Toll-like receptors (TLRs) are a family of conserved pattern-recognition receptors (PRRs) that detect and bind to evolutionarily conserved molecular motifs in pathogen-associated molecular patterns (PAMPs) of microbes or damage-associated molecular patterns (DAMPs) of injured tissues to induce innate and adaptive immune response (1–3). Other classes of PRRs include Nod-like receptors (NLRs), RIG-I-like receptors (RLRs), AIM2-like receptors (ALRs), C-type lectin receptors (CLRs), and intracellular DNA and RNA sensors such as cyclic GMP-AMP synthase (cGAS) (4, 5). Obviously, TLRs are expressed in innate immune cells such as macrophages, neutrophils, dendritic cells, natural killer cells, mast cells, eosinophils, basophils as well as adaptive immune cells, including B cells and specific T cells. They are also found in and on non-immune cells such as fibroblasts, epithelial and endothelial cells (6–8). TLRs are type I transmembrane proteins consisting of an extracellular domain (ECD) with leucine-rich repeats (LRRs) responsible for the detection of PAMPs and DAMPs, a transmembrane region, and an intracytoplasmic toll/interleukin 1 (IL-1) receptor (TIR) domain necessary for the initiation of the downstream signaling pathways (9) ( Figure 1 ). LRRs exist in different protein combinations depending on the type of receptor and the corresponding ligand recognition and signal transduction (10). This high variety of LRRs is attributable to its consensus sequence motif of L(X₂)LXL(X₂)NXL(X₂)L(X₇)L(X₂), where X can be any amino acid. Upon interaction of distinct PAMPs or DAMPs with the corresponding LRR, the extracellular domains of the respective TLRs form homo- or heterodimers with co-receptors or accessory molecules, which is essential for the activation of various signaling pathways and thus for an appropriate immune response (11). The LRR carboxy-terminal domain, characterized by the consensus motif CXC(X₂₃)C(X₁₇)C, separates the LRR region from the transmembrane region in TLRs (12). The TIR domain exhibits high homology to that of the IL-1 receptor family (13). Three highly conserved regions in TIR domains have been identified that mediate the protein-protein interactions between TLRs and adaptor proteins necessary for signal transduction. The various TLRs use different sets of adaptor proteins to determine signal transduction, with TLR4 being the only known TLR that uses all TIR domain-containing adaptor proteins, including the myeloid differentiation factor 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), also known as MyD88-adaptor like (Mal), TIR domain-containing adaptor inducing interferon beta (TRIF), and TRIF-related adaptor protein (TRAM) (14, 15).

In 1985, Anderson et al. originally identified Toll as a gene product essential for the embryonic dorsoventral axis formation in the fruit fly Drosophila melanogaster (16), and 11 years later, its critical antifungal function in this species was described (17). Just one year later, in 1997, the discovery of the mammalian homologue of the toll receptor (now designated TLR4), which plays a major role in the innate immune response by inducing the expression of pro-inflammatory genes, revolutionized our knowledge of the immune system and triggered an irresistible research on PRRs (18). To date, a total of 10 and 12 functional TLRs have been identified in humans and mice, respectively, with TLR1 - TLR9 being conserved in both species. A retroviral insertion into the TLR10 gene in mice impaired its functionality, and the human genome lost TLR11, TLR12, and TLR13 (19). Based on their cellular localization and respective PAMP ligands, human TLRs are classified into cell surface TLRs and intracellular TLRs ( Figure 2 ). TLR2/TLR1 and TLR2/TLR6 heterodimers, TLR4 and TLR5 homodimers, and the orphan TLR10 (incomplete understanding of dimerization) are expressed on the cell surface and primarily recognize microbial membrane components such as lipoteichoic acid (LTA) (TLR2), lipopeptides (LP) (TLR1, 2, 6) and lipopolysaccharide (LPS) (TLR4, 10) as well as flagellin (TLR5, 10) (20, 21). In addition, TLR2 detects zymosan glycan in fungi and like TLR4 glycophosphatidylinositol (GPI) anchors in protists. In contrast, human TLR3, TLR7, TLR8, and TLR9 homodimers are exclusively found in intracellular components such as lysosomes, endosomes, endolysosomes, and the endoplasmic reticulum, where they mainly target microbial nucleic acids (9, 22) ( Figure 3 ).

