Viral inflammation:Sensing, signaling, safeguarding: DENV and SARS-CoV-2

This thesis is dedicated to the study of the interactions of these viruses with human cells. It focuses on investigating DENV infection in host immune cells, along with the immune responses it initiates and how they develop. Furthermore, the work centers on the study of SARS-CoV-2 infection in various cell models, while also examining whether the infection can be inhibited with potential antiviral agents. This thesis revealed the critical role of Toll-like receptor 2 (TLR2) in severe DENV infections, particularly its association with classical monocytes and disease progression. Furthermore, it delved into antibody-dependent enhancement (ADE) in DENV infections, emphasizing TLR2's pivotal role in mediating viral entry and triggering inflammatory responses, along with the contributions of TLR8, TLR3, and SYK.Furthermore, this thesis presents compelling evidence of the antiviral properties of resveratrol and pterostilbene against SARS-CoV-2, offering promising therapeutic avenues. However, it issues a word of caution regarding the use of ivermectin and moxidectin for against SARS-CoV-2, underlining the limitations of in vitro results and their applicability to human respiratory epithelial cells.In addition, we have demonstrated that human lung microvascular endothelial cells are not susceptible and permissive to SARS-CoV-2 infection, and that virus exposure to endothelial cells does not lead to endothelial activation.Collectively, these chapters offer a comprehensive exploration of the complex interactions between viruses and the human immune system, yielding novel insights into potential therapeutic strategies and enhancing our broader understanding of viral diseases. These findings underscore the significance of both in vitro and in vivo research, providing valuable implications for the development of antiviral therapies and the management of viral diseases.

Characterization of soluble TLR2 and CD14 levels during acute dengue virus infection

Dengue virus infection results in a broad spectrum of diseases ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Hitherto, there is no consensus biomarker for the prediction of severe dengue disease in patients. Yet, early identification of patients who progress to severe dengue is pivotal for better clinical management. We have recently reported that an increased frequency of classical (CD14 ++CD16 -) monocytes with sustained high TLR2 expression in acutely infected dengue patients correlates with severe dengue development. Here, we hypothesized that the relatively lower TLR2 and CD14 expression in mild dengue patients is due to the shedding of their soluble forms (sTLR2 and sCD14) and that these could be used as indicators of disease progression. Therefore, using commercial sandwich ELISAs, we evaluated the release of sTLR2 and sCD14 by peripheral blood mononuclear cells (PBMCs) in response to in vitro dengue virus (DENV) infection and assessed their levels in acute-phase plasma of 109 dengue patients. We show that while both sTLR2 and sCD14 are released by PBMCs in response to DENV infection in vitro, their co-circulation in an acute phase of the disease is not always apparent. In fact, sTLR2 was found only in 20% of patients irrespective of disease status. In contrast, sCD14 levels were detected in all patients and were significantly elevated in DF patients when compared to DHF patients and age-matched healthy donors. Altogether, our results suggest that sCD14 may help in identifying patients at risk of severe dengue at hospital admittance. </p

Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro)vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ- and microvascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin−/VCAM-1− (–/–), E-selectin+/VCAM-1− (E-sel+), E-selectin+/VCAM-1+ (+/+), and E-selectin−/VCAM-1+ (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-κB and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a “quiescent” subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction

Mediators of Obesity Do Not Influence SARS-CoV-2 Infection or Activation of Primary Human Lung Microvascular Endothelial Cells In Vitro

