Circulating c-Met-Expressing Memory T Cells Define Cardiac Autoimmunity.

BACKGROUND: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)-expressing (c-Met+) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. METHODS: In-depth phenotyping of peripheral blood T cells, including c-Met+ T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. RESULTS: We show that c-Met+ T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met+ T cells are distinct from those of c-Met-negative (c-Met-) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met+ T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met+ T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met+ T cells. CONCLUSIONS: Our study demonstrates that the detection of circulating c-Met+ T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

A randomised, double-blind, placebo-controlled, multicentre clinical trial of AZD1656 in diabetic patients hospitalised with COVID-19: The ARCADIA Trial - implications for therapeutic immune modulation.

Background: A potential immunotherapeutic role for AZD1656 (a glucokinase activator) in the treatment of COVID-19 was hypothesized. The ARCADIA trial investigated the safety and efficacy of AZD1656 in diabetic patients admitted to hospital with COVID-19. Methods: The ARCADIA trial was a Phase II randomised, double-blind, placebo-controlled clinical trial. Adult diabetic patients, admitted with COVID-19, were recruited at 28 hospitals in the UK, Romania and Czech Republic and randomly assigned (1:1) to receive AZD1656 tablets (100mg twice a day), or matched placebo, for up to 21 days, in addition to usual care. All involved were masked to treatment allocation. The primary endpoint was clinical improvement measured at Day 14. The Full Analysis Set (FAS) included all patients who received at least one dose of assigned treatment. ARCADIA is complete and registered with ClinicalTrials.gov (NCT04516759). Findings: Between 29 September 2020 to 16 April 2021, 170 patients were screened and 156 patients were randomised, three of whom did not commence treatment. Of the remaining 153, 80 were assigned to AZD1656 and 73 were assigned to placebo and included in the Full Analysis Set (FAS). The primary analysis showed no statistically significant difference between groups (AZD1656: 76·3%; Placebo: 69·9%, p=0·19). There was no difference in the number of adverse events between groups (AZD1656: 35·7%; Placebo: 33·3%). Mortality was lower in the AZD1656 group compared to the placebo group (AZD1656: four (5%); Placebo: nine (12·3%), p=0·090)). At Day 7 there were zero deaths in the AZD1656 group compared to six deaths in the placebo group (p=0·011, post hoc). A difference between groups in time to hospital discharge was also seen (p=0·16). Immunophenotyping data suggested that AZD1656-treated patients had a less pro-inflammatory immune response and a better adaptive immune response than those treated with placebo. Interpretation: Although the trial did not achieve its primary endpoint, AZD1656 was associated with a decrease in deaths and a reduction in the duration of hospitalisation, as compared to Placebo. Immunophenotyping and immunochemistry indicated an immunomodulatory effect of AZD1656. The trial suggests a beneficial therapeutic effect of AZD1656 and identifies a new therapeutic concept: small molecule activation of endogenous homeostatic immune cells which themselves become the therapeutic agent within the body. Phase 2 trials of this size carry the risk of false positive results and confirmation of these results in a larger clinical trial is now required. Funding: UK Research and Innovation (UKRI) 'Innovate UK' programme and Excalibur Medicines Ltd.

Notch Signaling Regulates Muscle Stem Cell Homeostasis and Regeneration in a Teleost Fish.

Muscle regeneration is mediated by the activity of resident muscle satellite cells (muSCs) that express Pax7. In mouse Notch signaling regulates muSCs during quiescence and promotes muSC proliferation in regeneration. It is unclear if these roles of Notch in regulating muSC biology are conserved across vertebrates or are a mammalian specific feature. We have therefore investigated the role of Notch in regulating muSC homeostasis and regeneration in a teleost fish, the zebrafish. We have also tested whether muSCs show differential sensitivity to Notch during myotome development. In an absence of injury Notch is important for preventing muSC proliferation at the vertical myoseptum. In contrast, Notch signaling promotes proliferation and prevents differentiation in the context of injury. Notch is required for the proliferative response to injury at early and later larval stages, suggesting it plays a similar role in regulating muSCs at developing and adult stages. Our results reveal a conserved role for Notch signaling in regulating muSCs under homeostasis and for promoting proliferation during regeneration in teleost fish.

Differential regulation of epithelial growth by gingival and periodontal fibroblasts in vitro.

OBJECTIVES: To investigate the underlying molecular mechanisms by which gingival and periodontal ligament (PDL) fibroblasts regulate epithelial phenotype. BACKGROUND: Fibroblast populations regulate the epithelial phenotype through epithelial-mesenchymal interactions (EMI). Previous studies have proposed that maintenance of the junctional epithelium (JE) is dependent on the differential effects from gingival and PDL tissues. However, these cell populations are undefined and the signalling mechanisms which may regulate JE are unknown. METHODS: Immunohistochemical analyses were performed on formalin-fixed paraffin-embedded sections of dentogingival tissues to identify phenotypic differences in fibroblast populations. The effect of distinct fibroblasts on epithelial phenotype was studied via 3D organotypic cultures, consisting of an H400 epithelium supported by human gingival fibroblasts (HGF) or human periodontal ligament fibroblasts (HPDLF), embedded in collagen gel. To investigate the involvement of Wnt signalling in EMI, the Wnt antagonist rhDKK1 was added to HGF constructs. The gene expression of Wnt antagonists and agonists was tested via RNA extraction and qPCR. Specific gene silencing using RNA interference was performed on HPDLF/HGF constructs. RESULTS: Gingival fibroblasts were characterized by Sca1 expression, and PDL fibroblasts, characterized by Periostin and Asporin expression. Through the construction of 3D organotypic cultures, we showed that HGF supported epithelial multilayering, whilst HPDLF failed to support epithelial cell growth. Furthermore, HGF constructs treated with rhDKK1 resulted in a profound reduction in epithelial thickness. We identified SFRP4 to be highly specifically expressed in HPDLF, at both the mRNA and protein levels. A knockdown of SFRP4 in HPDLF constructs led to an increase in epithelial growth. CONCLUSION: The study demonstrates the presence of phenotypically distinct fibroblast populations within dentogingival tissues and that these specific populations have different influences on the epithelium. Our data suggest that a downregulation of Wnt signalling within PDL may be important in maintaining the integrity and anatomical position of the JE.

