**2. Type III interferon**

The success of pregnancy is dependent on a coordinated balance between the "invading" fetal trophoblast and a receptive maternal decidua in the placenta, maintaining a dynamic and responsive immune system. The longest period of the pregnancy, fetal growth, demands a symbiotic and tolerogenic environment, but congenital viral infections can disrupt this equilibrium. In order to avoid infection severity placenta actively modulates the immunologic profile of the maternalfetal interface [40, 41]. In this context, recent studies demonstrated that placenta responds to ZIKV infection by production of the newest interferon group type III interferons [21, 42, 43].

Type III interferon (IFN-λ 1–4) comprising a group of cytokines with action pathways under strengthen discovery [44–46], basically acting with shared inflammatory regulation and antiviral properties [47]. IFN-λs receptor was identified as a complex composed of two subunits: IFN-λR1 and IL-10R2, which is also a receptor subunit of the regulatory cytokines IL10, IL22, and IL26 [48]. In contrast with the classical pro-inflammatory type I interferons which receptors are expressed in almost all cell types, the IFNLR1/IL10RB complex is expressed primarily in cells of epithelial origin and few immune cells conferring selective IFN-λ responsiveness to them: neutrophils [49], myeloid dendritic cells (DCs) [50, 51] and plasmacytoid dendritic cells (pDC) [52]. Because of the restricted cell types producing IFN-λs, this cytokine acts locally as an immunologic barrier in organs with suppressing innate pro-inflammatory responses and limiting host damaging effects associated with inflammation [53]. Moreover, IFN-λs utilize mechanisms to suppress viral infections which induce a strong antiviral state following receptor binding with non-translational and translational processes [49, 54].

Between the different inflammatory regulation actions already described for IFN-λs, the suppression of neutrophil gains prominence because they are the immune cells that present higher expression.

of IFN-λR1 at the steady-state [55–57]. Neutrophils contribute to various stages of the reproductive process since conception and implantation, ensuring fetal wellbeing during pregnancy and finally contributing to parturition and postpartum maternal health. On the other hand, aberrant neutrophil activity is associated with severe pregnancy-related disorders such as pre-eclampsia, recurrent fetal loss or gestational diabetes mellitus [58–60]. In murine models, it was demonstrated

that neutrophil exposed to IFN-λ can induce antiviral interferon-stimulated genes (ISGs); and IFN-λ (but not IFN-β) specifically activated a translation-independent signaling pathway that diminished the production of reactive oxygen species and degranulation in neutrophils, which might permit a controlled development of the inflammatory process [49].

Studies utilizing a cellular model of collagen-induced arthritis demonstrated that IFN-λ2 was protective and could stop the progression of the disease, diminishing infiltration of neutrophils to the inflamed joints as well as the production of IL-1β upon treatment with pegylated recombinant IFN-λ2 [57]. *Ex vivo* experiments with cardiopathic patients` cells demonstrated that IFN-λ inhibits Neutrophil Extracellular Traps (NETs) [61]. NETosis has been appointed as critical agents during pregnancy, particularly involved an auto-inflammatory process involving the release of placental micro-debris in preeclampsia and recurrent fetal loss [62]. In collagen-induced arthritis murine models, it was demonstrated that IFN-λ exerts its anti-inflammatory effect by restricting recruitment of IL-1β–expressing neutrophils, which are important for amplification of inflammation, and reducing IL-17–producing Th17 and γδ T cells in the joints and inguinal lymph nodes, without affecting T cell proliferative responses [57].

IFN-λ is strongly associated with DCs activity inducing an effector adaptive immunity response [63, 64]. Studies with a mice model of influenza A virus infection demonstrated that IFN-λ directed acts in the migration and function of CD103(+) dendritic cells, also regulating DC IL-10 network [65]. Migratory CD103(+) DCs derived from skin, lung, and intestine, efficiently present exogenous antigens in their corresponding draining lymph nodes to specific CD8(+) T cells through a mechanism known as cross-presentation, demonstrating the IFN-λ importance for the development of specific CD8+ T cell responses [65, 66]. Moreover, IFN-λ contributes to the formation of tolerogenic DCs cell, contributing to control inflammatory responses and homeostasis by fostering the conversion of naive T cells into induced Foxp3(+) regulatory T cells [66]. In vitro studies demonstrated that IFN- λ directs DCs to a regulatory phenotype with diminished capacity to stimulate T cell proliferation in a PD-1/PD-L1 dependent manner with contribution from the imbalanced cytokine milieu, such as low IL-12 and IL-2 and/or high IL-10 production [50]. Another study using mixed lymphocyte cultures demonstrated that IFN- λ -treated DCs specifically induced IL-2-dependent proliferation of a CD4(+) CD25(+) Foxp3(+) T-cell subset with contact-dependent suppressive activity on T-cell proliferation initiated by fully mature DCs [51].

Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues, considered to be "professional" type I IFN-producing cells and produce 10- to 100-fold more IFN-α than other cell types in response to enveloped viruses. However, in vitro IFN-λ treatment of pDC resulted in increased virusinduced expression of both IFN-α and IFN-λ, indicating that pDC are high producers of IFN-λ1 and -λ2 in response to viral stimulation and the consequences of this high IFN-λ production by pDC should be further explored [52].

In human congenital ZIKV infections, it was demonstrated that ZIKV infection leads to a typical inflammatory response in the placenta, including the expression of anti-viral Type I interferon genes (*IFIT5*, *IFNA1*, and *IFNB*), type II interferon (*IFI16*), cytokine signaling (*IL22RA* and *IP10*), and interferon regulatory factors (*IRF7* and *IRF9*). Furthermore, the CZS cases present a gene expression profile with impaired *IFNL2* response, accompanied by an exacerbated type I IFN response; with an increased expression of *IFIT5*, parallel to a decrease in *ISG15* mRNA [67], which was already identified as negative modulator of type I IFN and protective against ZIKV ocular manifestations [68]. These results are corroborated by *in vitro*

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

#### **Figure 1.**

*Summary of Interferon lambda (IFN-*λ*) function during normal pregnancy (A), Healthy Congenital Zika infection (B), and Zika-Associated Birth Defects (C). (A) In normal pregnancy, trophoblasts exhibit a constitutive IFN-*λ *production, contributing to the general tolerogenic environment demanded by pregnancy (A1); Considering the peripheral blood tissue IFN-*λ *Interact with: (A2) neutrophils leading to a decrease in ROS and IL1*β*, and (A3) migratory CD103+Dendritic cells (DC) that present low levels of PD1, IL2 and IL12 together with high IL10. These CD103+DC foster the conversion of naive T cells into induced Foxp3(+) regulatory T cells (Treg) (A4). In the placenta, the constitutive IFN-*λ *is accompanied by decreased type I IFN pathway: low expression of IFIT5, IFNA, and IFNB, and high expression of type I IFN the negative regulator ISG15 (A5). In the lack of viral infection, the interferon regulatory factors IRF7 and IRF9 present low expression levels (A7). (B) In healthy congenital Zika infections, the placenta expresses high levels of IFN-*λ *to protect the fetus from congenital defects (B1). In this low damage antiviral response, high levels of IFN-*λ *elicits the production of ISGs and the decrease of ROS and IL1*β *by circulating neutrophils (B2), meanwhile the CD103+ DC presents an accented regulatory profile (B3), with induction of high specific anti-ZIKV response by Treg (B4) and TCD8+ cells (B5). In the placental level type, I interferon pathway shows a slight increase, together with the enhance of IRF7 and IRF9, forming a balanced antiviral response. (C) In Congenital Zika Syndrome (CZS) the lack of IFN-*λ *contributes to a damaging outcome (C1). Diminished levels of IFN-*λ *could not control the neutrophil activity, culminating in augmented ROS and IL1*β *(C2), and presence of aberrant activation forms as well as degranulation, migration, and NETosis (C3). Without IFN-*λ *the Dendritic Cells (DC) present a pr*ό*-inflammatory profile, with augmented PD1, IL2, and IL12 and diminished IL10 (C4). The placenta shows an exacerbated type I interferon response, which together with low IFN-*λ *levels (C5), leads to an imbalanced damaging antiviral response. 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).*

studies that showed induction of *IFNL1* expression by susceptible placental cells after ZIKV infection, acting as an antiviral agent [43], reinforcing that IFN-λs are protective factors in ZIKV congenital infections. Studies with *ex vivo* placental 3D cultures from a different trimester of healthy pregnant volunteers showed that IFN-λs are expressed mostly by deciduous (the maternal portion of the placenta), already indicating that mothers are the agents on the immunoregulation of CZS outcome (**Figure 1**) [21].

## **3. Innate regulatory cells - myeloid-derived suppressor cells (MDSC)**

Immunity during pregnancy is very important to be explored since successful pregnancy requires that immunoregulatory mechanisms are triggered to suppress activated fetal-specific T cells lymphocytes [36, 37]. Maternal immune cells can recognize paternal antigens on fetus. Thus, it has been very well described that

dysfunction of immune cells during pregnancy can lead to immunologic fetal rejection by mother, in which the consequences are related to abortion, preterm delivery, or other severe complications [35–37].

