Development of a Large-Scale Pathogen Screening Test for the Biosafety Evaluation of Canine Mesenchymal Stem Cells.

BACKGROUND: The action of mesenchymal stem cells (MSCs) is the subject of intense research in the field of regenerative medicine, including their potential use in companion animals, such as dogs. To ensure the safety of canine MSC batches for their application in regenerative medicine, a quality control test must be conducted in accordance with Good Manufacturing Practices (GMP). Based on guidance provided by the European Medicines Agency, this study aimed to develop and validate a highly sensitive and robust, nucleic acid-based test panel for the detection of various canine pathogens. Analytical sensitivity, specificity, amplification efficiency, and linearity were evaluated to ensure robust assessment. Additionally, viable spike-in controls were used to control for optimal nucleic acid extraction. The conventional PCR-based and real-time PCR-based pathogen assays were evaluated in a real-life setting, by direct testing MSC batches. RESULTS: The established nucleic acid-based assays displayed remarkable sensitivity, detecting 100-1 copies/reaction of template DNA. They also exhibited high specificity and efficiency. Moreover, highly effective nucleic acid isolation was confirmed by the sensitive detection of spike-in controls. The detection capacity of our optimized and validated methods was determined by direct pathogen testing of nine MSC batches that displayed unusual phenotypes, such as reduced cell division or other deviating characteristics. Among these MCS batches of uncertain purity, only one tested negative for all pathogens. The direct testing of these samples yielded positive results for important canine pathogens, including tick-borne disease-associated species and viral members of the canine infectious respiratory disease complex (CIRDC). Notably, samples positive for the etiological agents responsible for enteritis (CPV), leptospirosis (Leptospira interrogans), and neosporosis (Neospora caninum) were also identified. Furthermore, we conducted biosafety evaluation of 12 MSC batches intended for therapeutic application. Eleven MSC batches were found to be free of extraneous agents, and only one tested positive for a specific pathogen, namely, canine parvovirus. CONCLUSION: In this study, we established and validated reliable, highly sensitive, and accurate nucleic acid-based testing methods for a broad spectrum of canine pathogens.

TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages.

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-ß. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.

Pathophysiology and Emerging Molecular Therapeutic Targets in Heterotopic Ossification.

The term heterotopic ossification (HO) describes bone formation in tissues where bone is normally not present. Musculoskeletal trauma induces signalling events that in turn trigger cells, probably of mesenchymal origin, to differentiate into bone. The aetiology of HO includes extremely rare but severe, generalised and fatal monogenic forms of the disease; and as a common complex disorder in response to musculoskeletal, neurological or burn trauma. The resulting bone forms through a combination of endochondral and intramembranous ossification, depending on the aetiology, initiating stimulus and affected tissue. Given the heterogeneity of the disease, many cell types and biological pathways have been studied in efforts to find effective therapeutic strategies for the disorder. Cells of mesenchymal, haematopoietic and neuroectodermal lineages have all been implicated in the pathogenesis of HO, and the emerging dominant signalling pathways are thought to occur through the bone morphogenetic proteins (BMP), mammalian target of rapamycin (mTOR), and retinoic acid receptor pathways. Increased understanding of these disease mechanisms has resulted in the emergence of several novel investigational therapeutic avenues, including palovarotene and other retinoic acid receptor agonists and activin A inhibitors that target both canonical and non-canonical signalling downstream of the BMP type 1 receptor. In this article we aim to illustrate the key cellular and molecular mechanisms involved in the pathogenesis of HO and outline recent advances in emerging molecular therapies to treat and prevent HO that have had early success in the monogenic disease and are currently being explored in the common complex forms of HO.

The Kinesin Gene KIF26B Modulates the Severity of Post-Traumatic Heterotopic Ossification.

