Promyelocytic leukemia protein is a restriction factor for Junín virus independently of Z matrix protein.

The New World (NW) mammarenavirus Junín (JUNV) is the etiological agent of Argentine hemorrhagic fever, a human endemic disease of Argentina. Promyelocytic leukemia protein (PML) has been reported as a restriction factor for several viruses although the mechanism/s behind PML-mediated antiviral effect may be diverse and are a matter of debate. Previous studies have reported a nuclear to cytoplasm translocation of PML during the murine Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) infection. This translocation was found to be mediated by the viral Z protein. Here, we show that PML restricts JUNV infection in human A549 cells. However, in contrast to LCVM, JUNV infection enhances PML expression and PML is not translocated to the cytoplasm neither it colocalizes with JUNV Z protein. Our study demonstrates that a NW mammarenavirus as JUNV interacts differently with the antiviral protein PML than LCMV.

Proteomic Analysis Identifies Molecular Players and Biological Processes Specific to SARS-CoV-2 Exposure in Endothelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the severe pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), experienced in the 21st century. The clinical manifestations range from mild symptoms to abnormal blood coagulation and severe respiratory failure. In severe cases, COVID-19 manifests as a thromboinflammatory disease. Damage to the vascular compartment caused by SARS-CoV-2 has been linked to thrombosis, triggered by an enhanced immune response. The molecular mechanisms underlying endothelial activation have not been fully elucidated. We aimed to identify the proteins correlated to the molecular response of human umbilical vein endothelial cells (HUVECs) after exposure to SARS-CoV-2, which might help to unravel the molecular mechanisms of endothelium activation in COVID-19. In this direction, we exposed HUVECs to SARS-CoV-2 and analyzed the expression of specific cellular receptors, and changes in the proteome of HUVECs at different time points. We identified that HUVECs exhibit non-productive infection without cytopathic effects, in addition to the lack of expression of specific cell receptors known to be essential for SARS-CoV-2 entry into cells. We highlighted the enrichment of the protein SUMOylation pathway and the increase in SUMO2, which was confirmed by orthogonal assays. In conclusion, proteomic analysis revealed that the exposure to SARS-CoV-2 induced oxidative stress and changes in protein abundance and pathways enrichment that resembled endothelial dysfunction.

Platelets and extracellular traps in infections.

Platelets have a well-recognized role in hemostasis and thrombosis, and they are important amplifiers of inflammation and innate immune responses. The formation of DNA extracellular traps (ETs) is a complex cellular mechanism, which occurs in response to microbial infections and sterile inflammation, and results in the release of DNA complexed with histones and various granular proteins. ETs were first discovered in neutrophils (NETs); however, it is now accepted that other leukocytes, including eosinophils (EETs) and monocytes/macrophages (MoETs/METs), can also generate them. Moreover, several types of ETs have been described.Increasing evidence has demonstrated that platelets modulate the formation of ETs. This review summarizes recent findings about the physiopathological role of platelets in the formation of ETs during infection and future perspectives in the field.

Leptospira species promote a pro-inflammatory phenotype in human neutrophils.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. Neutrophils are key cells against bacterial pathogens but can also contribute to tissue damage. Because the information regarding the role of human neutrophils in leptospirosis is scant, we comparatively analysed the human neutrophil's response to saprophytic Leptospira biflexa serovar Patoc (Patoc) and the pathogenic Leptospira interrogans serovar Copenhageni (LIC). Both species triggered neutrophil responses involved in migration, including the upregulation of CD11b expression, adhesion to collagen, and the release of IL-8. In addition, both species increased levels of pro-inflammatory IL-1ß and IL-6 associated with the inflammasome and NFκB pathway activation and delayed neutrophil apoptosis. LIC was observed on the neutrophil surface and not phagocytized. In contrast, Patoc generated intracellular ROS associated with its uptake. Neutrophils express the TYRO3, AXL, and MER receptor protein tyrosine kinases (TAM), but only LIC selectively increased the level of AXL. TLR2 but not TLR4-blocking antibodies abrogated the IL-8 secretion triggered by both Leptospira species. In summary, we demonstrate that Leptospira species trigger a robust neutrophil activation and pro-inflammatory response. These findings may be useful to find new diagnostic markers and therapeutic strategies against leptospirosis.

Cross-talk between monocyte invasion and astrocyte proliferation regulates scarring in brain injury.