Clinical observations have shown that obesity is associated with the severe outcome of SARS-CoV-2 infection hallmarked by microvascular dysfunction in the lungs and other organs. Excess visceral fat and high systemic levels of adipose tissue (AT) derived mediators such as leptin and other adipokines have also been linked to endothelial dysfunction. Consequently, we hypothesized that AT-derived mediators may exacerbate microvascular dysfunction during of SARS-CoV-2 infection and tested this in a primary human lung microvascular endothelial (HLMVEC) cell model. Our results indicate that HLMVEC are not susceptible to SARS-CoV-2 infection since no expression of viral proteins and no newly produced virus was detected. In addition, exposure to the virus did not induce endothelial activation as evidenced by a lack of adhesion molecule, E-selectin, VCAM-1, ICAM-1, and inflammatory cytokine IL-6 induction. Incubation of endothelial cells with the pro-inflammatory AT-derived mediator, leptin, prior to virus inoculation, did not alter the expression of endothelial SARS-CoV-2 entry receptors and did not alter their susceptibility to infection. Furthermore, it did not induce inflammatory activation of endothelial cells. To verify if the lack of activated phenotype in the presence of adipokines was not leptin-specific, we exposed endothelial cells to plasma obtained from critically ill obese COVID-19 patients. Plasma exposure did not result in E-selectin, VCAM-1, ICAM-1, or IL-6 induction. Together our results strongly suggest that aberrant inflammatory endothelial responses are not mounted by direct SARS-CoV-2 infection of endothelial cells, even in the presence of leptin and other mediators of obesity. Instead, endothelial activation associated with COVID-19 is likely a result of inflammatory responses initiated by other cells. Further studies are required to investigate the mechanisms regulating endothelial behavior in COVID-19 and the mechanisms driving severe disease in obese individuals

Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air-Liquid Interface Cultured Human Primary Bronchial Epithelial Cells

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air–liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment

Viral inflammation:Sensing, signaling, safeguarding: DENV and SARS-CoV-2

This thesis is dedicated to the study of the interactions of these viruses with human cells. It focuses on investigating DENV infection in host immune cells, along with the immune responses it initiates and how they develop. Furthermore, the work centers on the study of SARS-CoV-2 infection in various cell models, while also examining whether the infection can be inhibited with potential antiviral agents. This thesis revealed the critical role of Toll-like receptor 2 (TLR2) in severe DENV infections, particularly its association with classical monocytes and disease progression. Furthermore, it delved into antibody-dependent enhancement (ADE) in DENV infections, emphasizing TLR2's pivotal role in mediating viral entry and triggering inflammatory responses, along with the contributions of TLR8, TLR3, and SYK.Furthermore, this thesis presents compelling evidence of the antiviral properties of resveratrol and pterostilbene against SARS-CoV-2, offering promising therapeutic avenues. However, it issues a word of caution regarding the use of ivermectin and moxidectin for against SARS-CoV-2, underlining the limitations of in vitro results and their applicability to human respiratory epithelial cells.In addition, we have demonstrated that human lung microvascular endothelial cells are not susceptible and permissive to SARS-CoV-2 infection, and that virus exposure to endothelial cells does not lead to endothelial activation.Collectively, these chapters offer a comprehensive exploration of the complex interactions between viruses and the human immune system, yielding novel insights into potential therapeutic strategies and enhancing our broader understanding of viral diseases. These findings underscore the significance of both in vitro and in vivo research, providing valuable implications for the development of antiviral therapies and the management of viral diseases.

Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air–Liquid Interface Cultured Human Primary Bronchial Epithelial Cells

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air–liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment

A Sobolev norm based distance measure for HARDI clustering : a feasibility study on phantom and real data

Dissimilarity measures for DTI clustering are abundant. However, for HARDI, the L2 norm has up to now been one of only few practically feasible measures. In this paper we propose a new measure, that not only compares the amplitude of diffusion profiles, but also rewards coincidence of the extrema. We tested this on phantom and real brain data. In both cases, our measure significantly outperformed the L2 norm

A Sobolev norm based distance measure for HARDI clustering : a feasibility study on phantom and real data

Dissimilarity measures for DTI clustering are abundant. However, for HARDI, the L2 norm has up to now been one of only few practically feasible measures. In this paper we propose a new measure, that not only compares the amplitude of diffusion profiles, but also rewards coincidence of the extrema. We tested this on phantom and real brain data. In both cases, our measure significantly outperformed the L2 norm

Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro) vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ-andmicrovascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin(-)/VCAM-1(-) (-/-), E-selectin(+)/VCAM-1(-) (E-sel+), E-selectin(+)/VCAM-1(+) (+/+), and E-selectin(-)/VCAM-1(+) (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-kappa B and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a "quiescent" subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction