The role of neutrophils and CD8 T cells in Zika virus infection

Infecções por flavivírus são causa de grande preocupação em todo o mudo devido às recorrentes epidemias e altos índices de mortalidade, sendo um grande problema de saúde pública. A relevância do estudo da infecção por ZIKV ganhou destaque devido ao enorme número de bebês nascidos com microcefalia, especialmente no Nordeste Brasileiro, tendo em maio de 2016 sido comprovada a relação causal entre a infecção por ZIKV durante a gestação e os casos de microcefalia. Sabendo-se que o ZIKV, assim como outros flavivírus, tem a capacidade de modular a resposta imune inata e adaptativa do hospedeiro, este projeto visou compreender o papel dos neutrófilos e de células T CD8+Foxp3+ no controle da replicação viral e na progressão da doença em modelo de infecção murina por ZIKV. Para isso, neutrófilos de sangue periférico de indivíduos saudáveis e neutrófilos de medula óssea de animais C57BL/6 e SJL foram utilizados para os experimentos in vitro e, para os experimentos in vivo, animais C57BL/6 IFNAR-/-, C57BL/6 WT, C57BL/6 Foxp3GFP e C57BL/6 CD8-/- foram infectados ou não com ZIKV. Foram realizadas análises para quantificação viral, expressão gênica e citometria de fluxo. Nossos resultados mostraram que ZIKV é capaz de infectar neutrófilos murinos e humanos in vitro sem interferir na meia-vida, produção de ROS e liberação de NETs destas células, além de não induzir expressão de marcadores de ativação celular como MHC-I, MHC-II, CD80, CD86 e IFNAR. Em consideração à expressão gênica, apenas Ifn-b, Axl, Nos2 e Tnf foram alterados. Adicionalmente, neutrófilos infectados com ZIKV foram detectados em sangue e placenta de animais infectados. A transferência de neutrófilos infectados com ZIKV in vitro para animais prenhes foi capaz de causar infecção sistêmica, além da transferência de partículas virais para os fetos, provando que os neutrófilos podem agir como cavalo de Tróia para replicação e disseminação viral na infecção por ZIKV. Por outro lado, a infecção de animais deficientes de células CD8+ apresentou um perfil oscilatório, tendo maior cópias virais no soro, enquanto que menor número de partículas virais no baço em comparação ao grupo controle. Além disso, a análise por PCR Array evidenciou alterações na expressão de genes relacionados ao reconhecimento viral, como o Tlr3 e Nod2 e resposta imune do hospedeiro, como Nfkb1, FasL e Ifn-a. A análise das populações de células do baço mostrou que a infecção por ZIKV induz o aumento da população de células T CD8+Foxp3+. Estas células induzidas durante a infecção por ZIKV expressam altos níveis de FasL e Ly6C e parecem não responder à infecção por ZIKV, enquanto que células T CD8+Foxp3+ de animais naïve induzem maior replicação viral em experimento de transferência adotiva. Tais achados nos levam a crer que existe uma tentativa do patógeno em modular a resposta imune inata e adaptativa no intuito de facilitar sua replicação e permanência no hospedeiro. Em suma, este trabalho buscou elucidar a imunobiologia da infecção pelo ZIKV na gestação, correlacionando com a susceptibilidade à Síndrome Congênita Causada pelo vírusFlavivirus infections are a cause of great concern around the world due to recurrent epidemics and high mortality rates, being a major public health problem. The relevance of the study of ZIKV infection was highlighted due to the huge number of babies born with microcephaly, especially in Northeast Brazil, and in May 2016 our group proved the causal relationship between ZIKV infection during pregnancy and cases of microcephaly. Knowing that ZIKV, as other flaviviruses, has the ability to modulate the host\'s innate and adaptive immune response, this project aimed to understand the role of neutrophils, and CD8+Foxp3+ T cells, in the control of viral replication and in disease progression in a murine ZIKV infection model. For this, peripheral blood neutrophils from healthy individuals and bone marrow neutrophils from C57BL/6 and SJL animals were used for in vitro experiments, and for in vivo experiments, C57BL/6 IFNAR-/-, C57BL/6 WT, C57BL/6 Foxp3GFP and C57BL/6 CD8-/- mice were infected with ZIKV, followed by the most varied analyzes for viral quantification, gene expression and cell populations of interest. Our results showed that ZIKV was able to infect murine and human neutrophils in vitro without interfering with the half-life, ROS production and NET release of these cells, in addition to not inducing expression of cell activation markers such as MHC-I, MHC-II, CD80, CD86 and IFNAR. Regarding gene expression, only Ifn-b, Axl, Nos2 and Tnf were altered. Additionally, ZIKV-infected neutrophils were detected in blood and placenta from infected mice, and the transfer of in vitro ZIKV-infected neutrophils to pregnant mice was capable to cause systemic infection, in addition to the transfer of viral particles to fetuses, proving that neutrophils can act as a Trojan horse for viral replication and dissemination in ZIKV infection. On the other hand, the infection of mice deficient in CD8+ cells presented an oscillatory profile, with higher viral copies in the serum, while a lower number of viral particles in the spleen compared to the control group. Furthermore, PCRArray analysis showed alterations in the expression of genes related to viral recognition, such as Tlr3 and Nod2 and host immune response Nfkb1, FasL and Ifn-a. The analysis of spleen cell populations showed that ZIKV infection induces an increase in the population of CD8+Foxp3+ T cells, the CD8+regulators. However, these cells induced during ZIKV infection express high levels of FasL and Ly6C and seems to be not responder to ZIKV infection, whereas CD8+Foxp3+ T cells from naïve mice induce greater viral replication in adoptive transfer experiment. Such findings lead us to believe that there is an attempt by the pathogen to modulate the innate and adaptive immune response, in order to facilitate its replication and permanence in the host. In summary, this work sought to elucidate the immunobiology of ZIKV infection during pregnancy, and to correlate it with the susceptibility to Congenital Syndrome Caused by the viru