The formation of pathological bone deposits within soft tissues, termed heterotopic ossification (HO), is common after trauma. However, the severity of HO formation varies substantially between individuals, from relatively isolated small bone islands through to extensive soft tissue replacement by bone giving rise to debilitating symptoms. The aim of this study was to identify novel candidate therapeutic molecular targets for severe HO. We conducted a genome-wide scan in men and women with HO of varying severity following hip replacement for osteoarthritis. HO severity was dichotomized as mild or severe, and association analysis was performed with adjustment for age and sex. We next confirmed expression of the gene encoded by the lead signal in human bone and in primary human mesenchymal stem cells. We then examined the effect of gene knockout in a murine model of osseous trans-differentiation, and finally we explored transcription factor phosphorylation in key pathways perturbed by the gene. Ten independent signals were suggestively associated with HO severity, with KIF26B as the lead. We subsequently confirmed KIF26B expression in human bone and upregulation upon BMP2-induced osteogenic differentiation in primary human mesenchymal stem cells, and also in a rat tendo-Achilles model of post-traumatic HO. CRISPR-Cas9 mediated knockout of Kif26b inhibited BMP2-induced Runx2, Sp7/Osterix, Col1A1, Alp, and Bglap/Osteocalcin expression and mineralized nodule formation in a murine myocyte model of osteogenic trans-differentiation. Finally, KIF26B deficiency inhibited ERK MAP kinase activation during osteogenesis, whilst augmenting p38 and SMAD 1/5/8 phosphorylation. Taken together, these data suggest a role for KIF26B in modulating the severity of post-traumatic HO and provide a potential novel avenue for therapeutic translation.

Tribbles in inflammation.

Inflammation is part of the physiological innate immune response to invading pathogens and tissue injury. However, unresolved inflammation leads to human disease. The tribbles (TRIB) family of pseudokinase proteins has been shown to modulate key inflammatory signalling pathways, including the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) networks. This review summarizes our current knowledge on TRIBs in the context of inflammation, both at the level of molecular mechanisms and in disease development.

Tribbles role in reproduction.

Tribbles (TRIB) proteins, a family of evolutionary conserved psuedokinase proteins, modulate various signalling pathways within the cell. The regulatory roles of TRIB make them an important part of a number of biological processes ranging from cell proliferation to metabolism, immunity, inflammation and carcinogenesis. Innate immune system plays a pivotal role during the regulation of reproductive processes that allows successful creation of an offspring. Its involvement initiates from fertilization of the oocyte by spermatozoon and lasts throughout early embryonic development, pregnancy and labour. Therefore, there is a close cooperation between the reproductive system and the innate immune system. Evidence from our lab has demonstrated that improper activation of the innate immune system can reduce embryo implantation, thus leading to infertility. Therefore, control mechanisms regulating the innate immune system function can be critical for successful reproductive events.

Oncosuppressive functions of tribbles pseudokinase 3.

Tribbles pseudokinase 3 (TRIB3) belongs to the tribbles family of pseudokinases. In this article, we summarize several observation obtained by our laboratories supporting that TRIB3 plays a crucial role in the anti-cancer activity of cannabinoids (a novel family of potential anti-cancer agents derived from marijuana) and that TRIB3 genetic inactivation enhances cancer generation and progression.

Identification of tribbles-1 as a novel binding partner of Foxp3 in regulatory T cells.

In a previous study, we identified TRIB1, a serine-threonine kinase-like molecule, as a biomarker of chronic antibody-mediated rejection of human kidneys when measured in peripheral blood mononuclear cells. Here, we focused our analysis on a specific subset of peripheral blood mononuclear cells that play a dominant role in regulating immune responses in health and disease, so-called CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). We isolated both human and murine Treg and non-Treg counterparts and analyzed TRIB1 and Foxp3 mRNA expression by quantitative PCR on the freshly isolated cells or following 24 h of activation. Physical interaction between the human TRIB1 and Foxp3 proteins was analyzed in live cell lines by protein complementation assay using both flow cytometry and microscopy and confirmed in primary freshly isolated human CD4(+)CD25(hi)CD127(-) Tregs by co-immunoprecipitation. Both TRIB1 and Foxp3 were expressed at significantly higher levels in Tregs than in their CD4(+)CD25(-) counterparts (p < 0.001). Moreover, TRIB1 and Foxp3 mRNA levels correlated tightly in Tregs (Spearman r = 1.0; p < 0.001, n = 7), but not in CD4(+)CD25(-) T cells. The protein complementation assay revealed a direct physical interaction between TRIB1 and Foxp3 in live cells. This interaction was impaired upon deletion of the TRIB1 N-terminal but not the C-terminal domain, suggesting an interaction in the nucleus. This direct interaction within the nucleus was confirmed in primary human Tregs by co-immunoprecipitation. These data show a direct relationship between TRIB1 and Foxp3 in terms of their expression and physical interaction and highlight Tribbles-1 as a novel binding partner of Foxp3 in Tregs.