One of the best characterized PAMPs to date is the endotoxin LPS, which is derived from the outer membrane of Gram-negative bacteria. The general structure of LPS is divided into lipid A, a core oligosaccharide, and an O side chain, with lipid A being the major PAMP of LPS (23, 24). LPS stimulation of human cells triggers a series of interactions involving the soluble LPS binding protein (LBP), the soluble glycosylphosphatidylinositol-anchored protein cluster of differentiation 14 (CD14), the soluble myeloid differentiation factor 2 (MD-2), and TLR4 (25, 26). LBP extracts LPS from the outer membrane of Gram-negative bacteria and transports it to CD14, which then transfers LPS to the TLR4/MD-2 receptor complex (14, 27). Interestingly, LPS can bind to MD-2 even in the absence of TLR4, which does not seem to be possible the other way around, as there is only evidence that TLR4 can enhance the binding of LPS to MD-2 (28, 29). Upon LPS recognition, the extracellular domains of TLR4 dimerize and adaptor proteins recruit through interactions with the TIR domains of TLR4. Initial recruitment of the adaptor proteins MyD88 and TRIAP induces the MyD88-dependent signaling pathway, which triggers an early activation of the nuclear factor kappa B (NF-κB) and the mitogen-activated protein kinases (MAPK) (30, 31). Sequential binding of the adaptor proteins TRIF and TRAM and subsequent dynamin-driven endosomal translocation of TLR4 initiates the MyD88-independent pathway, also known as the TRIF-dependent pathway, which culminates in the late-phase activation of NF-κB and the IFN-inducing transcription factor IFN regulatory factor 3 (IRF3). The MyD88-dependent pathway leads to the production of pro-inflammatory cytokines. In contrast, the TRIF-dependent pathway additionally results in the secretion of type I interferons (IFN) (32). The signal transduction of the MyD88-dependent and TRIF-dependent pathway is described in detail in the following sections and is graphically displayed in Figure 2 .

The MyD88-dependent pathway is characterized by the formation of the Myddosome consisting of MyD88, TRIAP, IRAK4, IRAK1, and IRAK2. MyD88 recruits IL-1 receptor-associated kinase-4 (IRAK4) through homotypic interaction of their death domains (14). IRAK4 is a member of the IRAK family, which shares a death domain and a kinase domain (33). IRAK4 knock-in mutation studies in mice revealed that the kinase activity of IRAK4 is crucial for TLR signaling (34, 35). Of note, IRAK4 also activates NF-κB and MAPK (36). IRAK4 sequentially engages and activates IRAK1 and IRAK2, which are then autophosphorylated at multiple sites (37), leading to the binding of tumor necrosis factor receptor (TNFR)-associated factor-6 (TRAF6) (38). IRAK1 activates the E3 ubiquitin ligase TRAF6, which together with the E2 ubiquitin-conjugating enzymes Ubc13 and Uev1A, induces the synthesis of Lys63 (K63)-linked polyubiquitin to degrade both TRAF6 and IRAK1 and to recruit the transforming growth factor beta (TGF-β)-activated kinase 1 (TAK1) and the adaptor molecules TAK-1 binding protein 2 and 3 (TAB2, TAB3) (11). Subsequent phosphorylation of TAK1 simultaneously induces the IκB kinase (IKK)-NF-κB and MAPK signaling cascades. The IKK complex consists of two catalytic subunits, IKKα and IKKβ, and the regulatory subunit NF-κB essential modulator (NEMO), also called IKKγ. Upon its association with TAK1 through ubiquitin chains, the IKK complex is phosphorylated, leading to the activation of IKKβ (9, 19). Following phosphorylation and proteasomal degradation of the NF-κB inhibitory protein, IκBα releases NF-κB. Released NF-κB then translocates into the nucleus to initiate transcription of pro-inflammatory genes (39). Activated TAK1 simultaneously phosphorylates the MAPK kinases (MMK) 3/6, 4/7, and 1/2, thereby initiating the activation of p38, Jun N-terminal kinases (JNKs), and extracellular signal−regulated protein kinase 1/2 (ERK1/2). Once activated, they phosphorylate the cyclic adenosine monophosphate (cAMP)-responsive element-binding protein (CREB) and activator protein 1 (AP-1) transcription factors, which consist of a heterodimer of c-Fos and c-Jun subunits. The final interaction of CREB and AP-1 with NF-κB facilitates the transcription of pro-inflammatory genes (9, 40).