Pregnancy: an immune challenge

Several studies demonstrate the importance of immunological aspects of pregnancy. During pregnancy, the embryo is implanted in the womb, where it will develop until the end of pregnancy. Amongst the immune aspects, the importance of the modulation of T lymphocytes, natural killers (NK) cells and many cytokines in maternal organism can be mentioned. The maternal tolerance to the fetus appears to be mediated by specific maternal hormones and by the expression of human leukocyte antigen G (HLA-G) - characteristic in pregnancy. Other studies suggest that fetal rejection and complications during pregnancy may occur because of the presence of minor histocompatibility antigens (mHAg), acquired by blood sharing of the mother with the fetus, and because of the presence of maternal antibodies against the sperm and against the fetus. The purpose of this review is to describe the immunological aspects that allow maternal tolerance to the fetus during pregnancy, as well as possible causes for rejection of the embryo and complications during pregnancy

Reactive Oxygen Species (ROS) Are Not a Key Determinant for Zika Virus-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells

Introduction: ZIKV is a highly neurotropic virus that can cause the death of infected neuroprogenitor cells through mitochondrial damage and intrinsic apoptotic signaling. In this context, the role of reactive oxygen species (ROS) in neuronal cell death caused by ZIKV still remains elusive. Objective: We aimed at evaluating the role of these cellular components in the death of human undifferentiated neuroblastoma cell line infected with ZIKV. Results: ZIKV infection resulted in the extensive death of SH-SY5Y cells with the upregulation of several genes involved in survival and apoptotic responses as well as the colocalization of mitochondrial staining with ZIKV Envelope (E) protein. Notably, levels of intracellular reactive oxygen species (ROS) were not altered during ZIKV infection in undifferentiated SH-SY5Y cells, and consistent with these results, the treatment of infected cells with the widely studied ROS scavenger N-acetylcysteine (NAC) did not prevent cell death in these cells. Conclusion: Altogether, our results suggest that excessive ROS production is not the main trigger of SH-SY5Y cells death in ZIKV infection

