SARS-CoV-2 infection results in upregulation of Plasminogen Activator Inhibitor-1 and Neuroserpin in the lungs, and an increase in fibrinolysis inhibitors associated with disease severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in coagulation activation although it is usually not associated with consumption coagulopathy. D-dimers are also commonly elevated despite systemic hypofibrinolysis. To understand these unusual features of coronavirus disease 2019 (COVID-19) coagulopathy, 64 adult patients with SARS-CoV-2 infection (36 moderate and 28 severe) and 16 controls were studied. We evaluated the repertoire of plasma protease inhibitors (Serpins, Kunitz, Kazal, Cystatin-like) targeting the fibrinolytic system: Plasminogen Activator Inhibitor-1 (PAI-1), Tissue Plasminogen Activator/Plasminogen Activator Inhibitor-1 complex (t-PA/PAI-1), α-2-Antiplasmin, Plasmin-α2-Antiplasmin Complex, Thrombin-activatable Fibrinolysis Inhibitor (TAFI)/TAFIa, Protease Nexin-1 (PN-1), and Neuroserpin (the main t-PA inhibitor of the central nervous system). Inhibitors of the common (Antithrombin, Thrombin-antithrombin complex, Protein Z [PZ]/PZ inhibitor, Heparin Cofactor II, and α2-Macroglobulin), Protein C ([PC], Protein C inhibitor, and Protein S), contact (Kallistatin, Protease Nexin-2/Amyloid Beta Precursor Protein, and α-1-Antitrypsin), and complement (C1-Inhibitor) pathways, in addition to Factor XIII, Histidine-rich glycoprotein (HRG) and Vaspin were also investigated by enzyme-linked immunosorbent assay. The association of these markers with disease severity was evaluated by logistic regression. Pulmonary expression of PAI-1 and Neuroserpin in the lungs from eight post-mortem cases was assessed by immunohistochemistry. Results show that six patients (10%) developed thrombotic events, and mortality was 11%. There was no significant reduction in plasma anticoagulants, in keeping with a compensated state. However, an increase in fibrinolysis inhibitors (PAI-1, Neuroserpin, PN-1, PAP, and t-PA/PAI-1) was consistently observed, while HRG was reduced. Furthermore, these markers were associated with moderate and/or severe disease. Notably, immunostains demonstrated overexpression of PAI-1 in epithelial cells, macrophages, and endothelial cells of fatal COVID-19, while Neuroserpin was found in intraalveolar macrophages only. These results imply that the lungs in SARS-CoV-2 infection provide anti-fibrinolytic activity resulting in a shift toward a local and systemic hypofibrinolytic state predisposing to (immuno)thrombosis, often in a background of compensated disseminated intravascular coagulation.

Upregulation of pulmonary tissue factor, loss of thrombomodulin and immunothrombosis in SARS-CoV-2 infection.

BACKGROUND: SARS-CoV-2 infection is associated with thrombotic and microvascular complications. The cause of coagulopathy in the disease is incompletely understood. METHODS: A single-center cross-sectional study including 66 adult COVID-19 patients (40 moderate, 26 severe disease), and 9 controls, performed between 04/2020 and 10/2020. Markers of coagulation, endothelial cell function [angiopoietin-1,-2, P-selectin, von Willebrand Factor Antigen (WF:Ag), von Willebrand Factor Ristocetin Cofactor, ADAMTS13, thrombomodulin, soluble Endothelial cell Protein C Receptor (sEPCR), Tissue Factor Pathway Inhibitor], neutrophil activation (elastase, citrullinated histones) and fibrinolysis (tissue-type plasminogen activator, plasminogen activator inhibitor-1) were evaluated using ELISA. Tissue Factor (TF) was estimated by antithrombin-FVIIa complex (AT/FVIIa) and microparticles-TF (MP-TF). We correlated each marker and determined its association with severity. Expression of pulmonary TF, thrombomodulin and EPCR was determined by immunohistochemistry in 9 autopsies. FINDINGS: Comorbidities were frequent in both groups, with older age associated with severe disease. All patients were on prophylactic anticoagulants. Three patients (4.5%) developed pulmonary embolism. Mortality was 7.5%. Patients presented with mild alterations in the coagulogram (compensated state). Biomarkers of endothelial cell, neutrophil activation and fibrinolysis were elevated in severe vs moderate disease; AT/FVIIa and MP-TF levels were higher in severe patients. Logistic regression revealed an association of D-dimers, angiopoietin-1, vWF:Ag, thrombomodulin, white blood cells, absolute neutrophil count (ANC) and hemoglobin levels with severity, with ANC and vWF:Ag identified as independent factors. Notably, postmortem specimens demonstrated epithelial expression of TF in the lung of fatal COVID-19 cases with loss of thrombomodulin staining, implying in a shift towards a procoagulant state. INTERPRETATION: Coagulation dysregulation has multifactorial etiology in SARS-Cov-2 infection. Upregulation of pulmonary TF with loss of thrombomodulin emerge as a potential link to immunothrombosis, and therapeutic targets in the disease. FUNDING: John Hopkins University School of Medicine.

