panel of macrophage-associated molecules. RESULTS: scRNAseq revealed macrophages to be a major constituent of VS microenvironment with three distinct subclusters based on gene expression. The subclusters were also defined by expression of CD163, CD68 and IL-1ß. AREG and PLAUR were expressed in the CD68+CD163+IL-1ß+ subcluster, PLCG2 and NCKAP5 were expressed in CD68+CD163+IL-1ß- subcluster and AUTS2 and SPP1 were expressed in the CD68+CD163-IL-1ß+ subcluster. RT-qPCR showed expression of several macrophage markers in VS of which CD14, ALOX15, Interleukin-1ß, INHBA and Colony Stimulating Factor-1R were found to have a high correlation with tumour volume. CONCLUSIONS: Macrophages form an important component of VS stroma. scRNAseq reveals three distinct subsets of macrophages in the VS tissue which may have differing roles in the pathogenesis of VS.

Hybrid predictive coding: Inferring, fast and slow.

Predictive coding is an influential model of cortical neural activity. It proposes that perceptual beliefs are furnished by sequentially minimising "prediction errors"-the differences between predicted and observed data. Implicit in this proposal is the idea that successful perception requires multiple cycles of neural activity. This is at odds with evidence that several aspects of visual perception-including complex forms of object recognition-arise from an initial "feedforward sweep" that occurs on fast timescales which preclude substantial recurrent activity. Here, we propose that the feedforward sweep can be understood as performing amortized inference (applying a learned function that maps directly from data to beliefs) and recurrent processing can be understood as performing iterative inference (sequentially updating neural activity in order to improve the accuracy of beliefs). We propose a hybrid predictive coding network that combines both iterative and amortized inference in a principled manner by describing both in terms of a dual optimization of a single objective function. We show that the resulting scheme can be implemented in a biologically plausible neural architecture that approximates Bayesian inference utilising local Hebbian update rules. We demonstrate that our hybrid predictive coding model combines the benefits of both amortized and iterative inference-obtaining rapid and computationally cheap perceptual inference for familiar data while maintaining the context-sensitivity, precision, and sample efficiency of iterative inference schemes. Moreover, we show how our model is inherently sensitive to its uncertainty and adaptively balances iterative and amortized inference to obtain accurate beliefs using minimum computational expense. Hybrid predictive coding offers a new perspective on the functional relevance of the feedforward and recurrent activity observed during visual perception and offers novel insights into distinct aspects of visual phenomenology.

A behavioral and modeling study of control algorithms underlying the translational optomotor response in larval zebrafish with implications for neural circuit function.

The optomotor response (OMR) is central to the locomotory behavior in diverse animal species including insects, fish and mammals. Furthermore, the study of the OMR in larval zebrafish has become a key model system for investigating the neural basis of sensorimotor control. However, a comprehensive understanding of the underlying control algorithms is still outstanding. In fish it is often assumed that the OMR, by reducing average optic flow across the retina, serves to stabilize position with respect to the ground. Yet the degree to which this is achieved, and how it could emerge from the intermittent burst dynamics of larval zebrafish swimming, are unclear. Here, we combine detailed computational modeling with a new approach to free-swimming experiments in which we control the amount of visual feedback produced by a given motor effort by varying the height of the larva above a moving grid stimulus. We develop an account of underlying feedback control mechanisms that describes both the bout initiation process and the control of swim speed during bouts. We observe that the degree to which fish stabilize their position is only partial and height-dependent, raising questions about its function. We find the relative speed profile during bouts follows a fixed temporal pattern independent of absolute bout speed, suggesting that bout speed and bout termination are not separately controlled. We also find that the reverse optic flow, experienced when the fish is swimming faster than the stimulus, plays a minimal role in control of the OMR despite carrying most of the sensory information about self-movement. These results shed new light on the underlying dynamics of the OMR in larval zebrafish and will be crucial for future work aimed at identifying the neural basis of this behavior.

Physical and Chemical Compatibility of Medications Commonly Used in Critically Ill Patients With Balanced Crystalloids: A Systematic Review.

OBJECTIVE: Evaluate available evidence of physical and/or chemical compatibility of commonly used medications in critically ill patients with balanced crystalloids. DATA SOURCES: Ovid MEDLINE, Embase, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews were queried from inception to September 2022. STUDY SELECTION AND DATA EXTRACTION: This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. English-language studies reporting physical and/or chemical compatibility data between 50 selected medications and balanced crystalloids were included. A previously designed tool to assess risk of bias was adapted for use. DATA SYNTHESIS: Twenty-nine studies encompassing 39 (78%) medications and 188 unique combinations with balanced crystalloids were included. Combinations included 35 (70%) medications with lactated Ringer's, 26 (52%) medications with Plasma-Lyte, 10 (20%) medications with Normosol, and one (2%) medication with Isolyte. Studies commonly evaluated physical and chemical compatibility (55.2%). More medications were evaluated via Y-site than admixture. Incompatibilities were identified in 18% of combinations comprising 13 individual drugs. RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: This systematic review evaluates the compatibility of select critical care medications with balanced crystalloid solutions. Results may be used as a tool to guide clinicians on balanced crystalloid compatibility, potentially increasing ubiquitous use and reducing patient exposure to normal saline. CONCLUSION AND RELEVANCE: Data are limited regarding chemical/physical compatibility of commonly used medications in critically ill patients with balanced crystalloids. Additional compatibility studies are warranted, particularly methodologically rigorous studies assessing Plasma-Lyte, Normosol, and Isolyte. Of the evaluated medications, there was a low frequency of incompatibilities with balanced crystalloids.

Induction and transcriptional regulation of the co-inhibitory gene module in T cells.

The expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated expression of co-inhibitory receptors on CD4+ T cells promotes autoimmunity, whereas sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and diseases such as cancer1,2. Here, using RNA and protein expression profiling at single-cell resolution in mouse cells, we identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, TIM-3, LAG-3 and TIGIT) but also many new surface receptors. We functionally validated two new co-inhibitory receptors, activated protein C receptor (PROCR) and podoplanin (PDPN). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in several physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors PRDM1 and c-MAF as cooperative regulators of the co-inhibitory module, and this was validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies regulators of T cell function with the potential to control autoimmunity and tumour immunity.

Stress testing the Centiloid: Precision and variability of PET quantification of amyloid pathology.

INTRODUCTION: Assessing the potential sources of bias and variability of the Centiloid (CL) scale is fundamental for its appropriate clinical application. METHODS: We included 533 participants from AMYloid imaging to Prevent Alzheimer's Disease (AMYPAD DPMS) and Alzheimer's Disease Neuroimaging Initiative (ADNI) cohorts. Thirty-two CL pipelines were created using different combinations of reference region (RR), RR and target types, and quantification spaces. Generalized estimating equations stratified by amyloid positivity were used to assess the impact of the quantification pipeline, radiotracer, age, brain atrophy, and harmonization status on CL. RESULTS: RR selection and RR type impact CL the most, particularly in amyloid-negative individuals. The standard CL pipeline with the whole cerebellum as RR is robust against brain atrophy and differences in image resolution, with 95% confidence intervals below ± 3.95 CL for amyloid beta positivity cutoffs (CL < 24). DISCUSSION: The standard CL pipeline is recommended for most scenarios. Confidence intervals should be considered when operationalizing CL cutoffs in clinical and research settings. HIGHLIGHTS: We developed a framework for evaluating Centiloid (CL) variability to different factors. Reference region selection and delineation had the highest impact on CL values. Whole cerebellum (WCB) and whole cerebellum plus brainstem (WCB+BSTM) as reference regions yielded consistent results across tracers. The standard CL pipeline is robust against atrophy and image resolution variation. Estimated within- and between-pipeline variability (95% confidence interval) in absolute CL units.

Investigating reliable amyloid accumulation in Centiloids: Results from the AMYPAD Prognostic and Natural History Study.

INTRODUCTION: To support clinical trial designs focused on early interventions, our study determined reliable early amyloid-ß (Aß) accumulation based on Centiloids (CL) in pre-dementia populations. METHODS: A total of 1032 participants from the Amyloid Imaging to Prevent Alzheimer's Disease-Prognostic and Natural History Study (AMYPAD-PNHS) and Insight46 who underwent [18F]flutemetamol, [18F]florbetaben or [18F]florbetapir amyloid-PET were included. A normative strategy was used to define reliable accumulation by estimating the 95th percentile of longitudinal measurements in sub-populations (NPNHS = 101/750, NInsight46 = 35/382) expected to remain stable over time. The baseline CL threshold that optimally predicts future accumulation was investigated using precision-recall analyses. Accumulation rates were examined using linear mixed-effect models. RESULTS: Reliable accumulation in the PNHS was estimated to occur at >3.0 CL/year. Baseline CL of 16 [12,19] best predicted future Aß-accumulators. Rates of amyloid accumulation were tracer-independent, lower for APOE ε4 non-carriers, and for subjects with higher levels of education. DISCUSSION: Our results support a 12-20 CL window for inclusion into early secondary prevention studies. Reliable accumulation definition warrants further investigations.

Osteonecrosis of the jaw after radiation followed by bevacizumab.

Osteonecrosis of the jaw is a recognized complication associated with bevacizumab. Here, we present a patient with squamous cell carcinoma of the tonsil who experienced minimal skin fibrosis following intensity-modulated radiation therapy. Subsequently, the patient developed rectal adenocarcinoma and encountered osteonecrosis of the jaw after receiving two cycles of bevacizumab. Close monitoring, accompanied by thorough examination to detect early signs of osteonecrosis of the jaw, should be considered for patients who have undergone radiation therapy in the head and neck region and are receiving bevacizumab or other medications known to be associated with osteonecrosis of the jaw.

Natural Induction: Spontaneous Adaptive Organisation without Natural Selection.

Evolution by natural selection is believed to be the only possible source of spontaneous adaptive organisation in the natural world. This places strict limits on the kinds of systems that can exhibit adaptation spontaneously, i.e., without design. Physical systems can show some properties relevant to adaptation without natural selection or design. (1) The relaxation, or local energy minimisation, of a physical system constitutes a natural form of optimisation insomuch as it finds locally optimal solutions to the frustrated forces acting on it or between its components. (2) When internal structure 'gives way' or accommodates a pattern of forcing on a system, this constitutes learning insomuch, as it can store, recall, and generalise past configurations. Both these effects are quite natural and general, but in themselves insufficient to constitute non-trivial adaptation. However, here we show that the recurrent interaction of physical optimisation and physical learning together results in significant spontaneous adaptive organisation. We call this adaptation by natural induction. The effect occurs in dynamical systems described by a network of viscoelastic connections subject to occasional disturbances. When the internal structure of such a system accommodates slowly across many disturbances and relaxations, it spontaneously learns to preferentially visit solutions of increasingly greater quality (exceptionally low energy). We show that adaptation by natural induction thus produces network organisations that improve problem-solving competency with experience (without supervised training or system-level reward). We note that the conditions for adaptation by natural induction, and its adaptive competency, are different from those of natural selection. We therefore suggest that natural selection is not the only possible source of spontaneous adaptive organisation in the natural world.