The TRIF-dependent pathway is characterized by the recruitment of TRAM and TRIF to form the so-called Triffosome (41). TRIF associates with TRAF6 and TRAF3. Activated TRAF6 recruits the kinase receptor-interacting protein 1 (RIP1), which subsequently engages the TAK1 and IKK complex, leading to the activation of NF-κB and MAPKs. Activated TRAF3 recruits the IKK-related kinase TANK-binding kinase 1 (TBK1) and the inhibitor of NF-κB kinase (IKKi), which in turn phosphorylates and activates IRF3, ultimately leading to the induction of IFN (42, 43).

Importantly, once activated, TLR4 is the only member of the TLR family that induces both the MyD88-dependent and TRIF-dependent signaling pathways. TLR2/TLR1, TLR2/TLR6, TLR5, TLR7, TLR8, and TLR9 initiate the MyD88-dependent pathway, whereas TLR3 induces the TRIF-dependent pathway (9, 11). Of note, upon Myddosome formation in response to TLR7, TLR8, or TLR9 activation, IRF7 binds and is directly activated by IRAK1 and IKKα. Like IRF3, IRF7 leads to the induction of IFN (44, 45). We already have a quite clear understanding of the ligand recognition, dimerization, signaling, and biological functions of human TLR1-9, but not of the orphan TLR10. While we know that TLR10 is localized on the cell surface, its ligands, homo- or heterodimerization, TIR domains and adaptor molecules, signal transduction and biological function are still unclear (21).

Endothelial cells (ECs) form the innermost layer of blood vessels, which include arteries, veins, and capillaries, and separate the circulating blood from the surrounding tissues (46) ( Figure 4 ). Since the blood pressure is much higher in the arteries than in veins, arteries require more layers of smooth muscle cells than veins, making them the thickest blood vessels. Capillaries consist of just one single layer of ECs and no layers of smooth muscle cells because they do not have to withstand any pressure (47).

Due to their unique position, they are the first cells exposed to circulating substances in the blood, under healthy and pathological conditions, and thus act as gatekeepers of vascular health and function (48). Under physiological conditions, the endothelium perform several functions to maintain organ homeostasis, including vasoregulation, selective vascular permeability, and provision of an anti-coagulant environment (49). Upon recognition of PAMPs and DAMPs by PRRs, including TLR1-10, NOD1, and RIG-1, ECs secrete various cytokines, chemokines, growth factors, and adhesion molecules to facilitate vasodilation, promote neutrophil trafficking to the endothelium, activate coagulation pathways, and increase vascular permeability (48, 50–52) ( Table 1 ). Of note, studies have highlighted that TLR2, TLR4, and TLR9 are the main drivers of the endothelial immune response (50, 59, 77, 78). In addition to that, by analyzing the baseline mRNA transcription levels of TLR1-10 in different endothelial cell lines, including human umbilical vein endothelial cells (HUVECs), human coronary artery endothelial cells (HCAECs), human microvascular endothelial cells (HMVECs) derived from the brain, liver, and lung, Khakpour et al. clearly demonstrated that TLR4 is the most highly expressed of all TLRs, suggesting that TLR4 is the central PRR in the acute and chronic endothelial immune response (50).