Sustained IL-2R signaling of limited duration by high-dose mIL-2/mCD25 fusion protein amplifies tumor-reactive CD8+ T cells to enhance antitumor immunity.

High-dose IL-2 induces cancer regression but its therapeutic use is limited due to high toxicities resulting from its broad cell targeting. In one strategy to overcome this limitation, IL-2 has been modified to selectively target the intermediate affinity IL-2R that broadly activates memory-phenotypic CD8+ T and NK cells, while minimizing Treg-associated tolerance. In this study, we modeled an alternative strategy to amplify tumor antigen-specific TCR transgenic CD8+ T cells through limited application of a long-acting IL-2 fusion protein, mIL-2/mCD25, which selectively targets the high-affinity IL-2R. Here, mice were vaccinated with a tumor antigen and high-dose mIL-2/mCD25 was applied to coincide with the induction of the high affinity IL-2R on tumor-specific T cells. A single high dose of mIL-2/mCD25, but not an equivalent amount of IL-2, amplified the frequency and function of tumor-reactive CD8+ T effector (Teff) and memory cells. These mIL-2/mCD25-dependent effects relied on distinctive requirements for TLR signals during priming of CD8+ tumor-specific T cells. The mIL-2/mCD25-amplified tumor-reactive effector and memory T cells supported long-lasting antitumor responses to B16-F10 melanoma. This regimen only transiently increased Tregs, yielding a favorable Teff-Treg ratio within the tumor microenvironment. Notably, mIL-2/mCD25 did not increase non-tumor-specific Teff or NK cells within tumors, further substantiating the specificity of mIL-2/mCD25 for tumor antigen-activated T cells. Thus, the selectivity and persistence of mIL-2/mCD25 in conjunction with a tumor vaccine supports antitumor immunity through a mechanism that is distinct from recombinant IL-2 or IL-2-based biologics that target the intermediate affinity IL-2R.

A New Delhi metallo-ß-lactamase (NDM)-positive isolate of Klebsiella pneumoniae causing catheter-related bloodstream infection in an ambulatory hemodialysis patient.

The New Delhi metallo-ß-lactamase (NDM) is a mediator of broad antimicrobial resistance among the Enterobacteriaceae and other gram-negative pathogens that cause opportunistic and nosocomial infections. In the decade since its discovery, NDM has spread worldwide and represents an increasing threat to public health. NDM is capable of hydrolyzing nearly all known ß-lactam antibiotics, including the carbapenems, and due to its zinc ion-dependent catalytic mechanism is unaffected by available ß-lactamase inhibitors. We report a case of catheter-related bloodstream infection caused by a pan-resistant, NDM-positive isolate of Klebsiella pneumoniae in an ambulatory end-stage renal disease patient started on hemodialysis approximately 8 weeks prior. The absence of any recent hospitalization indicates that the infection was likely acquired from a hemodialysis center in the United States. This case demonstrates the increasing prevalence of antimicrobial resistance mechanisms in ambulatory as well as inpatient healthcare settings, and highlights the particular risk of the outpatient hemodialysis facility as an optimal environment for colonization with multidrug- and pandrug-resistant pathogens.

Essential and non-overlapping IL-2Rα-dependent processes for thymic development and peripheral homeostasis of regulatory T cells.

IL-2R signaling is essential for regulatory T cell (Treg) function. However, the precise contribution of IL-2 during Treg thymic development, peripheral homeostasis and lineage stability remains unclear. Here we show that IL-2R signaling is required by thymic Tregs at an early step for expansion and survival, and a later step for functional maturation. Using inducible, conditional deletion of CD25 in peripheral Tregs, we also find that IL-2R signaling is indispensable for Treg homeostasis, whereas Treg lineage stability is largely IL-2-independent. CD25 knockout peripheral Tregs have increased apoptosis, oxidative stress, signs of mitochondrial dysfunction, and reduced transcription of key enzymes of lipid and cholesterol biosynthetic pathways. A divergent IL-2R transcriptional signature is noted for thymic Tregs versus peripheral Tregs. These data indicate that IL-2R signaling in the thymus and the periphery leads to distinctive effects on Treg function, while peripheral Treg survival depends on a non-conventional mechanism of metabolic regulation.