The pseudokinase tribbles homologue-3 plays a crucial role in cannabinoid anticancer action.

Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer. This effect relies, at least in part, on the up-regulation of several endoplasmic reticulum stress-related proteins including the pseudokinase tribbles homologue-3 (TRIB3), which leads in turn to the inhibition of the AKT/mTORC1 axis and the subsequent stimulation of autophagy-mediated apoptosis in tumor cells. Here, we took advantage of the use of cells derived from Trib3-deficient mice to investigate the precise mechanisms by which TRIB3 regulates the anti-cancer action of THC. Our data show that RasV(12)/E1A-transformed embryonic fibroblasts derived from Trib3-deficient mice are resistant to THC-induced cell death. We also show that genetic inactivation of this protein abolishes the ability of THC to inhibit the phosphorylation of AKT and several of its downstream targets, including those involved in the regulation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) axis. Our data support the idea that THC-induced TRIB3 up-regulation inhibits AKT phosphorylation by regulating the accessibility of AKT to its upstream activatory kinase (the mammalian target of rapamycin complex 2; mTORC2). Finally, we found that tumors generated by inoculation of Trib3-deficient cells in nude mice are resistant to THC anticancer action. Altogether, the observations presented here strongly support that TRIB3 plays a crucial role on THC anti-neoplastic activity. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Tribbles1 is host protective during in vivo mycobacterial infection.

Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production, and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown/knockout exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of proinflammatory factors il-1ß and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.

Aging-related defects in macrophage function are driven by MYC and USF1 transcriptional programs.

Macrophages are central innate immune cells whose function declines with age. The molecular mechanisms underlying age-related changes remain poorly understood, particularly in human macrophages. We report a substantial reduction in phagocytosis, migration, and chemotaxis in human monocyte-derived macrophages (MDMs) from older (>50 years old) compared with younger (18-30 years old) donors, alongside downregulation of transcription factors MYC and USF1. In MDMs from young donors, knockdown of MYC or USF1 decreases phagocytosis and chemotaxis and alters the expression of associated genes, alongside adhesion and extracellular matrix remodeling. A concordant dysregulation of MYC and USF1 target genes is also seen in MDMs from older donors. Furthermore, older age and loss of either MYC or USF1 in MDMs leads to an increased cell size, altered morphology, and reduced actin content. Together, these results define MYC and USF1 as key drivers of MDM age-related functional decline and identify downstream targets to improve macrophage function in aging.

Generation of a novel mouse model for the inducible depletion of macrophages in vivo.

Macrophages play an essential role in tissue homeostasis, innate immunity, inflammation, and wound repair. Macrophages are also essential during development, severely limiting the use of mouse models in which these cells have been constitutively deleted. Consequently, we have developed a transgenic model of inducible macrophage depletion in which macrophage-specific induction of the cytotoxic diphtheria toxin A chain (DTA) is achieved by administration of doxycycline. Induction of the DTA protein in transgenic animals resulted in a significant 50% reduction in CD68+ macrophages of the liver, spleen, and bone over a period of 6 weeks. Pertinently, the macrophages remaining after doxycycline treatment were substantially smaller and are functionally impaired as shown by reduced inflammatory cytokine production in response to lipopolysaccharide. This inducible model of macrophage depletion can now be utilized to determine the role of macrophages in both development and animal models of chronic inflammatory diseases.

Distinct control of MyD88 adapter-dependent and Akt kinase-regulated responses by the interleukin (IL)-1RI co-receptor, TILRR.