Scar formation after brain injury is still poorly understood. To further elucidate such processes, here, we examine the interplay between astrocyte proliferation taking place predominantly at the vascular interface and monocyte invasion. Using genetic mouse models that decrease or increase reactive astrocyte proliferation, we demonstrate inverse effects on monocyte numbers in the injury site. Conversely, reducing monocyte invasion using CCR2-/- mice causes a strong increase in astrocyte proliferation, demonstrating an intriguing negative cross-regulation between these cell types at the vascular interface. CCR2-/- mice show reduced scar formation with less extracellular matrix deposition, smaller lesion site and increased neuronal coverage. Surprisingly, the GFAP+ scar area in these mice is also significantly decreased despite increased astrocyte proliferation. Proteomic analysis at the peak of increased astrocyte proliferation reveals a decrease in extracellular matrix synthesizing enzymes in the injury sites of CCR2-/- mice, highlighting how early key aspects of scar formation are initiated. Taken together, we provide novel insights into the cross-regulation of juxtavascular proliferating astrocytes and invading monocytes as a crucial mechanism of scar formation upon brain injury.

Human Plasmacytoid Dendritic Cells Elicited Different Responses after Infection with Pathogenic and Nonpathogenic Junin Virus Strains.

The arenavirus Junin virus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. We characterized the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection with the C#1 strain (attenuated) and even more susceptible to infection with the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expression after infection with both JUNV strains.

Macrophages and galectin 3 play critical roles in CVB3-induced murine acute myocarditis and chronic fibrosis.

Macrophage influx and galectin 3 production have been suggested as major players driving acute inflammation and chronic fibrosis in many diseases. However, their involvement in the pathogenesis of viral myocarditis and subsequent cardiomyopathy are unknown. Our aim was to characterise the role of macrophages and galectin 3 on survival, clinical course, viral burden, acute pathology, and chronic fibrosis in coxsackievirus B3 (CVB3)-induced myocarditis. Our results showed that C3H/HeJ mice infected with CVB3 and depleted of macrophages by liposome-encapsulated clodronate treatment compared with infected untreated mice presented higher viral titres but reduced acute myocarditis and chronic fibrosis, compared with untreated infected mice. Increased galectin 3 transcriptional and translational expression levels correlated with CVB3 infection in macrophages and in non-depleted mice. Disruption of the galectin 3 gene did not affect viral titres but reduced acute myocarditis and chronic fibrosis compared with C57BL/6J wild-type mice. Similar results were observed after pharmacological inhibition of galectin 3 with N-acetyl-d-lactosamine in C3H/HeJ mice. Our results showed a critical role of macrophages and their galectin 3 in controlling acute viral-induced cardiac injury and the subsequent fibrosis. Moreover, the fact that pharmacological inhibition of galectin 3 induced similar results to macrophage depletion regarding the degree of acute cardiac inflammation and chronic fibrosis opens up the possibility of new pharmacological strategies for viral myocarditis.

Human platelets and their capacity of binding viruses: meaning and challenges?

Blood platelets are first aimed at ensuring primary hemostasis. Beyond this role, they have been acknowledged as having functions in the maintenance of the vascular arborescence and, more recently, as being also innate immune cells, devoted notably to the detection of danger signals, of which infectious ones. Platelets express pathogen recognition receptors that can sense bacterial and viral moieties. Besides, several molecules that bind epithelial or sub-endothelial molecules and, so forth, are involved in hemostasis, happen to be able to ligate viral determinants, making platelets capable of either binding viruses or even to be infected by some of them. Further, as platelets express both Fc-receptors for Ig and complement receptors, they also bind occasionally virus-Ig or virus-Ig-complement immune complexes. Interplays of viruses with platelets are very complex and viral infections often interfere with platelet number and functions. Through a few instances of viral infections, the present review aims at presenting some of the most important interactions from pathophysiological and clinical points of view, which are observed between human viruses and platelets.

DTU I isolates of Trypanosoma cruzi induce upregulation of Galectin-3 in murine myocarditis and fibrosis.