Distribution and population structure of endobacteria in arbuscular mycorrhizal fungi at North Atlantic dunes.

Arbuscular mycorrhizal fungi (AMF, Glomeromycotina), in addition to forming symbioses with the majority of land plants, harbor vertically transmitted endosymbiotic bacteria 'Candidatus Glomeribacter gigasporarum' (CaGg) and 'Candidatus Moeniiplasma glomeromycotorum' (CaMg). CaGg is a nonessential mutualist of AMF, whereas the lifestyle of CaMg is unknown. To start unraveling the interactions between AMF and their endosymbionts in nature, we examined diversity and distribution of AMF-associated endobacteria in North Atlantic dunes at Cape Cod. Of nearly 500 foredune AMF isolates successfully genotyped during a systematic study, 94% were classified as Gigasporaceae. Two percent of all AMF spores harbored CaGg, and 88% contained CaMg. CaGg was found only in the Gigasporaceae, whereas CaMg was present in Gigasporaceae, Acaulosporaceae, and Diversisporaceae. Incidence of CaGg across AMF was not affected by any of the environmental parameters measured, whereas distribution of CaMg in one of the fungal hosts was impacted by plant density. CaMg populations associated with AMF individuals displayed high levels of genetic diversity but no evidence of gene flow, suggesting that host physical proximity is not sufficient to facilitate horizontal transmission of CaMg. Finally, in addition to a novel lineage of CaGg, we discovered that AMF likely harbor Burkholderia-related bacteria with close phylogenetic affinity to free-living Burkholderia and endobacteria of other Mucoromycota fungi.

Initiation of inflammatory tumorigenesis by CTLA4 insufficiency due to type 2 cytokines.

Genetically predisposed CTLA4 insufficiency in humans is associated with gastric cancer development, which is paradoxical to the prototypical role of CTLA4 in suppressing antitumor immunity. CTLA4 is a critical immune checkpoint against autoimmune disorders. Autoimmunity has been implicated in protumor or antitumor activities. Here, we show that CTLA4 insufficiency initiates de novo tumorigenesis in the mouse stomach through inflammation triggered by host-intrinsic immune dysregulation rather than microbiota, with age-associated progression to malignancy accompanied by epigenetic dysregulation. The inflammatory tumorigenesis required CD4 T cells, but not the TH1 or TH17 subsets. Deficiencies in IL-4 and IL-13 or IL-4 receptor α broke the link between inflammation and initiation of tumorigenesis. This study establishes the causality of CTLA4 insufficiency in gastric cancer and uncovers a role of type 2 inflammation in initiating gastric epithelial transformation. These findings suggest possible improvement of immune therapies by blocking tumorigenic type 2 inflammation while preserving antitumor type 1 immunity.

Cytokine Signaling in the Development and Homeostasis of Regulatory T cells.

Cytokine signaling is indispensable for regulatory T-cell (Treg) development in the thymus, and also influences the homeostasis, phenotypic diversity, and function of Tregs in the periphery. Because Tregs are required for establishment and maintenance of immunological self-tolerance, investigating the role of cytokines in Treg biology carries therapeutic potential in the context of autoimmune disease. This review discusses the potent and diverse influences of interleukin (IL)-2 signaling on the Treg compartment, an area of knowledge that has led to the use of low-dose IL-2 as a therapy to reregulate autoaggressive immune responses. Evidence suggesting Treg-specific impacts of the cytokines transforming growth factor ß (TGF-ß), IL-7, thymic stromal lymphopoietin (TSLP), IL-15, and IL-33 is also presented. Finally, we consider the technical challenges and knowledge limitations that must be overcome to bring other cytokine-based, Treg-targeted therapies into clinical use.

Altered homeostasis and development of regulatory T cell subsets represent an IL-2R-dependent risk for diabetes in NOD mice.

