**3.1 From immature to mature cDCs in viral infection**

Immature DCs are considered the sentinels of the immune response. These cells are distributed in practically all the body where they have the capacity of interact with the invading virus. They carry out the function against viral infection by different mechanisms. They can be infected by viruses or they can respond to molecules produced and secreted by other virus infected cells. When they are infected, DCs can respond in various ways, firstly, DCs have different receptors distributed on cell surface, cytoplasm, and specialized endosomes. TLRs and C-type lectins receptors (CLRs) are present in cell surface and some TLRs in endosomes, while retinoic acid-inducible gene (RIG), melanoma differentiation-associated protein 5 (MDA5) and nucleotide-binding oligomerization domain 2 (NOD2) are only present in cytosol [20–22]. TLRs have N-terminal ectodomains (ECDs) which recognize molecules of viruses. This ECDs are constructed by a tandem motif of leucine-rich repeats (LRRs) and forms a horseshoe structures [23]. Binding of TLRs with their ligand depends on these structures [24]. However, diverse receptors respond to an extensive repertoire of viral PAMPs. These viral PAMPs can be glycoproteins present on the viral external surface, viral genome, or replication intermediates formed during viral replication [25].

Depending on the activated receptor, DCs can produce proinflammatory cytokines or IFN. During maturation process DCs interact with the antigen and upregulate MHC-II to present antigen to naïve CD4+ T cells. In addition, DCs produce diverse surface molecules such as CCR7 which is necessary in trafficking into lymphatic nodes and CD40, CD80, and CD86 which are co-stimulatory surface factors that enable them to activate T naïve cell to initiate the adaptive immune responses [26, 27].

#### **3.2 Differential PRR activation on dendritic cells**

DCs is the main cell used to establish an effective immune response. At present, four subsets with different functions have been identify in human. Each subset of DC has different markers and a functional distinction that enable them to participate in different states to orchestrate an antiviral immune response. Each type of DC expresses different receptors that can be membrane-associated molecules or free in the cytoplasm. Activation of these receptors ends in different cytokine-proinflammatory

production and interferon. Depending on cytokine produced, naïve CD4+ T cells is differentiated into T helper effector cell [14].

Myeloid DCs, called classical or conventional DCs (cDCs) detect viral proteins through expression of membrane surface receptors such TLR-4 and DC-specific intercellular adhesion molecule 3 (ICAM3)-grabbing non-integrin (DC-SIGN) (see **Figure 2**) [28]. DC-SIGN support the initial immune response between T cells and DCs, but when DC-SIGN have contact with viral glycoproteins results in activation of signal transduction pathways than cause modulation of immune responses [29]. The signaling pathway triggered by DC-SIGN recruits Ras and the subsequent phosphorylation of the kinase RAF1 which is mediated by p21-activated kinases (PAKs) and Src Kinases. The activation of RAF1 induces phosphorylation of nuclear factor (NF)-κB increasing the transcriptional activation from IL-18, IL-10 and IL-12 promoter [29, 30].

The association of viral proteins through concave surface of TLR4-ECD induces two different pathways [31]. Myeloid differentiation primary response 88 (MyD88)-Dependent Pathway initiates with the recruitment of MyD88 adapter and subsequent activation of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Then TRAF6 activates the NF-κB essential modulator (NEMO), which is the regulatory subunit IKK complex and activates NF-κB causing its translocation to the nucleus, where induces gene expression such as IL-6 and IL-12 [21]. MyD88-Independent pathway recruits TIR-domain-containing adapter-inducing interferon-β (TRIF) [32]. TRIF activates TRAF3 and finally induce interferon regulatory transcription factor (IRF-3) activation and the subsequent IFN-β expression [21].

In addition to membrane surface receptors cDCs also have endosomal TLRs such as TLR-3 and TLR-7/TLR-8 which sense dsRNA and ssRNA respectively. Each receptor has a specific signaling pathways [14]. TLR-3 sense viral dsRNA through its largely uniform and flat horseshoe structure of TLR-ECD [33]. TLR3 has the same MyD88-Independent pathway with the activation of TRAF3 and subsequent IFN-β expression [32]. Viral ssRNA are sense by TLR-7 and TLR-8, these receptors activate MyD88 pathway with the recruitment of TRAF6 and TRAF3. Finally, activation of IRF-3 and IRF-7 induces IFN-β and IFN-α expression respectively (see **Figure 2A**) [21, 34].

In addition to DC-SIGN and TLRs, the viral genome can be exposed in the cytoplasm during the replicative processes or during direct penetration into the cell. NOD2 and RNA helicases such melanoma differentiation-associated protein 5 (MDA5) and RIG-1 detect dsRNA in the cytoplasm [35]. Interferon promoter

#### **Figure 2.**

*Signaling pathway and cytokines production of DCs during viral infection. (A) Myeloid DCs and (B) Plasmacytoid DCs. Description in the text (figure created by Muñoz-Carrillo* et al*., with BioRender.com).*

stimulator-1 (IPS-1) interacts with MDA5, RIG-1 and NOD2 *via* caspase activation and recruitment (CARD) domain. IPS-1 localizes in mitochondria and interacts with TRAF3. TRAF family member associated NF-κB activator (TANK) is recruited from TAF3 and interacts with TANK Binding Kinase 1 (TBK1) and the kinase IKKε [36–38]. Finally, TBK1 and IKKε interact *via* their C termini with NFκB activating kinase (NAK)-associated protein 1 (NAP1) [39]. This signaling pathway activates NFκB, IRF-3 and IRF-7 to express IL-12, IFN-β and IFN-α [38, 39].

On the other hand, pDCs not express DC-SIGN but express CD4 that can sense glycoproteins of viruses as human immunodeficiency virus (HIV). The viruses can enter through direct fusion with the cell membrane or through receptor-mediated endocytosis and activates different signaling pathways (see **Figure 2B**) [40, 41]. The endosomal receptors TLR-7 and TLR-9 are selectively express in pDCs and sense RNA or DNA respectively. This engage activates downstream signaling pathway [42]. TLR-9 and TLR-7 activates IRF-3 and IRF-7 like in cDCs signaling with final IFN-β and IFN-α expression respectively [43]. TLR-9 signaling pathways include the recruitment of Interleukin-1 receptor-associated kinase 4 (IRAK4) through its death domain. Activated IRAK4 interacts with IRAK2. This complex associates with TRAF6 to final activation and nucleus translocation of NF-κB and leads TNF-α and IL-6 production [17, 44, 45]. pDCs can also be infected by direct penetration of virus and the viral genome can be sense by RIG-1, MDA5 and NOD2. The signaling in the pDCs is through IPS-1 pathways as the same way that on cDCs [20, 22]. This pathway activates NFκB, IRF-3 and IRF-7 to express IL-12, IFN-β and IFN-α respectively [38, 39].

Other subsets of DCs are the LCs and Interstitial DCs (IDCs), these kinds of DCs are commonly the first DCs that have contact with some virus [46]. LCs are localized in mucosal stratified squamous epithelium and skin epidermis. LCs express different CLR: CD207 or Langerin. Moreover, LC has a low expression of TLR4 and expression of TLR-3, −7 and − 8 [14, 47]. LCs activated finally express IL-8, IL-6, TNF-α [48]. On the other hand, the IDCs are localized in the epidermis and express similar receptors that cDCs like DC-SIGN and TLR-3, -4, -7 and -8 and have similar signaling pathways [14].

Activation of the antiviral response generated by immune system depends largely on the activation of dendritic cells. Each subtype of this family of antigenpresenting cells have an important role by processing viral antigens that trigger different signaling pathways through their distinct receptors. The consequence of this signaling pathway results in the expression of various cytokines involved in the activation of immune cells. For this reason, a better knowledge about how different immune cells subtypes can induce distinct pathways is required for a better vision of whole antiviral response.