Inflammatory responses are controlled through members of the interleukin-1 receptor (IL-1R)/Toll-like receptor superfamily. Our earlier work demonstrates that the IL-1 receptor type 1 (IL-1RI) co-receptor, Toll-like and IL-1 receptor regulator (TILRR), amplifies IL-1 activation of NF-κB and inflammatory genes. Here we show that TILRR similarly promotes IL-1-induced anti-apoptotic signals and reduces caspase-3 activity. Further, the TILRR-induced effects on cell survival and inflammatory responses are controlled through distinct parts of the IL-1RI regulatory Toll IL-1 receptor (TIR) domain. Alanine-scanning mutagenesis identified a functional TILRR mutant (R425A), which blocked increases in cell survival and upstream activation of Akt but had no effect on amplification of MyD88-dependent inflammatory responses. A second mutant (D448A) blocked TILRR potentiation of MyD88-dependent signals and inflammatory activation but had no impact on cell survival. Secondary structure predictions suggested that the mutations induce distinct alterations in the α-helical structure of the TILRR core protein. The results indicate a role for TILRR in selective amplification of NF-κB responses through IL-1RI and suggest that the specificity is determined by changes in receptor conformation and adapter protein recruitment.

The tribbles gene family and lipoprotein metabolism.

PURPOSE OF REVIEW: The success of high throughput sequencing programmes, including the Human Genome Project led to the 'identification' of a large number of novel genes of completely unknown function. Since then, many of these genes have been subject to functional studies focussed on uncovering their biological importance. Recent advances in genome-wide screening of DNA sequence variants as well as focussed genetic studies identified a number of candidate loci contributing to the development of complex diseases, including those affecting lipid homeostasis. An excellent example for the convergence of genetics and experimental biology is the tribbles gene family which was among those identified both in recent genetic studies and were implicated in dysregulation of lipid levels experimentally. Thus, there is a need now to take a step back and reconcile these findings accumulated over recent years. RECENT FINDINGS: Allelic variants of tribbles proteins have been associated with the control of fatty acid synthesis and insulin resistance as well as regulating plasma triglyceride and HDL cholesterol levels. Several mechanisms of molecular action have been proposed for the tribbles mediated control of these processes, including the regulation of signalling events, protein turnover and transcription, sometimes with conflicting evidence emerging. SUMMARY: This review attempts to synthesize knowledge obtained on the biology of the tribbles protein family in the context of lipid metabolism as well as discussing the recently emerging genetic evidence for the importance of these proteins in human disease.

Markers of the ageing macrophage: a systematic review and meta-analysis.

Introduction: Ageing research is establishing macrophages as key immune system regulators that undergo functional decline. Due to heterogeneity between species and tissue populations, a plethora of data exist and the power of scientific conclusions can vary substantially. This meta-analysis by information content (MAIC) and systematic literature review (SLR) aims to determine overall changes in macrophage gene and protein expression, as well as function, with age. Methods: PubMed was utilized to collate peer-reviewed literature relating to macrophage ageing. Primary studies comparing macrophages in at least two age groups were included. Data pertaining to gene or protein expression alongside method used were extracted for MAIC analysis. For SLR analysis, data included all macrophage-specific changes with age, as well as species, ontogeny and age of groups assessed. Results: A total of 240 studies were included; 122 of which qualified for MAIC. The majority of papers focussed on changes in macrophage count/infiltration as a function of age, followed by gene and protein expression. The MAIC found iNOS and TNF to be the most commonly investigated entities, with 328 genes and 175 proteins showing consistent dysregulation with age across the literature. Overall findings indicate that cytokine secretion and phagocytosis are reduced and reactive oxygen species production is increased in the ageing macrophage. Discussion: Collectively, our analysis identifies critical regulators in macrophage ageing that are consistently dysregulated, highlighting a plethora of targets for further investigation. Consistent functional changes with age found here can be used to confirm an ageing macrophage phenotype in specific studies and experimental models.

Tribbles Genes in Gastric Cancer: A Tumor-Suppressive Role for TRIB2.

Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients' outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype.

The pseudokinase TRIB3 controls adipocyte lipid homeostasis and proliferation in vitro and in vivo.

OBJECTIVE: In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. METHODS: We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. RESULTS: Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. CONCLUSIONS: Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.

TRIB1 regulates tumor growth via controlling tumor-associated macrophage phenotypes and is associated with breast cancer survival and treatment response.

Molecular mechanisms that regulate tumor-associated macrophage (TAM) phenotype and function are incompletely understood. The pseudokinase TRIB1 has been reported as a regulator of macrophage phenotypes, both in mouse and human systems. Methods: Bioinformatic analysis was used to investigate the link between TRIB1 expression in breast cancer and therapeutic response to chemotherapy. In vivo models of breast cancer included immune-competent mice to characterize the consequences of altered (reduced or elevated) myeloid Trib1 expression on tumor growth and composition of stromal immune cell populations. Results: TRIB1 was highly expressed by TAMs in breast cancer and high TRIB1 expression correlated with response to chemotherapy and patient survival. Both overexpression and knockout of myeloid Trib1 promote mouse breast tumor growth, albeit through different molecular mechanisms. Myeloid Trib1 deficiency led to an early acceleration of tumor growth, paired with a selective reduction in perivascular macrophage numbers in vivo and enhanced oncogenic cytokine expression in vitro. In contrast, elevated levels of Trib1 in myeloid cells led to an increased late-stage mammary tumor volume, coupled with a reduction of NOS2 expressing macrophages and an overall reduction of macrophages in hypoxic tumor regions. In addition, we show that myeloid Trib1 is a previously unknown, negative regulator of the anti-tumor cytokine IL-15, and that increased myeloid Trib1 expression leads to reduced IL-15 levels in mammary tumors, with a consequent reduction in the number of T-cells that are key to anti-tumor immune responses. Conclusions: Together, these results define a key role for TRIB1 in chemotherapy responses for human breast cancer and provide a mechanistic understanding for the importance of the control of myeloid TRIB1 expression in the development of this disease.

Enhanced neutrophil extracellular trap formation in COVID-19 is inhibited by the protein kinase C inhibitor ruboxistaurin.

Background: Neutrophil extracellular traps (NETs) are web-like DNA and protein lattices which are expelled by neutrophils to trap and kill pathogens, but which cause significant damage to the host tissue. NETs have emerged as critical mediators of lung damage, inflammation and thrombosis in coronavirus disease 2019 (COVID-19) and other diseases, but there are no therapeutics to prevent or reduce NETs that are available to patients. Methods: Neutrophils were isolated from healthy volunteers (n=9) and hospitalised patients with COVID-19 at the acute stage (n=39) and again at 3-4 months post-acute sampling (n=7). NETosis was measured by SYTOX green assays. Results: Here, we show that neutrophils isolated from hospitalised patients with COVID-19 produce significantly more NETs in response to lipopolysaccharide (LPS) compared to cells from healthy control subjects. A subset of patients was captured at follow-up clinics (3-4 months post-acute sampling), and while LPS-induced NET formation is significantly lower at this time point, it remains elevated compared to healthy controls. LPS- and phorbol myristate acetate (PMA)-induced NETs were significantly inhibited by the protein kinase C (PKC) inhibitor ruboxistaurin. Ruboxistaurin-mediated inhibition of NETs in healthy neutrophils reduces NET-induced epithelial cell death. Conclusion: Our findings suggest ruboxistaurin could reduce proinflammatory and tissue-damaging consequences of neutrophils during disease, and since it has completed phase III trials for other indications without safety concerns, it is a promising and novel therapeutic strategy for COVID-19.

Tribbles 3 deficiency promotes atherosclerotic fibrous cap thickening and macrophage-mediated extracellular matrix remodelling.

Tribbles 3 (TRIB3) modulates lipid and glucose metabolism, macrophage lipid uptake, with a gain-of-function variant associated with increased cardiovascular risk. Here we set out to examine the role of this pseudokinase in atherosclerotic plaque development. Human endarterectomy atherosclerotic tissue specimens analysed by immunofluorescence showed upregulated TRIB3 in unstable plaques and an enrichment in unstable regions of stable plaques. Atherosclerosis was induced in full body Trib3KO and Trib3WT littermate mice by injecting mPCSK9 expressing adeno-associated virus and western diet feeding for 12 weeks. Trib3KO mice showed expanded visceral adipose depot while circulatory lipid levels remained unaltered compared to wildtype mice. Trib3KO mice aortae showed a reduced plaque development and improved plaque stability, with increased fibrous cap thickness and collagen content, which was accompanied by increased macrophage content. Analysis of both mouse and human macrophages with reduced TRIB3 expression showed elongated morphology, increased actin expression and altered regulation of genes involved in extracellular matrix remodelling. In summary, TRIB3 controls plaque development and may be atherogenic in vivo. Loss of TRIB3 increases fibrous cap thickness via altered metalloproteinase expression in macrophages, thus inhibiting collagen and elastic fibre degradation, suggesting a role for TRIB3 in the formation of unstable plaques.