Monocyte-Mediated Thrombosis Linked to Circulating Tissue Factor and Immune Paralysis in COVID-19.

BACKGROUND: SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown. METHODS: In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19. RESULTS: In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression. CONCLUSIONS: Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.

Immune-Mediated Glycocalyx Remodeling in Hospitalized COVID-19 Patients.

PURPOSE: Vascular and immune dysfunction are hallmarks of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections and coronavirus disease 2019 (COVID-19). Although our understanding of the pathogenesis of COVID-19 has rapidly evolved, much of the focus has been on the immune mechanisms underlying COVID-19. In addition to immune dysfunction, vascular injury is also associated with COVID-19 and is a major driver of clinical deterioration in SARS-CoV-2 infections. The glycocalyx (GAC), a sugar-based shell that surrounds all mammalian cells, is an important regulator of vascular and immune responses. In sepsis, vascular dysfunction contributes to acute respiratory distress syndrome (ARDS) by altering vessel integrity, promoting thrombosis, and accelerating inflammation, all of which are also present in COVID-19. Observational studies in sepsis have found an association between levels of circulating GAC degradation products with both organ dysfunction and mortality. Although vascular dysfunction is a hallmark of COVID-19, it remains unclear whether GAC disruption occurs in COVID-19 and if GAC disruption contributes to the clinical progression of COVID-19. METHODS: In this prospective cohort study, we measured the GAC components syndecan-1 (SDC1) and hyaluronan (Hyal) along with inflammatory cytokines in 12 hospitalized COVID-19 patients and 8 healthy controls (HC). RESULTS: In agreement with other studies, we found that inflammatory cytokines are elevated in hospitalized COVID-19 patients compared with HC [median (IQR), all units picograms per milliliter: IL-6 4.65 (3.32-9.16) vs 0.69 (0.55-0.89), p < 0.001; TNFα 4.49 (1.87-8.03) vs 0.04 (0.04-0.84), p < 0.001]. Additionally, we found that the GAC components SDC1 and Hyal are also elevated in COVID-19 patients [median (IQR), all units picograms per milliliter: SDC1: 247.37 (101.43-458.26) vs 84.8 (52.88-123.59), p = 0.036; Hyal: 26.41 (16.4-35.1) vs 3.01 (1.66-4.61), p < 0.001]. CONCLUSION: We propose that GAC markers offer insights into the pathobiology of COVID-19, potentially guide therapeutic approaches, and could aid in early risk stratification that is particularly beneficial in phasic diseases such as COVID-19.

Glycocalyx Disruption Triggers Human Monocyte Activation in Acute Heart Failure Syndromes.

PURPOSE: Acute heart failure (AHF) syndromes manifest increased inflammation and vascular dysfunction; however, mechanisms that integrate the two in AHF remain largely unknown. The glycocalyx (GAC) is a sugar-based shell that envelops all mammalian cells. Much GAC research has focused on its role in vascular responses, with comparatively little known about how the GAC regulates immune cell function. METHODS: In this study, we sought to determine if GAC degradation products are elevated in AHF patients, how these degradation products relate to circulating inflammatory mediators, and whether the monocyte GAC (mGAC) itself modulates monocyte activation. Inflammatory markers and GAC degradation products were profiled using ELISAs. Flow cytometry was used to assess the mGAC and RNA-seq was employed to understand the role of the mGAC in regulating inflammatory activation programs. RESULTS: In a cohort of hospitalized AHF patients (n = 17), we found that (1) the GAC degradation product heparan sulfate (HS) was elevated compared with age-matched controls (4396 and 2903 ng/mL; p = 0.01) and that (2) HS and soluble CD14 (a marker of monocyte activation) levels were closely related (Pearson's r = 0.65; p = 0.002). Mechanistically, Toll-like receptor (TLR) activation of human monocytes results in GAC remodeling and a decrease in the mGAC (71% compared with no treatment; p = 0.0007). Additionally, we found that ex vivo enzymatic removal of HS and disruption of the mGAC triggers human monocyte activation and amplifies monocyte inflammatory responses. Specifically, using RNA-seq, we found that enzymatic degradation of the mGAC increases transcription of inflammatory (IL6, CCL3) and vascular (tissue factor/F3) mediators. CONCLUSION: These studies indicate that the mGAC is dynamically remodeled during monocyte activation and that mGAC remodeling itself may contribute to the heightened inflammation associated with AHF.

