TLR3 is required for survival following Coxsackievirus B3 infection by driving T lymphocyte activation and polarization: The role of dendritic cells.

Type B coxsackievirus (CVB) is a common cause of acute and chronic myocarditis, meningitis and pancreatitis, often leading to heart failure and pancreatic deficiency. The polarization of CD4+ T lymphocytes and their cytokine milieu are key factors in the outcome of CVB-induced diseases. Thus, sensing the virus and driving the adaptive immune response are essential for the establishment of a protective immune response. TLR3 is a crucial virus recognition receptor that confers the host with resistance to CVB infection. In the current study, we found that TLR3 expression in dendritic cells plays a role in their activation upon CVB3 infection in vitro, as TLR3-deficient dendritic cells up-regulate CD80 and CD86 to a less degree than WT cells. Instead, they up-regulated the inhibitory molecule PD-L1 and secreted considerably lower levels of TNF-α and IL-10 and a higher level of IL-23. T lymphocyte proliferation in co-culture with CVB3-infected dendritic cells was increased by TLR3-expressing DCs and other cells. Furthermore, in the absence of TLR3, the T lymphocyte response was shifted toward a Th17 profile, which was previously reported to be deleterious for the host. TLR3-deficient mice were very susceptible to CVB3 infection, with increased pancreatic injury and extensive inflammatory infiltrate in the heart that was associated with uncontrolled viral replication. Adoptive transfer of TLR3+ dendritic cells slightly improved the survival of TLR-deficient mice following CVB3 infection. Therefore, our findings highlight the importance of TLR3 signaling in DCs and in other cells to induce activation and polarization of the CD4+ T lymphocyte response toward a Th1 profile and consequently for a better outcome of CVB3 infection. These data provide new insight into the immune-mediated mechanisms by which CVBs are recognized and cleared in order to prevent the development of myocarditis and pancreatitis and may contribute to the design of therapies for enteroviral infections

Mice deficient in IL-17 are resistant to CVB3 infection.

<p>WT, IL-17 KO and IL-23R KO mice were i.p. infected with 10⁶ CVB3. (A) Survival was monitored. (B) Heart (top panel) and pancreas (bottom panel) sections were stained with hematoxylin and eosin at 12 dpi. Data are means of n = 5 + SE for each group, and are representative of two independent experiments. Gehan-Breslow-Wilcoxon test was used to compare survival curves, which showed p = 0.0175 for WT vs IL-23R KO.</p

TLR3 modulates the proliferation and cytokine production of CD4⁺ and CD8⁺ T lymphocytes after stimulation with CVB3-infected dendritic cells.

<p>WT and TLR3 KO DCs were differentiated in culture from bone marrow cells with 20 ng/ml GM-CSF for 7 days. Cells were infected with CVB3 (MOI: 10) or incubated with medium and co-cultured with isolated WT or TLR3 KO T lymphocytes from CVB3-infected mice (5 dpi) at a ratio of 10 lymphocytes/1 DC. In some wells, anti-CD3 and anti-CD28 were added for the polyclonal stimulation of lymphocytes. (A) Lymphocytes were stained with CFSE, and proliferation was assessed after 3 days of co-culture via CFSE dilution in the CD4⁺ or CD8⁺ populations. (B) Intracellular cytokines were measured by flow cytometry after re-stimulation with PMA and ionomycin in the presence of Golgi Stop. DC: dendritic cells; T: T lymphocytes. Data are means of n = 3 + SE (3 uninfected DCs and T cells from 3 infected mice) for each condition (medium, CVB3 and anti-CD3 + anti-CD28) and are representative of two independent experiments. Two-way ANOVA followed by Bonferroni’s test was used for all comparisons. P<0.001: ***; P<0.01:**; and P<0.05:*; NS: non-significant. All analyzed parameters in CVB3-infected condition are significantly different between WT DC + WT T cell vs KO DC + KO T cell.</p

Dendritic cell activation after in vivo CVB3 infection is altered by TLR3.

<p>WT and TLR3 KO mice were i.p. infected with 10⁶ CVB3. Cells from the spleen and the mediastinal (MLN) and pancreatic (PLN) lymph nodes were isolated at the indicated times, and activation markers were evaluated by flow cytometry. MFI: Median fluorescence intensity. The MFI was measured in the CD11c⁺ population. Data are means of n = 3–5 + SE for each group and each time point, and are representative of three independent experiments. Two-way ANOVA followed by Bonferroni’s test was used for all comparisons. P<0.05:*.</p

TLR3 is essential for controlling tissue inflammation and viral load after CVB3 infection.

