Activity Sensing of Coagulation and Fibrinolytic Proteases.

The blood coagulation cascade is a complex physiological process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.

The Long Non-Coding Antisense RNA JHDM1D-AS1 Regulates Inflammatory Responses in Human Monocytes.

Monocytes are key players in innate immunity, with their ability to regulate inflammatory responses and combat invading pathogens. There is a growing body of evidence indicating that long non-coding RNA (lncRNA) participate in various cellular biological processes, including the innate immune response. The immunoregulatory properties of numerous lncRNAs discovered in monocytes remain largely unexplored. Here, by RNA sequencing, we identified a lncRNA JHDM1D-AS1, which was upregulated in blood monocytes obtained from patients with sepsis relative to healthy controls. JHDM1D-AS1 expression was induced in primary human monocytes exposed to Toll-like receptor ligands, such as lipopolysaccharide (LPS), or bacteria. The inducibility of JHDM1D-AS1 expression in monocytes depended, at least in part, on nuclear factor-κB activation. JHDM1D-AS1 knockdown experiments in human monocyte-derived macrophages revealed significantly enhanced expression of inflammatory mediators, before and after exposure to LPS, relative to control cells. Specifically, genes involved in inflammatory responses were upregulated (e.g., CXCL2, CXCL8, IL1RN, TREM1, TNF, and IL6), whereas genes involved in anti-inflammatory pathways were downregulated (e.g., SOCS1 and IL10RA). JHDM1D-AS1 overexpression in a pro-monocytic cell line revealed diminished pro-inflammatory responses subsequent to LPS challenge. Collectively, these findings identify JHDM1D-AS1 as a potential anti-inflammatory mediator induced in response to inflammatory stimuli.

The Platelet Lipidome Is Altered in Patients with COVID-19 and Correlates with Platelet Reactivity.

BACKGROUND: Activated platelets have been implicated in the proinflammatory and prothrombotic phenotype of coronavirus disease 2019 (COVID-19). While it is increasingly recognized that lipids have important structural and signaling roles in platelets, the lipidomic landscape of platelets during infection has remained unexplored. OBJECTIVE: To investigate the platelet lipidome of patients hospitalized for COVID-19. METHODS: We performed untargeted lipidomics in platelets of 25 patients hospitalized for COVID-19 and 23 noninfectious controls with similar age and sex characteristics, and with comparable comorbidities. RESULTS: Twenty-five percent of the 1,650 annotated lipids were significantly different between the groups. The significantly altered part of the platelet lipidome mostly comprised lipids that were less abundant in patients with COVID-19 (20.4% down, 4.6% up, 75% unchanged). Platelets from COVID-19 patients showed decreased levels of membrane plasmalogens, and a distinct decrease of long-chain, unsaturated triacylglycerols. Conversely, platelets from patients with COVID-19 displayed class-wide higher abundances of bis(monoacylglycero)phosphate and its biosynthetic precursor lysophosphatidylglycerol. Levels of these classes positively correlated with ex vivo platelet reactivity-as measured by P-selectin expression after PAR1 activation-irrespective of disease state. CONCLUSION: Taken together, this investigation provides the first exploration of the profound impact of infection on the human platelet lipidome, and reveals associations between the lipid composition of platelets and their reactivity. These results warrant further lipidomic research in other infections and disease states involving platelet pathophysiology.

Structural anomalies in a published NMR-derived structure of IRAK-M.

Signaling by Toll-Like Receptors and the Interleukin-1 Receptor (IL1-R) involves intracellular binding of MyD88, followed by assembly of IL1-R Associated Kinases (IRAKs) into the so-called Myddosome. Using NMR, Nechama et al. determined the structure of the IRAK-M death domain monomer (PDBid: 5UKE). With this structure, they performed a docking study to model the location of IRAK-M in the Myddosome. Based on this, they present a molecular basis for selectivity of IRAK-M towards IRAK1 over IRAK2 binding. When we attempted to use 5UKE as a homology modeling template, we noticed that our 5UKE-based models had structural issues, such as disallowed torsion angles and solvent exposed tryptophans. We therefore analyzed the NMR ensemble of 5UKE using structure validation tools and we compared 5UKE with homologous high-resolution X-ray structures. We identified several structural anomalies in 5UKE, including packing issues, frayed helices and improbable side chain conformations. We used Yasara to build a homology model, based on two high resolution death domain crystal structures, as an alternative model for the IRAK-M death domain (atomic coordinates, modeling details and validation are available at https://swift.cmbi.umcn.nl/gv/service/5uke/). Our model agrees better with known death domain structure information than 5UKE and also with the chemical shift data that was deposited for 5UKE.

The Long Pentraxin PTX3 Controls Klebsiella Pneumoniae Severe Infection.

Klebsiella pneumoniae is a common pathogen in human sepsis. The emergence of multidrug-resistant K. pneumoniae strains represents a major clinical challenge in nosocomial and community acquired infections. The long pentraxin PTX3, a key component of humoral innate immunity, is involved in resistance to selected pathogens by promoting opsonophagocytosis. We investigated the relevance of PTX3 in innate immunity against K. pneumoniae infections using Ptx3-/- mice and mouse models of severe K. pneumoniae infections. Local and systemic PTX3 expression was induced following K. pneumoniae pulmonary infection, in association with the up-regulation of TNF-α and IL-1ß. PTX3 deficiency in mice was associated with higher bacterial burden and mortality, release of pro-inflammatory cytokines as well as IL-10 in the lung and systemically. The analysis of the mechanisms responsible of PTX3-dependent control of K. pneumoniae infection revealed that PTX3 did not interact with K. pneumoniae, or promote opsonophagocytosis. The comparison of susceptibility of wild-type, Ptx3-/-, C3-/- and Ptx3-/- /C3-/- mice to the infection showed that PTX3 acted in a complement-independent manner. Lung histopathological analysis showed more severe lesions in Ptx3-/- mice with fibrinosuppurative, necrotizing and haemorrhagic bronchopneumonia, associated with increased fibrin deposition in the lung and circulating fibrinogen consumption. These findings indicate that PTX3 contributes to the control of K. pneumoniae infection by modulating inflammatory responses and tissue damage. Thus, this study emphasizes the relevance of the role of PTX3 as regulator of inflammation and orchestrator of tissue repair in innate responses to infections.

Role of triggering receptor expressed on myeloid cells-1/3 in Klebsiella-derived pneumosepsis.

Triggering receptor expressed on myeloid cells (TREM)-1 and -2 can affect Toll-like receptor-mediated activation of immune cells. Klebsiella pneumoniae is a common cause of pneumonia-derived sepsis. Here we studied the role of TREM-1/3 and TREM-2 in the host response during Klebsiella pneumonia. Macrophages lacking either TREM-1/3 or TREM-2 were tested for their responsiveness toward K. pneumoniae and for their capacity to internalize this pathogen in vitro. TREM-1/3- and TREM-2-deficient mice were infected with K. pneumoniae via the airways, and their responses were compared with those in wild-type mice. TREM-1/3-deficient macrophages produced lower cytokine levels upon exposure to K. pneumoniae, whereas TREM-2-deficient macrophages released higher cytokine concentrations. TREM-2-deficient, but not TREM-1/3-deficient, macrophages showed a reduced capacity to phagocytose K. pneumoniae. TREM-1/3-deficient mice showed an impaired host defense during Klebsiella pneumonia, as reflected by worsened survival and increased bacterial growth and dissemination. In contrast, TREM-2 deficiency did not affect disease outcome. Although TREM-1/3 and TREM-2 influence macrophage responsiveness to K. pneumoniae in vitro, only TREM-1/3 contribute to the host response during Klebsiella pneumonia in vivo, serving a protective role.

Pulmonary tuberculosis induces a systemic hypercoagulable state.

OBJECTIVES: Human tuberculosis (TB) remains an important cause of death globally. Bangladesh is one of the most affected countries. We aimed to investigate the impact of pulmonary TB on pro- and anticoagulant mechanisms. METHODS: This prospective study was conducted in Chittagong, Bangladesh. We performed an in-depth analysis of coagulation activation and inhibition in plasma obtained from 64 patients with primary lung TB and 11 patients with recurrent lung TB and compared these with 37 healthy controls. Additionally, in nine patients coagulation activation was studied in bronchoalveolar lavage fluid (BALF) harvested from the site of infection and compared with BALF from a contralateral unaffected lung subsegment. RESULTS: Relative to uninfected controls, primary and recurrent TB were associated with a systemic net procoagulant state, as indicated by enhanced activation of coagulation (elevated plasma levels of thrombin-antithrombin complexes, D-dimer and fibrinogen) together with impaired anticoagulant mechanisms (reduced plasma levels of antithrombin, protein C activity, free protein S, and protein C inhibitor). Activation of coagulation did not correlate with plasma concentrations of established TB biomarkers. Coagulation activation could not be detected at the primary site of infection in a subset of TB patients. CONCLUSIONS: Pulmonary TB is associated with a systemic hypercoagulable state.

Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.

BACKGROUND & AIMS: Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. METHODS: We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. RESULTS: Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1ß before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1ß were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1ß stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. CONCLUSIONS: IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI.

Lipopolysaccharide inhibits Th2 lung inflammation induced by house dust mite allergens in mice.

The complex biology of asthma compels the use of more relevant human allergens, such as house dust mite (HDM), to improve the translation of animal models into human asthma. LPS exposure is associated with aggravations of asthma, but the mechanisms remain unclear. Here, we studied the effects of increasing LPS doses on HDM-evoked allergic lung inflammation. To this end, mice were intranasally sensitized and challenged with HDM with or without increasing doses of LPS (0.001-10 µg). LPS dose-dependently inhibited HDM-induced eosinophil recruitment into the lungs and mucus production in the airways. LPS attenuated the production of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in HDM-challenged lungs, while enhancing the HDM-induced release of IL-17, IL-33, IFN-γ, and TNF-α. The shift toward a Th1 inflammatory response was further illustrated by predominant neutrophilic lung inflammation after LPS administration at higher doses. LPS did not influence HDM-induced plasma IgE concentrations. Although LPS did not significantly affect the activation of coagulation or complement in HDM-challenged lungs, it reduced HDM-initiated endothelial cell activation. This study is the first to provide insights into the effects of LPS in an allergic lung inflammation model making use of a clinically relevant allergen without a systemic adjuvant, revealing that LPS dose-dependently inhibits HDM-induced pulmonary Th2 responses.