**1. Introduction**

Toll-like receptors (TLRs) are one of the most studied groups of pattern-recognition receptors (PRRs), which recognize pathogen-associated molecular patterns and damageassociated molecular patterns. TLRs provide protection against both external and internal threats by initiating a pro-inflammatory response as well as activating and guiding the adaptive immune system to mount effector responses that will eliminate and/or ameliorate the problem [1]. Dysfunction or dysregulation of TLR responses can have dire consequences for the host, such as increased susceptibility to infection or excessive life-threatening inflammation. Thus, understanding how these receptors operate holds grea<sup>t</sup> potential for guiding preventive measures as well as the development of better treatments and new drugs for infectious and autoimmune diseases, cancer, and cardiovascular disease [2]. Through the years, some TLRs have received more attention than others, often due to the availability of easily accessible models and their convenience for study, but with technological and

**Citation:** Nilsen, K.E.; Skjesol, A.; Frengen Kojen, J.; Espevik, T.; Stenvik, J.; Yurchenko, M. TIRAP/Mal Positively Regulates TLR8-Mediated Signaling via IRF5 in Human Cells. *Biomedicines* **2022**, *10*, 1476. https://doi.org/10.3390/ biomedicines10071476

Academic Editor: Krisztina Nikovics

Received: 13 May 2022 Accepted: 17 June 2022 Published: 22 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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scientific advances, this gap is closing. TLR8 provides a classic example of a less-studied TLR as it is non-functional in rodents and, thus, application of murine models/KOs is limited [3].

TLR8 is expressed by several immune cells, in particular monocytes, macrophages, myeloid dendritic cells, and neutrophils [4–7]. Localized at the endosomes, TLR8 recognizes ribonuclease T2 degradation products of single-stranded RNA (ssRNA) of various origin: viral, bacterial, protozoan, and (possibly human) endogenous RNA [5,8–12]. With this repertoire of potential ligands, TLR8 might be relevant for the defense against a broad range of infections, as well as for driving autoimmune diseases [3,6,11]. TLR8 ligand-binding in the endosomal lumen induces conformational changes that result in the dimerization of the cytosolic TIR domains. This allows myeloid differentiation primary response gene 88 (MyD88) to bind, followed by the recruitment of interleukin-1 receptor-associated kinase 4 (IRAK4) and interleukin-1 receptor-associated kinase 1 (IRAK1), resulting in the formation of the active Myddosome complex. The signal is transduced via TNF-receptor-associated factor 6 (TRAF6) and transforming growth-factor-β-activated kinase 1 (TAK1), which activates downstream mitogen-activated protein kinase (MAPK) cascades and inhibitor of nuclear-factor kappa B kinase subunit beta (IKKβ), culminating in the activation of transcription factors such as nuclear-factor kappa B (NF-κB) and activator-protein 1 (AP-1) [1]. In human primary macrophages, TLR8-activated TAK1 also signals via IKKβ to induce the nuclear translocation of interferon regulatory factor 5 (IRF5), which is critical for the expression of interferon β (*IFNβ*) and interleukin-12 subunit alpha (*IL-12A*) genes [13].

The phosphoinositide 3-kinase (PI3K)-Akt serine/threonine kinase pathway is wellknown to be involved in the regulation of metabolism and survival, and its dysregulation is closely linked to tumor development [14]. It has also been implicated in regulating TLRmediated responses, although the reports are divergent, with evidence of both pro- and anti-inflammatory effects [15]. Aksoy et al., found that in human monocyte-derived DCs, PI3K negatively regulated the expression of *IFNβ* in TRIF-dependent signaling downstream of TLR3 and TLR4 [16], whereas Guiducci et al., found PI3K–Akt necessary for nuclear translocation of IRF7 and expression of TLR7 and TLR9-induced type I IFNs in human plasmacytoid predendritic cells [17]. These studies highlight the importance of taking cell-type specific differences into account when investigating the role of PI3K–Akt in TLR signaling.

TLR2 and TLR4 require a Toll-internleukin-1-receptor (TIR) domain containing adaptor protein/MyD88 adaptor-like (TIRAP) to attract MyD88 to the signaling complex. For some time, the restricted role of TIRAP for these plasma-membrane-localized TLRs seemed apparent, given the phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-binding motif in the N-terminal domain of TIRAP, which attracts this adaptor protein to the plasma membrane where PI(4,5)P2 is abundant [18,19]. However, Bonham et al., have shown that TIRAP is also capable of binding other phosphoinositides, PI(3)P and PI(3,5)P2, on endosomal membranes, and mediates signaling from endosomal TLR7 and TLR9 in murine cells [20]. Recently, some evidence of TIRAP involvement in signaling from TLR7 and TLR9 in human cells was provided by Leszczynska et al., and Zyzak et al. [21,22]. In both studies, TIRAP is suggested to regulate *IFNβ* expression by regulating ERK1/2 (MAPK3/MAPK1) activation. Whether TIRAP could contribute to signaling from TLR8, the third member of the TLR7 subgroup of TLRs, has not ye<sup>t</sup> been explored.

Here we show that TIRAP is regulating the expression and secretion of TLR8-induced IFNβ and IL-12A cytokines in human primary monocytes and monocyte-derived macrophages (MDMs). We propose that TIRAP is recruited to the activated Myddosome, from where it connects to Akt activation, contributing to the nuclear translocation of IRF5 and subsequent expression of *IFNβ* and *IL-12A* genes. In addition, TIRAP can enhance TLR8 signaling via the TAK1-pathway, thus modulating the expression of other cytokines such as TNF.

### **2. Materials and Methods**

### *2.1. Cells and Reagents*

Human buffy coats and serum were from the blood bank at St. Olavs Hospital (Trondheim, Norway), with approval by the Regional Committee for Medical and Health Research Ethics (REC) in Central Norway (no. 2009/2245). Primary human monocytes were isolated from the buffy coat by adherence, as previously described [23]. Monocytes were maintained in RPMI1640 (Sigma, Merck, Darmstadt, Germany), supplemented with 30% of pooled human serum. MDMs (used in the *TIRAP* silencing experiments) were obtained by differentiating cells for 12 days in RPMI1640 with 10% human serum and 20 ng/mL rhM-CSF (#216-MC-025, R&D Systems, Minneapolis, MN, USA). Ultrapure K12 LPS from *E. coli*, thiazoloquinoline compound CL075, and synthetic diacylated lipoprotein FSL-1 (Pam2CGDPKHPKSF) were from InvivoGen (San Diego, CA, USA). For stimulation of the primary cells, LPS and FSL-1 were used at concentration 100 ng/mL, CL075—2 μg/mL. IRAK4 inhibitor PF-06426779 (Merck, Darmstadt, Germany), selective allosteric pan-Akt inhibitors MK-2206 (#1032350-13-2, Axon Medchem, Groningen, Netherlands), Miransertib) and ATP-competitive pan-Akt inhibitor Capivasertib ((#1313881-70-7 and #1143532-39-1, MedChemExpress, Sollentuna, Sweden) were diluted in DMSO at concentration 5 mM and stored at −80 ◦C; working solutions were prepared in cell-culture media immediately before use. Preparation of THP-1 *TIRAP* KO cells using LentiCRISPRv2 plasmid [24] is described in supplementary materials and methods (available online).