<p>WT and TLR3 KO mice were i.p. infected with 10⁸ or 10⁶ CVB3. (A) Survival was monitored for 25 days. After infection with 10⁶ CVB3, (B) the weights of the heart and pancreas at 12 dpi are shown as a percentage of body weight; (C) heart (top panel) and pancreas (bottom panel) sections were stained with hematoxylin and eosin; and (D) viral load was determined from PCR of the heart and pancreas at the indicated times. Data are expressed as number of virus copies/μg RNA. Data are means of n = 3–6 + SE for each group and each time point. Unpaired <i>Student t</i> test was used to compare tissue weight and Two-way ANOVA followed by Bonferroni’s test was used for all comparisons of viral load. P<0.01:**; and P<0.05:*. Gehan-Breslow-Wilcoxon test was used to compare survival curves, which showed p = 0.0006 for WT vs TLR3 KO (10⁸), and p = 0.03 for WT vs TLR3 KO (10⁶).</p

Adoptive transfer of TLR3⁺ dendritic cells increases TNF-α production by T lymphocytes and slightly improves the survival of CVB3-infected mice.

<p>WT and TLR3 KO mice were i.p. infected with either 10⁷ or 10⁶ CVB3. WT bone marrow-derived DCs (2 x 10⁶) were adoptively transferred i.v. to TLR3 KO mice 1 day post-infection. (A) At 12 dpi with 10⁶ CVB3, spleen cells were isolated and stimulated with PMA and ionomycin in the presence of Golgi Stop for 6 h. Intracellular cytokines were measured by flow cytometry. (B) Survival was monitored. Data are means of n = 3–6 + SE for each group, and are representative of three independent experiments. One-way ANOVA followed by Bonferroni’s test was used to compare the three groups in (A). *: p<0.05. Gehan-Breslow-Wilcoxon test showed no significant difference between the survival curves in (B).</p

TLR3 skews the T lymphocyte response towards Th1 and inhibits the Th17 profile after CVB3 infection.

<p>WT and TLR3 KO mice were i.p. infected with 10⁶ CVB3. Spleen cells were isolated at the indicated times and stimulated with PMA and ionomycin in the presence of Golgi Stop for 6 h. Intracellular cytokines were measured by flow cytometry. (A) Percentage and (B) Total number of cytokines-producing T cells/spleen. Data are means of n = 4–5 + SE for each group and each time point, and are representative of three independent experiments. Two-way ANOVA followed by Bonferroni’s test was used for all comparisons. P<0.001:***; P<0.01:**; and P<0.05:*.</p

Protein C Pretreatment Protects Endothelial Cells from SARS-CoV-2-Induced Activation

SARS-CoV-2 can induce vascular dysfunction and thrombotic events in patients with severe COVID-19; however, the cellular and molecular mechanisms behind these effects remain largely unknown. In this study, we used a combination of experimental and in silico approaches to investigate the role of PC in vascular and thrombotic events in COVID-19. Single-cell RNA-sequencing data from patients with COVID-19 and healthy subjects were obtained from the publicly available Gene Expression Omnibus (GEO) repository. In addition, HUVECs were treated with inactive protein C before exposure to SARS-CoV-2 infection or a severe COVID-19 serum. An RT-qPCR array containing 84 related genes was used, and the candidate genes obtained were evaluated. Activated protein C levels were measured using an ELISA kit. We identified at the single-cell level the expression of several pro-inflammatory and pro-coagulation genes in endothelial cells from the patients with COVID-19. Furthermore, we demonstrated that exposure to SARS-CoV-2 promoted transcriptional changes in HUVECs that were partly reversed by the activated protein C pretreatment. We also observed that the serum of severe COVID-19 had a significant amount of activated protein C that could protect endothelial cells from serum-induced activation. In conclusion, activated protein C protects endothelial cells from pro-inflammatory and pro-coagulant effects during exposure to the SARS-CoV-2 virus

Low SARS‐CoV‐2 seroprevalence in a cohort of Brazilian sickle cell disease patients: Possible effects of emphasis on social isolation for a population initially considered to be at very high risk

Abstract Despite being initially considered at higher risk for severe COVID‐19, sickle cell disease (SCD) patients have mostly presented clinical severity similar to the general population. As their vulnerability to become infected remains uncertain, we assessed the seroreactivity for SARS‐CoV‐2 to estimate the prevalence of infection and possible phenotypic and socioeconomic determinants for their contagion. Serologic evaluation was performed on 135 patients with an overall prevalence of 11%; positivity was associated with older age and use of public transportation. We speculate that social distancing instructions recommended by our clinic may have contributed to lower levels of infection, but potential protection factors need further investigation