Chagas heart disease is a major public concern since 30% of infected patients develop cardiac alterations. The relationship between Trypanosoma cruzi discrete typing units (DTUs) and the biological properties exhibited by the parasite population has yet to be elucidated. In this study, we analysed the expression of α-smooth muscle actin (α-SMA) and galectin-3 (Gal-3) associated with cardiac extracellular matrix (ECM) remodelling a murine chronic cardiomyopathy induced by Tc I genotypes. We found the induction of myocarditis was associated with the upregulation of Col I, α-SMA, Gal-3, IFN-γ and IL-13, as analysed by q-PCR. In myocardial areas of fibrosis, the intensity of myocarditis and significant ECM remodelling correlated with the presence of Col I-, Gal-3- and α-SMA-positive cells. These results are promising for the further efforts to evaluate the relevance of Gal-3 in Chagas heart disease, since this galectin was proposed as a prognosis marker in heart failure patients.

Characterization of LIC11207, a novel leptospiral protein that is recognized by human convalescent sera and prevents apoptosis of polymorphonuclear leukocytes.

We report the study of a predicted outer-membrane leptospiral protein encoded by the gene lic11207. This protein is conserved in several pathogenic leptospiral strains but is absent in the saprophyte Leptospira biflexa. This putative outer-membrane protein has a domain of unknown function (DUF) 1565 found in several phylogenetically diverse bacteria and in the archaeon Methanosarcina acetivorans. The gene was cloned and expressed in Escherichia coli BL21 (SI) strain using the expression vector pDEST17. The 34 kDa recombinant protein was tagged with N-terminal hexahistidine and purified by metal-charged chromatography. The purified protein was used to assess: reactivity with human convalescent sera; in vivo expression; ability to activate endothelial cells (EC); and ability to modulate the apoptosis of polymorphonuclear cells (PMNs). The LIC11207 coding sequence was identified in vivo in the hamster renal tubules during experimental infection with Leptospira interrogans. The rLIC11207 showed significant antigenicity against human convalescent sera when compared with sera from healthy donors. The recombinant protein did not alter the surface expression of E-selectin or intercellular adhesion molecule 1 (ICAM-1) in EC and failed to induce the release of von Willebrand factor (vWF). Interestingly, rLIC11207 delayed apoptosis of PMNs suggesting a possible role of this protein during the infection.

Theiler's Murine Encephalomyelitis Virus Replicates in Primary Neuron Cultures and Impairs Spine Density Formation.

In this study, we examined infection with the highly neurovirulent GDVII, the less neurovirulent DA strains, and with a mutant DA, which lacks the L* protein (L*-1) involved in viral persistence and demyelinating disease, to analyze the direct effects of Theiler's murine encephalomyelitis virus (TMEV) replication using primary cultures of mouse brain hippocampal neurons. All viruses replicate in cultured neurons, with GDVII having the highest titers and L*-1 the lowest. Accordingly, all were positive for viral antigen staining 3 days postinfection (dpi), and DA and L*-1 were also positive after 12 dpi. NeuN + immunostaining showed an early and almost complete absence of positive cells in cultures infected with GDVII, an approximately 50% reduction in cultures infected with DA, and fewer changes in L*-1 strains at 3 dpi. Accordingly, staining with chloromethyltetramethylrosamine orange (Mitotracker OrangeTM) as a parameter for cell viability showed similar results. Moreover, at 1 dpi, the strain DA induced higher transcript levels of neuroprotective genes such as IFN-Iß, IRF7, and IRF8. At 3 dpi, strains GDVII and DA, but not the L*-1 mutant, showed lower PKR expression. In addition, confocal analysis showed that L*-1-infected neurons exhibited a decrease in spine density. Treatment with poly (I:C), which is structurally related to dsRNA and is known to trigger IFN type I synthesis, reduced spine density even more. These results confirmed the use of mouse hippocampal neuron cultures as a model to study neuronal responses after TMEV infection, particularly in the formation of spine density.

Activation of apoptotic signalling events in human embryonic stem cells upon Coxsackievirus B3 infection.

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate to a wide range of specialized cells and hold great promise as models for human development and disease, as well as for drug discovery and cell-replacement therapies. Group B Coxsackie viruses (CVBs) produce acute myocarditis, pancreatitis, non-septic meningitis and encephalitis in neonates, children and young adults. Moreover, CVBs can produce spontaneous miscarriage after early embryo infection. It was reported that hESCs express CVBs receptors and are susceptible to CVB3 infection. Apoptosis is one of the hallmarks of CVBs infection although details regarding CVB3 involvement in the apoptotic processes remain elusive. In order to evaluate the mechanisms of cell death induced by CVB3 in these pluripotent cells, we infected HUES-5 (H5) and WA01 (H1) hESC lines with CVB3. After validating the maintenance of stemness in these hESC lines when grown as confluent monolayers in feeder-free conditions, we analysed several aspects of programmed cell death triggered by CVB3. In all cases, we detected chromatin condensation, DNA fragmentation and caspase-9 and 3 cleavages. Moreover, we observed the presence of cleaved PARP product which was preceded by the appearance of p17, the catalytically active fragment of caspase-3. Mitochondrial function assays revealed a MOI dependent decrease in cell viability at 24 h post-infection (pi). No appreciable modifications in Bcl-2, Bcl-X(L) and Bax protein levels were observed upon CVB3 infection during 5-24 h observation period. However, a marked decrease in pro-apoptotic Bad abundance was detected without changes in its mRNA levels. In this study we found that the hESCs are highly susceptible to CVB3 infection and display elevated apoptosis rates, thus emerging as suitable human non-transformed in vitro models to study CVB3-induced apoptosis and resulting relevant to understand CVBs pathogenesis.

Junín virus infection of human hematopoietic progenitors impairs in vitro proplatelet formation and platelet release via a bystander effect involving type I IFN signaling.

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus (JUNV), a member of the arenaviridae family. Although a recently introduced live attenuated vaccine has proven to be effective, AHF remains a potentially lethal infection. Like in other viral hemorrhagic fevers (VHF), AHF patients present with fever and hemorrhagic complications. Although the causes of the bleeding are poorly understood, impaired hemostasis, endothelial cell dysfunction and low platelet counts have been described. Thrombocytopenia is a common feature in VHF syndromes, and it is a major sign for its diagnosis. However, the underlying pathogenic mechanism has not yet been elucidated. We hypothesized that thrombocytopenia results from a viral-triggered alteration of the megakaryo/thrombopoiesis process. Therefore, we evaluated the impact of JUNV on megakaryopoiesis using an in vitro model of human CD34+ cells stimulated with thrombopoietin. Our results showed that CD34+ cells are infected with JUNV in a restricted fashion. Infection was transferrin receptor 1 (TfR1)-dependent and the surface expression of TfR1 was higher in infected cultures, suggesting a novel arenaviral dissemination strategy in hematopoietic progenitor cells. Although proliferation, survival, and commitment in JUNV-infected cultures were normal, viral infection impaired thrombopoiesis by decreasing in vitro proplatelet formation, platelet release, and P-selectin externalization via a bystander effect. The decrease in platelet release was also TfR1-dependent, mimicked by poly(I:C), and type I interferon (IFN alpha/beta) was implicated as a key paracrine mediator. Among the relevant molecules studied, only the transcription factor NF-E2 showed a moderate decrease in expression in megakaryocytes from either infected cultures or after type I IFN treatment. Moreover, type I IFN-treated megakaryocytes presented ultrastructural abnormalities resembling the reported thrombocytopenic NF-E2(-/-) mouse phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels.

Decay-accelerating factor 1 deficiency exacerbates Trypanosoma cruzi-induced murine chronic myositis.

INTRODUCTION: Murine infection with Trypanosoma cruzi (Tc) has been used to study the role of T-cells in the pathogenesis of human inflammatory idiopathic myositis. Absence of decay-accelerating factor 1 (Daf1) has been shown to enhance murine T-cell responses and autoimmunity. METHODS: To determine whether Daf1 deficiency can exacerbate Tc-induced myositis, C57BL/6 DAF(+/+) and DAF(-/-) mice were inoculated with 5 × 10(4) trypomastigotes, and their morbidity, parasitemia, parasite burden, histopathology, and T-cell expansion were studied in the acute and chronic stages. RESULTS: DAF(-/-) mice had lower parasitemia and parasite burden but higher morbidity, muscle histopathology, and increased number of CD44(+) (activated/memory phenotype) splenic CD4(+) and CD8(+) T-cells. CONCLUSIONS: An enhanced CD8(+) T-cell immune-specific response may explain the lower parasitemia and parasite burden levels and the increase in histopathological lesions. We propose that Tc-inoculated DAF(-/-) mice are a useful model to study T-cell mediated immunity in skeletal muscle tissues.

Molecular determinants of disease in coxsackievirus B1 murine infection.

To understand better how different genomic regions may confer pathogenicity for the coxsackievirus B (CVB), two intratypic CVB1 variants, and a number of recombinant viruses were studied. Sequencing analysis showed 23 nucleotide changes between the parental non-pathogenic CVB1N and the pathogenic CVB1Nm. Mutations present in CVB1Nm were more conserved than those in CVB1N when compared to other CVB sequences. Inoculation in C3H/HeJ mice showed that the P1 region is critical for pathogenicity in murine pancreas and heart. The molecular determinants of disease for these organs partially overlap. Several P1 region amino acid differences appear to be located in the decay-accelerating factor (DAF) footprint CVBs. CVB1N and CVB1Nm interacted with human CAR, but only CVB1N seemed to interact with human DAF, as determined using soluble receptors in a plaque-reduction assay. However, the murine homolog Daf-1 did not interact with any virus assessed by hemagglutination. The results of this study suggest that an unknown receptor interaction with the virus play an important role in the pathogenicity of CVB1Nm. Further in vivo studies may clarify this issue.

Role of inducible nitric oxide synthase in the pathogenesis of experimental leptospirosis.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is a radical effector molecule of the innate immune system that can directly inhibit pathogen replication. In order to study subsequent iNOS kidney expression in experimental leptospirosis, Golden Syrian hamsters and C3H/HeJ mice were infected intraperitoneally with 10(2) or 10(7) virulent Leptospira interrogans serovar Copenhageni (LIC) strain Fiocruz L1-130. Results showed increased levels of iNOS mRNA and protein in kidneys of infected animals when compared to that in mock-infected animals. To get a deeper insight into the role of iNOS in experimental leptospirosis, both subject species were treated or not treated with 4-aminopyridine (4-AP, 0.3mg/kg), an iNOS inhibitor. Treatment of infected hamsters with 4-AP accelerated the mortality rate to 100% by one day and increased the mortality rate from 20 to 60% in mice at 14 days post-infection. In kidney tissues, 4-AP treatment increased the bacterial burden, as demonstrated through leptospiral DNA quantification by real-time PCR, and aggravated tubulointerstitial nephritis. In addition, iNOS inhibition reduced the specific humoral response against LIC when compared to that in untreated infected animals. According to these results, iNOS expression and the resulting NO have an important role in leptospirosis.

Increased immunogenicity to LipL32 of Leptospira interrogans when expressed as a fusion protein with the cholera toxin B subunit.

Leptospirosis is one of the most widespread zoonosis in the world. The development of a recombinant leptospira vaccine remains a challenge. In this study, we cloned the Leptospira interrogans open reading frame (ORF) coding the external membrane protein LipL32, an immunodominant antigen found in all pathogenic leptospira, downstream of the highly immunogenic cholera toxin B subunit (CTB) ORF. Expression and assembly of the CTB-LipL32 fusion protein into oligomeric structures of pentameric size were observed in soluble fractions by Western blot analysis. The CTB-LipL32 protein demonstrated strong affinity for monosialotetrahexosylgaglioside (GM1-ganglioside) in an enzyme-linked immunosorbent assay (ELISA), suggesting that the antigenic sites for binding and proper folding of the pentameric CTB structure were conserved. Furthermore, antisera against LipL32 also recognized the CTB-LipL32 fusion protein, suggesting that LipL32 also conserved its antigenic sites, a fact confirmed by an ELISA assay showing soluble CTB-LipL32 recognition by sera from convalescent patients. In addition, soluble CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32. The data presented here provide support for CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.

Evaluation of the expression and protective potential of Leptospiral sphingomyelinases.

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.

The immunoregulatory glycan-binding protein galectin-1 triggers human platelet activation.

Platelet activation is a critical process during inflammation, thrombosis, and cancer. Here, we show that galectin-1, an endogenous lectin with immunoregulatory properties, plays a key role in human platelet activation and function. Galectin-1 binds to human platelets in a carbohydrate-dependent manner and synergizes with ADP or thrombin to induce platelet aggregation and ATP release. Furthermore, galectin-1 induces F-actin polymerization, up-regulation of P-selectin, and GPIIIa expression; promotes shedding of microvesicles; and triggers conformational changes in GPIIb/IIIa. In addition, exposure to this lectin favors the generation of leukocyte-platelet aggregates. A further mechanistic analysis revealed the involvement of Ca(2+) and cyclic nucleotide-dependent pathways in galectin-1-mediated control of platelet activation. Finally, expression of endogenous galectin-1 in human platelets contributes to ADP-induced aggregation. Our study reveals a novel unrecognized role for galectin-1 in the control of platelet physiology with potential implications in thrombosis, inflammation, and metastasis.