Misregulation of Nucleoporins 98 and 96 leads to defects in protein synthesis that promote hallmarks of tumorigenesis.

Nucleoporin 98KD (Nup98) is a promiscuous translocation partner in hematological malignancies. Most disease models of Nup98 translocations involve ectopic expression of the fusion protein under study, leaving the endogenous Nup98 loci unperturbed. Overlooked in these approaches is the loss of one copy of normal Nup98 in addition to the loss of Nup96 - a second Nucleoporin encoded within the same mRNA and reading frame as Nup98 - in translocations. Nup98 and Nup96 are also mutated in a number of other cancers, suggesting that their disruption is not limited to blood cancers. We found that reducing Nup98-96 function in Drosophila melanogaster (in which the Nup98-96 shared mRNA and reading frame is conserved) de-regulates the cell cycle. We found evidence of overproliferation in tissues with reduced Nup98-96, counteracted by elevated apoptosis and aberrant signaling associated with chronic wounding. Reducing Nup98-96 function led to defects in protein synthesis that triggered JNK signaling and contributed to hallmarks of tumorigenesis when apoptosis was inhibited. We suggest that partial loss of Nup98-96 function in translocations could de-regulate protein synthesis, leading to signaling that cooperates with other mutations to promote tumorigenesis.

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup.

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse beta-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the "library model."

Analysis of the complete mitochondrial DNA from Anopheles funestus: an improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution.

Virtually no information regarding timing of deep lineage divergences within mosquito family (Culicidae) exists, which poses an important problem in the postgenomic era. To address this issue, the complete 15,354 bp mitochondrial genome of Anopheles funestus was assembled from both mtDNA and cDNA sequences generated from transcripts of the mtDNA-encoded protein and rRNA genes. Analysis of the transcript information allowed an improved genome annotation, revealing that the translation initiation codon for the cox1 gene is TCG, rather than atypical, longer codons proposed in several other insects. The 5'ends of nad1 and nad5 transcripts begin with TTG and GTG triplets, respectively, which apparently serve as the translation initiators for those genes. We used all the A. funestus mtDNA gene sequences and three other publicly available mosquito mtDNA genomes for the estimation of divergence time points within Culicidae. The maximum likelihood date estimates for the splits between Anopheles and Aedes (approximately 145-200 Mya), between Anopheles subgenera Cellia and Anopheles (approximately 90-106 Mya), and between lineages within subgenus Anopheles (approximately 70-85 Mya) inferred from protein-coding genes are roughly twice as high as the dates based on RNA gene sequences. Although existing evidence does not unequivocally favor one of the alternatives, fossil-based predictions of the age of the family Culicidae are in better agreement with dates inferred from protein-coding genes.

Inversions and gene order shuffling in Anopheles gambiae and A. funestus.

In tropical Africa, Anopheles funestus is one of the three most important malaria vectors. We physically mapped 157 A. funestus complementary DNAs (cDNAs) to the polytene chromosomes of this species. Sequences of the cDNAs were mapped in silico to the A. gambiae genome as part of a comparative genomic study of synteny, gene order, and sequence conservation between A. funestus and A. gambiae. These species are in the same subgenus and diverged about as recently as humans and chimpanzees. Despite nearly perfect preservation of synteny, we found substantial shuffling of gene order along corresponding chromosome arms. Since the divergence of these species, at least 70 chromosomal inversions have been fixed, the highest rate of rearrangement of any eukaryote studied to date. The high incidence of paracentric inversions and limited colinearity suggests that locating genes in one anopheline species based on gene order in another may be limited to closely related taxa.