The cytokine interleukin-2 (IL-2) is critical for the functions of regulatory T cells (Tregs). The contribution of polymorphisms in the gene encoding the IL-2 receptor α subunit (IL2RA), which are associated with type 1 diabetes, is difficult to determine because autoimmunity depends on variations in multiple genes, where the contribution of any one gene product is small. We investigated the mechanisms whereby a modest reduction in IL-2R signaling selectively in T lymphocytes influenced the development of diabetes in the NOD mouse model. The sensitivity of IL-2R signaling was reduced by about two- to threefold in Tregs from mice that coexpressed wild-type IL-2Rß and a mutant subunit (IL-2RßY3) with reduced signaling (designated NOD-Y3). Male and female NOD-Y3 mice exhibited accelerated diabetes onset due to intrinsic effects on multiple activities in Tregs Bone marrow chimera and adoptive transfer experiments demonstrated that IL-2RßY3 Tregs resulted in impaired homeostasis of lymphoid-residing central Tregs and inefficient development of highly activated effector Tregs and that they were less suppressive. Pancreatic IL-2RßY3 Tregs showed impaired development into IL-10-secreting effector Tregs The pancreatic lymph nodes and pancreases of NOD-Y3 mice had increased numbers of antigen-experienced CD4+ effector T cells, which was largely due to impaired Tregs, because adoptively transferred pancreatic autoantigen-specific CD4+ Foxp3- T cells from NOD-Y3 mice did not accelerate diabetes in NOD.SCID recipients. Our study indicates that the primary defect associated with chronic, mildly reduced IL-2R signaling is due to impaired Tregs that cannot effectively produce and maintain highly functional tissue-seeking effector Treg subsets.

Developmental Progression and Interrelationship of Central and Effector Regulatory T Cell Subsets.

Resting central Tregs (cTregs) and activated effector Tregs (eTregs) are required for self-tolerance, but the heterogeneity and relationships within and between phenotypically distinct subsets of cTregs and eTregs are poorly understood. By extensive immune profiling and deep sequencing of TCR-ß V regions, two subsets of cTregs, based on expression of Ly-6C, and three subsets of eTregs, based on distinctive expression of CD62L, CD69, and CD103, were identified. Ly-6C(+) cTregs exhibited lower basal activation, expressed on average lower affinity TCRs, and less efficiently developed into eTregs when compared with Ly-6C(-) cTregs. The dominant TCR Vßs of Ly-6C(+) cTregs were shared by eTregs at a low frequency. A single TCR clonotype was also identified that was largely restricted to Ly-6C(+) cTregs, even under conditions that promoted the development of eTregs. Collectively, these findings indicate that some Ly-6C(+) cTregs may persist as a lymphoid-specific subset, with minimal potential to develop into highly activated eTregs, whereas other cTregs readily develop into eTregs. In contrast, subsets of CD62L(lo) eTregs showed higher clonal expansion and were more highly interrelated than cTreg subsets based on their TCR-ß repertoires, but exhibited varied immune profiles. The CD62L(lo) CD69(-) CD103(-) eTreg subset displayed properties of a transitional intermediate between cTregs and more activated eTreg subsets. Thus, eTreg subsets appear to exhibit substantial flexibility, most likely in response to environmental cues, to adopt defined immune profiles that are expected to optimize suppression of autoreactive T cells.

Molecular evolution patterns reveal life history features of mycoplasma-related endobacteria associated with arbuscular mycorrhizal fungi.

The mycoplasma-related endobacteria (MRE), representing a recently discovered lineage of Mollicutes, are widely distributed across arbuscular mycorrhizal fungi (AMF, Glomeromycota). AMF colonize roots of most terrestrial plants and improve plant mineral nutrient uptake in return for plant-assimilated carbon. The role of MRE in the biology of their fungal hosts is unknown. To start characterizing this association, we assessed partitioning of MRE genetic diversity within AMF individuals and across the AMF phylogeographic range. We further used molecular evolution patterns to make inferences about MRE codivergence with AMF, their lifestyle and antiquity of the Glomeromycota-MRE association. While we did not detect differentiation between MRE derived from different continents, high levels of diversity were apparent in MRE populations within AMF host individuals. MRE exhibited significant codiversification with AMF over ecological time and the absence of codivergence over evolutionary time. Moreover, genetic recombination was evident in MRE. These patterns indicate that, while MRE transmission is predominantly vertical, their complex intrahost populations are likely generated by horizontal transmission and recombination. Based on predictions of evolutionary theory, we interpreted these observations as a suggestion that MRE may be antagonists of AMF. Finally, we detected a marginally significant signature of codivergence of MRE with Glomeromycota and the Endogone lineage of Mucoromycotina, implying that the symbiosis between MRE and fungi may predate the divergence between these two groups of fungi.