Then, maternal-fetal tolerance involves the regulation of mother's immune system to tolerate the semi allogeneic fetus expressing paternal antigens without immune rejection. Even though, some studies showed that regulatory T cells are the main cells which plays an important role in suppressing activated T cells during gestation; since then innate immunity system is poorly investigated [69–71].

Considering infections during pregnancy, it is also important to know that changes on maternal immune responses are required to induce limited immunosuppression without loss of host defense, in which a balance between activated and immunosuppressed cells needs to be regular [35].

Myeloid-derived suppressor cells (MDSC) are a heterogeneous mixture of immature myeloid cells, been part of innate immune cells, having a crucial role in immunomodulatory mechanisms during pregnancy [36, 72, 73]. There are two subtypes of MDSC, a monocytic and granulocytic. Phenotype is characterized by expression of CD33 and CD11b in humans, CD14 by monocytic MDSC and CD15 by granulocytic MDSC cells but lacks the maturation marker HLA-DR. But both subtypes share the characteristic of immune-suppressive function inhibiting activated NK and T cell expansion [73, 74].

Normally, immature myeloid cells as MDSC are scarcely found in peripheral blood, and their maturation includes macrophages, dendritic cells, and granulocytes formation. Nevertheless, the MDSC are also recognized by their role in some pathological conditions, like cancer, sepsis, stress, autoimmune disorders and infectious diseases [38, 75, 76].

Several studies have been reported that a decrease of MDSC during pregnancy may lead to poor outcomes, as miscarriage [77]. Also, it has been shown that progesterone levels increase MDSC during pregnancy in mice, as well as high levels of TNF and IL-1β, pro-inflammatory cytokines [38, 78].

In murine models, it was demonstrated that MDSC can produce TGF-β and IL-10, as immunosuppressive cytokines, similarly to regulatory T cells. Adding to that, MDSC can suppress T cell activation and function by arginase-1 (Arg-1) secretion, as well as nitric oxide synthase and indoleamine 2,3 dioxygenase aimed to deplete nutrients for T cell proliferation, as I-arginine (I-Arg). According to Ismail 2018, arginine is also involved in replication, and virulence of several agents, as viruses and bacteria. Then, it is suggested that an accumulation of MDSC in placenta could influence an increase of arginase activity, and it would serve for a dual purpose, inhibiting the adaptive immune system whilst also providing potential protection against infection by arginine auxotrophic pathogens [79].

Nitric oxide (NO) has been related to embryo successful implantation during early pregnancy, but excessive NO production by decidual macrophages seems to be harmful and was linked with early pregnancy loss [37, 80, 81]. Another study suggests that in early pregnancy in decidua CD33+ cells express nitric oxide synthase, playing an important role to maintained pregnancy during this phase, while in later pregnancy CD33+ cells lose the expression of this enzyme [35, 37].

Kostlin-Gille *et al* 2019 showed that hypoxia condition is important to normal placenta development and its driven by a hypoxia-inducible factor 1 (HIF-1), a key regulator responsible for initiate transcription of several genes. The subunit HIF-1α is highly expressed in placenta during early gestation period, characterized by low oxygen pressure conditions. This study used myeloid HIF-1 knockout mice to evaluate the role of HIF-1α on myeloid-derived suppressor cell function, showing that HIF-1α deficiency in myeloid cells leads to diminished suppressive activity of MDSC in uterus from pregnant mice, but the expression of chemokine receptor or

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

integrins was not altered. Despite MDSC recruitment to uterus was not altered, it was observed a lower MDSC accumulation as well as an increase of MDSC apoptosis, contributing to an elevated abortion rate in knockout mice [73].

Regarding Zika virus, there are few studies showing the presence of MDSC on women blood and during pregnancy, and considering the facts, it will be very important to know any relationship of their presence with congenital syndrome, as observed in 2016, Brazil [82, 83]. A study with 10 non-pregnant women with Zika infection showed that frequencies of circulating MDSC did not change over time [84]. Another study with pregnant monkeys infected with Zika virus showed that an imbalance on blood frequencies of MDSC and activated CD8 T cells in the acute phase may lead to poor outcome to the fetus. Adding to that, the high frequency of MDSC on placenta from pregnant monkeys showed a positive effect on pregnancy outcome, even more if a drug antiviral treatment was used [85].

Furthermore, it is worth to note that immune signature, sometimes is the key factor to explain some diseases progressions. Despite Dengue viruses is more related to signals and symptoms with Zika virus infection [86, 87], some similarities with hepatitis C virus (HCV) were also noted, and mechanisms of immune evasion have been described, as inhibition of interferon pathway, allowing virus life cycle for a long-term period, up to 100 days [88, 89]. To note, ZIKV infection is also classified as an immune-mediated viral disease, like Dengue and other viruses [86, 87, 90]. Disease progression in HCV patients to chronic infection has been associated to an increase of MDSC phenotype in peripheral blood mediated by viral proteins [38]. Wang et al., 2017 examined Japanese encephalitis virus (JEV) infection leading acute encephalopathy depending on suppression of adaptive immune response, especially T follicular helper cells, mediated by enhanced MDSC populations, such as an involvement of MDSC on splenic B cells reduction, and in lower levels of total IgM JEV-specific neutralizing antibodies in mice models [39]. Burrack et al., also suggests that MDSC has an important suppressive T cells activity and may contribute to reduce the immune-mediated disease during Chikungunya infection [90].

Otherwise, the immunosuppressive activity triggered by RNA viruses, MDSC has been associated with metabolic regulation of immunopathology induced by DNA viruses, like hepatitis B virus (HBV) [91]. Pallett et al., 2015 showed that frequencies of MDSC on liver from HBV patients without liver damage, monitored by levels of liver transaminase enzymes, were higher in comparison with patients with liver damage, showing a protective effect for patients with immune-mediated viral disease, as hepatitis B [91].

In the new coronavirus pandemic (COVID-19), the MDSC have been reported to play an important role in the early phase of symptoms, increasing their frequency on blood in the first days of signals and symptoms, and it was related to poor outcome in severe acute respiratory syndrome in hospitalized patients. Pregnancy is a risk factor for COVID-19 severity, given the Brazilian high mortality rate of 12.7% in June 2020 withing pregnant, which may be associated with the change of the immunity [92–94].

Although few studies involving MDSC frequencies on blood during Zika infection were published yet, those cell type needs to be investigated, even though in animal models for medical science breakthroughs. The technique to characterize this cell phenotype is simpler than to characterize regulatory T cells, once the procedure does not require intracellular staining [95].

If those MDSC are crucial to maintaining a healthy pregnancy, any adverse effects, as Zika virus infection could trigger an imbalance between MDSC and T cells. This dysfunction may induce a deactivation of functional MDSC on blood and placenta with failure to attempt to eliminate viral infection. In addition, T cell function during ZIKV infection is known to be delayed throughout interferences

### *Cell Interaction - Molecular and Immunological Basis for Disease Management*

#### **Figure 2.**

*Myeloid-derived suppressor cell (MDSC) activation and regulation triggered by normal pregnancy and by Zika virus infection. Summary of MDSC functionality during normal pregnancy (A) and during acute phase of Zika virus infection (B) as suggested by others into an innate immunity dysregulation observed in abnormal pregnancies on monkeys [35, 37, 38, 73, 77–81, 85]. Hormone and cytokines produced in normal pregnancy induce an equilibrium in peripheral blood maintaining frequency of MDSC elevated (1.A), as well as levels of IL-10 and TGF-beta. Meanwhile, circulating levels of T cell frequencies are reduced and controlled. In placenta, Hofbauer cells (macrophages) are responsible for immune surveillance also intermediating the cross-talking between fetus-maternal interface, with equilibrium of MDSC and T cells to maintain a healthy pregnancy. In abnormal pregnancy, also suggestive for Zika virus infection during pregnancy of non-human primates, the equilibrium is broken. Once ZIKV is circulating, there is a reduction of MDSC frequency (B), compromising pregnancy immunosuppression, with elevation of activated T cells, attempting to virus elimination. In the placental parenchyma, MDSC has a reduction in their frequency. This scenario also suggests an immune dysfunction in fetus-maternal environment, diminishing functional macrophages (Hofbauer cells), which are infected by virus. All events together can induce several poor outcomes (abortion, neurological disorders). Black arrows filled with white color represent the frequency of cells (up = high, down = low). Grey arrows represent levels of cytokines (up = high, down = low). Red dashed arrows represent the direction of function/induction events that have been known during Zika infection during pregnancy. Figure was created using Biorender software (https://www.biorender.com).*

on interferon pathway, as described above. Then, this scenario may contribute to immune evasion of ZIKV, in which viral replication on maternal-fetal environment is unavoidable, inducing poor outcomes during pregnancy: fetal death, congenital syndrome, abortion, neurological disorders, etc. (**Figure 2**).
