**4. Programmed cell death: A host innate immune protection or a virus evasion strategy**

It has been described that a protective response by innate immune cells to viruses is triggered by several distinct mechanisms including apoptosis, necrosis, paraptosis, pyroptosis, autophagy cell death, and others. Each one is depending on several aspects of infection, including where the microorganism was detected, susceptible target-cells, through signaling systems discharging the death signal, and its intensity. During the innate immune response to infections, programmed cell death may occur as a direct pathogenic mechanism of viral spread and escape from the immune system or represents an appropriate host response to limit pathogen replication. Apoptosis of lymphocytes and monocytes also plays an important role in the control of inflammatory responses, as well as in the development of maternal-fetal tolerance [96–99].

#### *Innate Immunity Modulation during Zika Virus Infection on Pregnancy: What We Still Need… DOI: http://dx.doi.org/10.5772/intechopen.94861*

Type 1 programmed cell death, also known as apoptosis, is defined by internucleosomal DNA fragmentation, marked irreversible apoptotic characteristic indicating chromatin condensation, degradation of cytoskeleton and nuclear proteins, protein crosslinking, apoptotic bodies' formation baring ligands for receptors of phagocytic cells and, finally, the uptake by these phagocytes [97–99]. Type 2, or autophagic cell death, presents unique characteristics organelles formation including autophagosomes and autophagolysosomes in the dying cell, sources of self-degradation, and recycling [100].

Two pathways can regulate the apoptosis program in different aspects: extrinsic and intrinsic. Extrinsic pathway is activated by a transduction signal through death receptors, in which TNF, Fas ligand, or TRAIL bind to their respective receptors, such as TNF receptor family: TNFR1, Fas (CD95/APO-1) and TRAIL-R1/2. A complex signal mediated by this binding leads to an enzymatic cascade of cell degradation, and at this point caspase-3 is activated promoting DNA damage [101]. Intrinsic pathway involves intracellular mitochondria, which its membrane is the local for many Bcl-2 family members and their activity in inducing / inhibiting the mitochondrial apoptosis program implies in those proteins lead to membrane collapse as well as a transition from mitochondrial permeability promoting apoptosis process [96, 101–105].

Taking together, type 2, or autophagic cell death, consists of a conserved catabolic process that contributes to degradation and recycling of many intracellular substances, through lysosome activity. In this sense, many studies have shown its importance in immune responses, including degradation of microbes, direct viral peptides MHC class I presentation [106] and even altering T-cell signaling and tolerance [107, 108]. At first, autophagy is necessary to keep the cell alive under stress conditions that precede their demise. Such kind of cell death could be achieved by several mechanisms, including prolonged hypoxia or digestion of vital factors, regulatory molecules or essential organelles. In a stress situation, caused by virus, an infected cell can induce intracellular signals of autophagy, inhibiting cell proliferation, arresting cell cycle and eventually leading to cell death [106–111].

In the acute ZIKV infection during pregnancy, macrophages and dendritic cells are involved in inflammatory cytokines production, in which CARD9 expression, an important regulator of caspase activity playing an important role in cell apoptosis regulation, is elevated allowing that pattern recognition receptors (PRR) induce pro-inflammatory cytokines cascade, as the first step on CZS, as suggested [67]. According to Quicke et al., Hofbauer cells infected with ZIKV in placenta induces IFN type I activation, reactive oxygen species production, as well as pro-inflammatory cytokines, but with minimal cell death, showing a scape of innate immune response [23]. Recently, Cao et al., showed that ZIKV could activate and increase an autophagic process in pregnant mice, suggesting an imbalance of trophoblastic cells in placenta, and relation with fetal loss [112]. Corroborating, Ribeiro et al. using a human model of placenta explants for in vitro infection demonstrated tissue injury as consequence of the association between fetal pro-inflammatory responses mediated by IL-1β, IL-6 and TNF and extrinsic caspase 3 dependent apoptosis (TNF-TNFR pathway). Together data suggest that ZIKV infection corroborates to placenta innate immune and hormonal dysfunction, increasing loss barrier integrity [42] Thus, this inflammatory status could trigger cell death and barrier loss, allowing ZIKV cross placenta and infect fetuses' neural stem cells (**Figure 3**) [23, 113–115]. Interesting, autophagosomes are present in neural stem cells and it could facilitate ZIKV replication [116], although inflammation generated as well as the cytopathic effect itself culminate in extensive caspase-dependent neuronal cell death.

#### **Figure 3.**

*Programmed cell death activation during normal pregnancy and abnormal pregnancy induced by Zika virus. Normal pregnancy equilibrium is driven by regulation of number of innate immune cells in placenta leading by programmed cell death. In this situation, caspase activity starts on CARD9 expression with cytokines production by Hofbauer cells (1.A), which oxide nitric (NO) regulates trophoblasts autophagy (2.A, 3.A). Products of Hofbauer cells activity in the surveillance in placental parenchyma contributing to extrinsic (Fas/Fas-L) and intrinsic pathway (BCL2/BAX) activation in fetus brain with low expression of proinflammatory cytokines, regulating number of neural stem cells and microglia by apoptosis (4.A), maintaining the healthy pregnancy. Acute ZIKV infection during pregnancy suggests that macrophages and DCs are involved in pro-inflammatory cytokines production, in which CARD9 is upregulated, increasing caspase activity, allowing pro-inflammatory cytokines and reactive species cascade (1.B, 2.B), exacerbating autophagy in placenta (3.B). Taking together this innate immune dysfunction, fetus brain is affected by high activation of apoptosis pathway (4.B), provoking a cascade of cell death with an abrupt reduction of neural cells, causing severe damage [113–115]. Grey arrows represent the production or expression levels (up = high, down = low). Double arrows represent a high magnitude of production or expression. Red dashed arrows represent the direction of function/induction events that have been known and those suggested. Figure created using Biorender software (https://www.biorender.com).*

Corroborating, Lum et al. has shown that ZIKV mainly infects fetal microglia and induces high levels of pro-inflammatory cytokines that could be harmful to the fetus [117]. In addition, the analysis of in vitro culture, fetal brain histology and *ex vivo* studies with children presenting evidence of congenital infections demonstrated that, in fact, ZIKV promotes microglial activation, suggesting viral disseminating, neuronal death and an abnormal increase of astrocytes due to neurons destruction [117].

Thus, once in fetus central nervous system, ZIKV may contribute to extrinsic (Fas/Fas-L) and intrinsic (Bcl-2) pathways activation for programmed cell death, reducing number of neuronal cells. Thus, the risk of congenital syndrome is eminent, mainly in the first trimester, as well documented (**Figure 3**) [67, 118–123]. Some studies with fetuses' autopsies and infants with microcephaly have been demonstrated a broad spectrum of microscopic neuropathological abnormalities and brain damage, with direct virus cytopathic effects in neural glial cells. In this way, these data support the strong association with apoptotic cell death and microcalcifications [13, 23, 124].