TLR4 signaling is a complex, highly dynamic, and tightly regulated network of two distinct pathways that initiate the innate immune response. Persistent TLR4 signaling is responsible for chronic and acute inflammatory disorders, including sepsis (133), atherosclerosis (134, 135), rheumatoid arthritis (136), acute and chronic lung injury (137), sickle cell disease (63, 110, 138), neurodegenerative diseases (139, 140), and cancer (141, 142). For instance, sickle cell disease is a chronic inflammatory condition with hemolysis, vaso-occlusion, and ischemia-reperfusion due to the heme-induced MD-2/TLR4 activation leading to the production of pro-inflammatory mediators and a persistent activation of leukocytes, platelets, and endothelial cells (63, 110, 111, 138). Therefore, modulation of the TLR4 signaling pathway is a promising strategy to specifically target these pathologies. Common approaches used to study TLR4 signaling are primarily based on genetic manipulation through gain or loss-of-function mutations of the TLR4 or treatment with the bacterial endotoxin LPS. However, these strategies are often associated with the generation of irreversible phenotypes in the target cells or unintended cytotoxicity and signaling crosstalk due to off-target or pleiotropic effects. Furthermore, ligands are often unable to penetrate complex tissues, spheroids, or organoids, resulting in surface activation only. On top of that, the use of reagents is associated with complex operational design, high costs, and sources or errors (isolation impurities, batch variations, pipetting errors, instability upon solvation, etc.) (143–145). Here, optogenetics offers an alternative strategy to control and monitor cellular signaling in an unprecedented spatiotemporally precise, dose-dependent, and non-invasive manner ( Figure 10 ). It is based on utilizing light-sensitive protein domains of microbial or plant photoreceptors, integrated into effector proteins, to direct them with light stimuli. Hence, light induction allows activation, inactivation, stabilization, destabilization, or localization of signaling pathways depending on the protein type and setup (146–148). Initial optogenetic applications used naturally derived photosensitive opsins to investigate and control neuronal activity and later to study brain circuits. This allowed to replace conventional strategies, most of which were highly invasive, slow in kinetics, and imprecise in targeting specific neurons (149–152). Since then, optogenetics has revolutionized the study of cell biological processes, including signaling pathways, protein movement, or metabolic processes, and was even voted for the Method of the Year 2010 (153, 154). The present available repertoire of light-sensitive domains allows for the formation of protein complexes in response to blue (155–157), red (158, 159), or green (160) light. An important system involves the light oxygen voltage (LOV) domain isolated from the yellow-green algae Vaucheria frigida. This xanthophyte contains two distinct aureochromes, aureochrome 1 (AUREO1) and aureochrome 2 (AUREO2), each consisting of a LOV domain and a basic leucine zipper (bZIP) domain. AUREO1 controls blue light-induced cell branching, whereas AUREO2 mediates the development of a sex organ (161, 162). LOV domains are a subfamily of the Per-ARNT-Sim (PAS) family and shear a common PAS domain fold comprising of a five-stranded antiparallel β-sheet and four α-helices (163). The LOV domain derived from the AUREO1 of Vaucheria frigida (VfAU1-LOV), noncovalently binds a flavin chromophore that, upon blue light (λmax ≈ 470 nm) absorption, induces a photochemical reaction that leads to the formation of a covalent adduct between the conserved cysteine and the flavin ring. By fusing this blue light-sensing protein domain to the TLR4 and stably incorporating it into endothelial and pancreatic adenocarcinoma cells, Stierschneider et al. developed two physiologically relevant in vitro cell culture models in which the TLR4 can be turned on with blue light (470 nm) and turned off in the dark. These newly established optogenetic endothelial and pancreatic adenocarcinoma cell lines allow TLR4-specific studies of the underlying molecular and regulatory mechanisms in inflammation and cancer as well as high content screening for compounds that block TLR4 signaling with spatiotemporal precision (148, 164).

In ECs, activation of TLR4 by LPS ultimately leads to the release of cytokines, chemokines, adhesion molecules, coagulation and permeability factors, that are necessary for an immediate immune response to invading pathogens and tissue injury (12, 165). However, their systemic secretion is a major driver of autoimmune, acute and chronic inflammatory diseases (19). Therefore, negative regulation of TLR4 signaling pathways is a promising strategy to specifically target these pathologies (166). The search for such modulation options requires cell culture models with fast and unambiguous TLR4 signaling. Compared to available standard cell culture models that rely on genetic manipulation of TLR4 or the treatment with agonists such as LPS, optogenetic cell lines with light-inducible TLR4 provide these requirements. Hence, they are predestined for receptor-specific fundamental studies as well as for high content screening of biological active agents that negatively interfere with the TLR4 signaling pathway in inflammation.

CW: Funding acquisition, Project administration, Supervision, Writing – review & editing. AS: Conceptualization, Visualization, Writing – original draft.