Mapping the distribution of stem/progenitor cells across the mouse middle ear during homeostasis and inflammation.

The middle ear epithelium is derived from neural crest and endoderm, which line distinct regions of the middle ear cavity. Here, we investigate the distribution of putative stem cell markers in the middle ear, combined with an analysis of the location of label-retaining cells (LRCs) to create a map of the middle ear mucosa. We show that proliferating cells and LRCs were associated with specific regions of the ear epithelium, concentrated in the hypotympanum at the base of the auditory bulla and around the ear drum. Sox2 was widely expressed in the endodermally derived ciliated pseudostratified epithelium of the hypotympanum. This part of the middle ear showed high levels of Wnt activity, as indicated by the expression of Axin2, a readout of Wnt signalling. Keratin 5 showed a more restricted expression within the basal cells of this region, with very little overlap between the Sox2- and keratin 5-positive epithelium, indicating that these genes mark distinct populations. Little expression of Sox2 or keratin 5 was observed in the neural crest-derived middle ear epithelium that lined the promontory, except in cases of otitis media when this epithelium underwent hyperplasia. This study lays the foundation for furthering our understanding of homeostasis and repair in the middle ear.

Dissecting the role of Wnt signaling and its interactions with FGF signaling during midbrain neurogenesis.

Interactions between FGF and Wnt/ bcat signaling control development of the midbrain. The nature of this interaction and how these regulate patterning, growth and differentiation is less clear, as it has not been possible to temporally dissect the effects of one pathway relative to the other. We have employed pharmacological and genetic tools to probe the temporal and spatial roles of FGF and Wnt in controlling the specification of early midbrain neurons. We identify a ß-catenin (bcat) independent role for GSK-3 in modulating FGF activity and hence neuronal patterning. This function is complicated by an overlap with bcat-dependent regulation of FGF signaling, through the regulation of sprouty4. Additionally we reveal how attenuation of Axin protein function can promote fluctuating levels of bcat activity that are dependent on FGF activity. This highlights the complex nature of the interactions between FGF and Wnt/ bcat and reveals that they act at multiple levels to control each others activity in the midbrain.

Specification of sensory neurons occurs through diverse developmental programs functioning in the brain and spinal cord.

BACKGROUND: Vertebrates possess two populations of sensory neurons located within the central nervous system: Rohon-Beard (RB) and mesencephalic trigeminal nucleus (MTN) neurons. RB neurons are transient spinal cord neurons whilst MTN neurons are the proprioceptive cells that innervate the jaw muscles. It has been suggested that MTN and RB neurons share similarities and may have a common developmental program, but it is unclear how similar or different their development is. RESULTS: We have dissected RB and MTN neuronal specification in zebrafish. We find that RB and MTN neurons express a core set of genes indicative of sensory neurons, but find these are also expressed by adjacent diencephalic neurons. Unlike RB neurons, our evidence argues against a role for the neural crest during MTN development. We additionally find that neurogenin1 function is dispensable for MTN differentiation, unlike RB cells and all other sensory neurons. Finally, we demonstrate that, although Notch signalling is involved in RB development, it is not involved in the generation of MTN cells. CONCLUSIONS: Our work reveals fundamental differences between the development of MTN and RB neurons and suggests that these populations are non-homologous and thus have distinct developmental and, probably, evolutionary origins.

A bi-modal function of Wnt signalling directs an FGF activity gradient to spatially regulate neuronal differentiation in the midbrain.

FGFs and Wnts are important morphogens during midbrain development, but their importance and potential interactions during neurogenesis are poorly understood. We have employed a combination of genetic and pharmacological manipulations in zebrafish to show that during neurogenesis FGF activity occurs as a gradient along the anterior-posterior axis of the dorsal midbrain and directs spatially dynamic expression of the Hairy gene her5. As FGF activity diminishes during development, Her5 is lost and differentiation of neuronal progenitors occurs in an anterior-posterior manner. We generated mathematical models to explain how Wnt and FGFs direct the spatial differentiation of neurons in the midbrain through Wnt regulation of FGF signalling. These models suggested that a negative-feedback loop controlled by Wnt is crucial for regulating FGF activity. We tested Sprouty genes as mediators of this regulatory loop using conditional mouse knockouts and pharmacological manipulations in zebrafish. These reveal that Sprouty genes direct the positioning of early midbrain neurons and are Wnt responsive in the midbrain. We propose a model in which Wnt regulates FGF activity at the isthmus by driving both FGF and Sprouty gene expression. This controls a dynamic, posteriorly retracting expression of her5 that directs neuronal differentiation in a precise spatiotemporal manner in the midbrain.