Genome-wide analysis of alternative transcripts in human breast cancer.

Transcript variants play a critical role in diversifying gene expression. Alternative splicing is a major mechanism for generating transcript variants. A number of genes have been implicated in breast cancer pathogenesis with their aberrant expression of alternative transcripts. In this study, we performed genome-wide analyses of transcript variant expression in breast cancer. With RNA-Seq data from 105 patients, we characterized the transcriptome of breast tumors, by pairwise comparison of gene expression in the breast tumor versus matched healthy tissue from each patient. We identified 2839 genes, ~10 % of protein-coding genes in the human genome, that had differential expression of transcript variants between tumors and healthy tissues. The validity of the computational analysis was confirmed by quantitative RT-PCR assessment of transcript variant expression from four top candidate genes. The alternative transcript profiling led to classification of breast cancer into two subgroups and yielded a novel molecular signature that could be prognostic of patients' tumor burden and survival. We uncovered nine splicing factors (FOX2, MBNL1, QKI, PTBP1, ELAVL1, HNRNPC, KHDRBS1, SFRS2, and TIAR) that were involved in aberrant splicing in breast cancer. Network analyses for the coordinative patterns of transcript variant expression identified twelve "hub" genes that differentiated the cancerous and normal transcriptomes. Dysregulated expression of alternative transcripts may reveal novel biomarkers for tumor development. It may also suggest new therapeutic targets, such as the "hub" genes identified through the network analyses of transcript variant expression, or splicing factors implicated in the formation of the tumor transcriptome.

Autoimmunity as a double agent in tumor killing and cancer promotion.

Cancer immunotherapy through manipulation of the immune system holds great potential for the treatment of human cancers. However, recent trials targeting the negative immune regulators cytotoxic T-lymphocyte antigen 4, programed death 1 (PD-1), and PD-1 receptor ligand (PD-L1) demonstrated that clinically significant antitumor responses were often associated with the induction of autoimmune toxicity. This finding suggests that the same immune mechanisms that elicit autoimmunity may also contribute to the destruction of tumors. Given the fact that the immunological identity of tumors might be largely an immunoprivileged self, autoimmunity may not represent a wholly undesirable outcome in the context of cancer immunotherapy. Rather, targeted killing of cancer cells and autoimmune damage to healthy tissues may be intricately linked through molecular mechanisms, in particular inflammatory cytokine signaling. On the other hand, since chronic inflammation is a well-recognized condition that promotes tumor development, it appears that autoimmunity can be a "double agent" in mediating either pro-tumor or antitumor effects. This review surveys the tumor-promoting and tumoricidal activities of several prominent cytokines: IFN-γ, TNF-α, TGF-ß, IL-17, IL-23, IL-4, and IL-13, produced by three major subsets of T helper cells that interact with innate immune cells. Many of these cytokines exert divergent and seemingly contradictory effects on cancer development in different human and animal models, suggesting a high degree of context dependence in their functions. We hypothesize that these inflammatory cytokines could mediate a feedback loop of autoimmunity, antitumor immunity, and tumorigenesis. Understanding the diverse and paradoxical roles of cytokines from autoimmune responses in the setting of cancer will advance the long-term goal of improving cancer immunotherapy, while minimizing the hazards of immune-mediated tissue damage and the possibility of de novo tumorigenesis, through proper monitoring and preventive measures.

Evolutionary stability in a 400-million-year-old heritable facultative mutualism.

Many eukaryotes interact with heritable endobacteria to satisfy diverse metabolic needs. Some of these interactions are facultative symbioses, in which one partner is not essential to the other. Facultative symbioses are expected to be transitional stages along an evolutionary trajectory toward obligate relationships. We tested this evolutionary theory prediction in Ca. Glomeribacter gigasporarum, nonessential endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We found that heritable facultative mutualisms can be both ancient and evolutionarily stable. We detected significant patterns of codivergence between the partners that we would only expect in obligate associations. Using codiverging partner pairs and the fungal fossil record, we established that the Glomeromycota-Glomeribacter symbiosis is at least 400 million years old. Despite clear signs of codivergence, we determined that the Glomeribacter endobacteria engage in recombination and host switching, which display patterns indicating that the association is not evolving toward reciprocal dependence. We postulate that low frequency of recombination in heritable endosymbionts together with host switching stabilize facultative mutualisms over extended evolutionary times.