Extracellular Vesicles Shed By Trypanosoma cruzi Potentiate Infection and Elicit Lipid Body Formation and PGE2 Production in Murine Macrophages

During the onset of Trypanosoma cruzi infection, an effective immune response is necessary to control parasite replication and ensure host survival. Macrophages have a central role in innate immunity, acting as an important trypanocidal cell and triggering the adaptive immune response through antigen presentation and cytokine production. However, T. cruzi displays immune evasion mechanisms that allow infection and replication in macrophages, favoring its chronic persistence. One potential mechanism is the release of T. cruzi strain Y extracellular vesicle (EV Y), which participate in intracellular communication by carrying functional molecules that signal host cells and can modulate the immune response. The present work aimed to evaluate immune modulation by EV Y in C57BL/6 mice, a prototype resistant to infection by T. cruzi strain Y, and the effects of direct EV Y stimulation of macrophages in vitro. EV Y inoculation in mice prior to T. cruzi infection resulted in increased parasitemia, elevated cardiac parasitism, decreased plasma nitric oxide (NO), reduced NO production by spleen cells, and modulation of cytokine production, with a reduction in TNF-α in plasma and decreased production of TNF-α and IL-6 by spleen cells from infected animals. In vitro assays using bone marrow-derived macrophages showed that stimulation with EV Y prior to infection by T. cruzi increased the parasite internalization rate and release of infective trypomastigotes by these cells. In this same scenario, EV Y induced lipid body formation and prostaglandin E2 (PGE2) production by macrophages even in the absence of T. cruzi. In infected macrophages, EV Y decreased production of PGE2 and cytokines TNF-α and IL-6 24 h after infection. These results suggest that EV Y modulates the host response in favor of the parasite and indicates a role for lipid bodies and PGE2 in immune modulation exerted by EVs

Image_1_Extracellular Vesicles Shed By Trypanosoma cruzi Potentiate Infection and Elicit Lipid Body Formation and PGE2 Production in Murine Macrophages.PDF

<p>During the onset of Trypanosoma cruzi infection, an effective immune response is necessary to control parasite replication and ensure host survival. Macrophages have a central role in innate immunity, acting as an important trypanocidal cell and triggering the adaptive immune response through antigen presentation and cytokine production. However, T. cruzi displays immune evasion mechanisms that allow infection and replication in macrophages, favoring its chronic persistence. One potential mechanism is the release of T. cruzi strain Y extracellular vesicle (EV Y), which participate in intracellular communication by carrying functional molecules that signal host cells and can modulate the immune response. The present work aimed to evaluate immune modulation by EV Y in C57BL/6 mice, a prototype resistant to infection by T. cruzi strain Y, and the effects of direct EV Y stimulation of macrophages in vitro. EV Y inoculation in mice prior to T. cruzi infection resulted in increased parasitemia, elevated cardiac parasitism, decreased plasma nitric oxide (NO), reduced NO production by spleen cells, and modulation of cytokine production, with a reduction in TNF-α in plasma and decreased production of TNF-α and IL-6 by spleen cells from infected animals. In vitro assays using bone marrow-derived macrophages showed that stimulation with EV Y prior to infection by T. cruzi increased the parasite internalization rate and release of infective trypomastigotes by these cells. In this same scenario, EV Y induced lipid body formation and prostaglandin E₂ (PGE₂) production by macrophages even in the absence of T. cruzi. In infected macrophages, EV Y decreased production of PGE₂ and cytokines TNF-α and IL-6 24 h after infection. These results suggest that EV Y modulates the host response in favor of the parasite and indicates a role for lipid bodies and PGE₂ in immune modulation exerted by EVs.</p

AHR is a Zika virus host factor and a candidate target for antiviral therapy

Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity