TLRs and Other Molecules Signaling Crosstalk in Diseases

Abstract

Inflammatory diseases affect human health and the quality of life, causing heavy medical burdens in our society. Multiple pathogen-related molecular patterns, risk-related molecular patterns, and inflammatory cytokines exist in the inflammatory environment; these molecules activate immune cells and trigger inflammatory responses through pattern recognition receptors and cytokine receptors. Inflammatory molecules can activate immune cells alone or together through signaling crosstalk. For example, macrophages pretreated with interferon γ enhance Toll-like receptor 4 signal-induced gene expression through epigenetic remodeling. However, there are multiple forms of interactions between inflammatory molecules, including synergistic effects and antagonistic effects. At present, the forms of crosstalk between inflammatory molecules and TLRs that participate in immune cell activation and inflammatory disease progression and their detailed mechanisms are not fully discovered yet. In this chapter, we will enumerate the interaction between different immune molecules and TLRs and discuss how the interactions affect the process of inflammatory disease development and progression.

1. Introduction

During infection and sterile inflammatory conditions, the host’s immune response is triggered by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors (PRRs). Therefore, PRRs are critical during infection and noninfection-associated inflammatory diseases. There are four major families of PRRs involved in this process: Toll-like receptors (TLRs), RIG-like receptors (RLRs), NOD-like receptors (NLRs), and C-type lectin receptors (CLRs). These PRRs have overlapping ligand specificities and converge on common downstream signaling pathways, such as the NF-κB pathway [1, 2]. TLRs are membrane-bound receptors, which can travel between cell surface membranes and intracellular compartments, including endosomes, endolysosomes, phagosomes, and phagolysosomes [2, 3]. TLR1, 2, 4, 5, 6, and TLR10 are located in the cell surface membrane, while TLR3, 7, 8, 9, 11, 12, and 13 are located in the intracellular compartments [2, 4, 5, 6]. TLRs recognize a wide range of PAMPs, including bacterial and viral components; RLRs are cytoplasmic receptors that primarily detect viral RNA, while NLRs detect various intracellular PAMPs and DAMPs. CLRs are expressed on the surface of immune cells and recognize carbohydrates in pathogens [2, 7, 8, 9].

Upon activation, PRRs initiate signaling cascades that activate transcription factors, such as nuclear factor-kappa B (NF-κB), thereby inducing the production of proinflammatory cytokines and other immune mediators [2]. This coordinated immune response helps to eliminate the invading pathogens and restore tissue homeostasis. While there are numerous examples of positive and synergistic interactions between different PRRs and inflammatory molecules, it is also essential for the host to have mechanisms in place to prevent excessive immune activation and inflammation. Notably, PRRs can also exhibit negative regulation of each other, especially in the context of different pathogen infections. For instance, prior viral infection has been shown to increase susceptibility to subsequent bacterial infection [10, 11]. To maintain immune balance, various mechanisms come into play, including upregulation of inhibitory pathways, induction of anti-inflammatory mediators, and internalization and degradation of receptors. Additionally, PRRs and inflammatory molecules can directly antagonize each other at the signaling level. These regulatory mechanisms collectively contribute to preventing excessive immune responses and maintaining immune homeostasis [12]. In this chapter, we summarized the positive and synergistic interactions between TLRs and certain molecules, but also reviewed the negative regulatory or antagonistic effects of signaling crosstalk between TLRs and certain molecules. These signaling crosstalks are involved in and contribute to various pathogenic conditions. The understanding of the mechanisms of various paradigms of signaling crosstalks between TLRs and other molecules would benefit to develop new therapeutic strategies for disease control.

2. Signaling crosstalk between TLRs and other molecules in diseases

2.1 IFN signaling training to augment TLR response

Interferon γ (IFN-γ) is a very important cytokine for host defense against pathogen invasion and tumorigenesis, with the ability to modulate the immunological status in both infected cells and tumor cells [13, 14, 15]. IFN-γ is also involved in inflammatory diseases, where it activates inflammatory immune cells, especially monocytes and macrophages. IFN-γ combined with lipopolysaccharide (LPS) activate IFN-γ receptor- and TLR4-mediated signaling crosstalk to polarize macrophages toward an ‘M1-like’ state, characterized by increased proinflammatory responses and resistance to anti-inflammatory factors [16, 17]. In addition, cells pretreated with IFN-γ for 24 hours (hrs) or 48 hrs are very sensitive to TLRs signaling for inflammatory gene expression [18, 19]. In vivo, IFN-γ pretreatment increases LPS and TLR9 ligand CpG-induced responses and improves the capability of host cells to control pathogen infection [20]. Type I IFN (IFN-1) signaling modulates monocyte fate from tumor-associated macrophages to active monocytes, which can activate CD8⁺ T cells for tumor immunity [21]. Similar to IFN-γ, IFN-1 can also train monocytes/macrophages, but only under a concentration higher than 10 ng/ml in vitro [22]. Monocytes/macrophages primed with high concentration of IFN-1 are highly sensitive to TLR ligands, such as LPS (TLR4 ligand), ORN8L (TLR8 ligand), Pam3CSK4 (TLR2 ligand) and Poly (I:C) (TLR3 ligand), thereby inducing massive inflammatory response and cytokine storm responsible for tissue damage and dysfunction during viral infection, such as coronavirus disease 2019 (COVID-19), influenza pneumonia, etc. [22, 23]. IFN-1 signaling is the hallmark of and contributes to the development of autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) [24, 25, 26]. Signaling crosstalk between IFN-1 and self-DAMPs-mediated TLR activation underlies the pathogenesis and severity of inflammatory disorders [27].

Through the mechanism study of the model of IFN and TLR4 signaling crosstalk, we now know that both IFN-1 and IFN-γ-mediated training seem to be not through modulating TLR signaling pathway activities but to be through epigenetic chromatin remodeling of monocyte/macrophages to decrease the threshold of the cell sensitivity to TLR ligands for inflammatory gene expression [19, 22]. Epigenetic remodeling is a sophisticated process. How does IFN signaling initiates the epigenetic remodeling process and changes the chromatin accessibility of inflammatory genes is still under heavy study. In addition, IFN signaling also selectively alters macrophage metabolic pathways by suppressing TLR4-mediated gene activation [18, 22]. While IFN-γ abrogates the LPS-induced IL-10 negative feedback loop by completely suppressing IL-10 expression [18, 19], IFN-1 does not suppress LPS-induced IL-10 expression [22]. But, whether IFN-1 affects IL-10 auto-loop to augment inflammatory gene expression induced by TLRs is unclear yet. These findings suggest that similar but different mechanisms underpin IFN-1 and IFN-γ training.

2.2 IFN-1, TNF, and TLR signaling crosstalk

Tumor Necrosis Factor (TNF) plays an important role in host defense and inflammation and contributes to various inflammatory diseases and tumors. However, pretreatment of the human macrophages with TNF for a minimum of 12 h suppresses TLR4-mediated inflammatory gene expression and diminishes protection from LPS-induced cell death [28, 29]. This phenomenon is referred to as TNF cross-tolerance, which resembles classical endotoxin tolerance (which will be discussed in the other part of this chapter). The TNF cross-tolerance is mediated by the inactivation of glycogen synthase kinase 3β (GSK-3β) and the subsequent accumulation of NF-κB inhibitor alpha (IκBα), which prevents the translocation of active NF-κB to the nucleus and the induction of proinflammatory cytokines. TNF-induced tolerance may contribute to the regulation of sterile inflammation in conditions such as surgery, ischemia/reperfusion, and acute coronary syndrome, as well as to the control of chronic inflammation in autoimmune diseases [30, 31, 32]. A subgroup of genes substantially expressed by TNF treatment can be superinduced by secondary LPS challenge, referred to as the synergistic effect of TNF and TLR signaling crosstalk [29]. In the context of the TNF and TLR4 signaling crosstalk example, we believe that, in a substantial signaling crosstalk situation, synergistic effect and tolerizing effect co-exist for different subgroups of gene expression. The synergistic genes induced by TNF and TLR4 signaling crosstalk are TNF late response genes, which are epigenetic primed by TNF and are mainly involved in IFN signaling and lipid-metabolic processes [29]. Individuals with inflammatory diseases such as sepsis and SLE are exposed to TNF and TLR ligands. Strikingly, monocytes from septic patients mimic what happens during TNF and TLR4 signaling crosstalk for gene expression patterns [29]. However, the gene expression patterns in SLE differ from those regulated by the combination of TNF plus TLR4. IFN-1 s drive the pathogenesis of SLE and contribute to training immunity [22, 33]. Thus, the altered gene expression profile in SLE is modified by IFN-1 s in the presence of TNF and TLR signaling. Indeed, IFN-1 s block TNF-mediated tolerization of TLR4-induced inflammatory gene expression through epigenetic regulatory mechanisms [29]. In addition, the efficacy of TNF-induced feedback inhibitory mechanisms may be compromised by other cytokines like IFN-γ or genetic variants in genes involved in tolerance regulation, for example, TNF alpha-induced protein 3 (TNFAIP3, encodes A20) [34, 35].

2.3 Heme and TLR signaling crosstalk activates PANoptosis and pathology

During infection or inflammatory disorders, cell death occurs, and multiple DAMPs are released in the process of inflammatory cell death. The presence of PAMPs and DAMPs mimics infection and cell lytic diseases. The signaling crosstalk between PAMPs and DAMPs may affect the progress of the disease [36]. Heme is one important DAMP that is released into the bloodstream when red blood cells are damaged or lysed during infectious and inflammatory diseases [37, 38]. Heme can activate TLR4 signaling and inflammatory responses [38, 39]. Recently, it has been demonstrated that heme can coordinate with TLR2, TLR4, and TLR7 signaling, but not TLR3 signaling to activate a late inflammatory cell death-PANoptosis [36], which is a type of cell death caused by the combination of either apoptosis, pyroptosis or necroptosis [40]. The late PANoptosis induced by heme and PAMPs might be attributed to the need for time for the activated cells to produce enough amounts of critical protein interferon regulatory factor 1 (IRF1) and NLR family pyrin domain containing 12 (NLRP12). Although how does the combination of heme with certain TLRs initiates the signaling cascades to induce the late PANoptosis is unclear, heme and TLRs costimulation induced IRF1 and the generation of reactive oxygen species (ROS), which were responsible for NLRP12 expression; NLRP12 thereby interacted with and activated Caspase-8, RIPK3, and NRLP3 for apoptosis, necroptosis and pyroptosis [36]. Injection of phenylhydrazine (PHZ) to mice can induce hemolysis, which increases the level of heme in serum. The combination of PHZ and a nonlethal dose of LPS-induced kidney damage in mice, while the deficiency of Nlrp12 could significantly protect mice from lethality and kidney damage [36]. High levels of free heme exist in various pathological conditions [38, 41]. The signaling crosstalk between heme and TLRs is likely implicated in numerous inflammatory and infectious diseases. Targeting the critical signaling nodes such as NLRP12 will be beneficial to the control of disease progression.

2.4 Chemokines participate in regulating cytosolic TLR signaling

The natural ligands of cytosolic TLR3/7/8/9 are nucleic acids (NAs), which are derived from microbial DNA/RNA or extracellular NAs that are released by dying or netting cells. These NAs are negatively charged molecules, which can form nanoparticles with cationic proteins via charge:charge interactions, such as LL37 and HMGB1. The formed nanoparticles facilitate the internalization of NAs into the endosome for cytosolic TLR recognition [42, 43, 44, 45, 46, 47]. Thus, some of the cationic proteins are involved in and amplify cytosolic TLR responses to trigger and to exacerbate inflammatory diseases [48, 49, 50, 51]. Chemokine (C-X-C Motif) ligand 4 (CXCL4), CXCL10, CXCL12, CXCL14, and C-C motif chemokine ligand 5 (CCL5) are such cationic proteins that can form nanoparticles with TLR9 NA ligand CpG to increase the internalization of CpG in plasmacytoid dendritic cells (pDCs), thereby superinducing type I IFN, independently of their known chemokine receptors [27, 52]. CXCL4, for example, is the most studied chemokine that physically interacts with CpG to form nanoparticles. The nanoparticles can be taken up by pDCs through clathrin-mediated endocytosis. Besides, the nanoparticles formed between CXCL4 and CpG mediate transcriptional and epigenetic changes in pDCs that are responsible for the superinduction of IFN-1 [27].

CXCL4, activating a C-X-C motif chemokine receptor 3 (CXCR3)-independent inflammatory response in human monocytes, also forms nanoparticles with TLR8 oligoribonucleotides ligand (ORN8L) and promotes the internalization of ORN8L into the endosome of human monocytes [51]. The signals induced by both CXCL4 and ORN8L lead to a massive TBK1/IKKε activation, which then activates IRF5 and IRF3 to induce strong inflammatory gene and IFN-1 expression. The signaling crosstalk between CXCL4 and TLR8 also triggers NLRP3 inflammasome activation, IL-1β secretion, and pyroptosis. In addition, both CXCL4 and TLR8 signaling themselves can induce epigenetic remodeling; the combination of CXCL4 and TLR8 signaling mounts the epigenetic remodeling in human monocytes, which increases the accessibilities of NF-κB, AP-1, and IRFs to the opened chromatins of inflammatory genes [51, 53]. In addition, the signaling crosstalk between CXCL4 and TLR8 activates RIPK3 to modulate inflammatory gene expression and IFN response through activating PI3K-AKT serine/threonine kinase (AKT)-X-box binding protein 1 (XBP1)/NFE2 Like BZIP transcription factor 2 (NRF2) axis and through regulating signal transducer and activator of transcription 1 (STAT1) activation [54]. Both TLR8 and CXCL4 are highly expressed and associated with various inflammatory diseases including SSc and rheumatoid arthritis (RA) [33, 55]. Thus, the signaling crosstalk between CXCL4 and TLR8 ought to contribute to inflammatory disorders. Except for CXCL4, NET-associated RNA in complex with cationic LL37 amplifies TLR8 response in peripheral blood mononuclear cells (PBMCs) [56, 57]. With these examples and the evidence of charge:charge interactions between cationic chemokines and NAs, it is reasonable to deduce that chemokine CXCL10, CXCL12, CXCL14, and CCL5 might likewise contribute to TLR8 response.

2.5 TLR7/9 and B cell receptor-Signaling crosstalk in autoimmune diseases

B cells are lymphocytes that play an important role in adaptive immunity by producing antibodies against pathogens and tumor cells. In autoimmune diseases such as SLE, aberrant activation of B cells by sensing DNA and RNA self-antigens produces autoantibodies that cause inflammation, tissue damage, and dysfunction. B cell activation relies on B cell receptor (BCR)-mediated signaling, co-stimulatory molecules-mediated signaling, and cytokines. In autoimmune conditions, the ligands of TLR7 and TLR9 are endogenous NAs, which are more likely to form complexes with cationic protein or antibodies, facilitating the internalization of NAs for TLR7 and TLR9 activation. It has been shown that such BCR as rheumatoid factor (RF) AM14 BCR specifically can bind with low affinity to IgG2a to facilitate the internalization of its bound endogenous or synthetic, highly purified NA [58]. Similar to myeloid cells, B cells also experience tolerance after TLR signaling [59]. Nevertheless, simultaneous activation of BCR signaling and TLR signaling overcome TLR tolerized B cells [59]. TLR7 and TLR9 can activate B cells and cooperate with BCR signaling to induce substantial high amounts of cytokine expression and RNA-associated and DNA-associated autoantibody production, respectively [58]. TLR9 activation of atypical memory B cells from hepatitis C virus (HCV) infected patients triggers TNF secretion and rheumatoid factor-type IgMs; in addition, the TLR9 activated atypical memory B cells promote type 1 effector T cell activation and reduce regulatory T cells proliferation, which are associated with HCV infection caused with autoimmune disorder cryoglobulinemia vasculitis [60]. In lupus-susceptible mice, TLR7 is responsible for the generation of antibodies to RNA-containing antigens [58, 61]. In these mice, however, loss of TLR9 exacerbates autoimmune diseases with increased serum IgG and IFN-1 [58, 61]. Further, it has been demonstrated that TLR7 and TLR9 in B cells play pathogenic and protective roles in the balance of the autoimmune disease progression, respectively [62]. The opposite function of TLR7 and TLR9 in autoimmune diseases might be explained by the fact that TLR9 can directly suppress TLR7 signaling through direct or indirect physical interactions between the TLRs [63, 64].

2.6 TLRs interactions affect their signaling in immune response

Heme and TLRs signaling crosstalk to induce PANoptosis; the signaling crosstalk also exists in the combination of different TLRs to activate PANoptosis. Intriguingly, the combinations of TLR2 signaling with TLR7 signaling fail to induce PANoptosis [36]. The reason for this is still elusive but could basically exclude the contribution of IFN-1 signaling in the system, as TLR7 is professionally responsible for IFN-1 expression [22]. However, TLR7 and TLR3 or TLR4 signaling crosstalk could induce PANoptosis [36]. The concurrent activation of various TLRs, such as TLR2 and TLR4 sensing bacterial wall components, along with TLR3 and TLR7 detecting bacterial RNAs, closely mimics the effects of a live bacterial infection. This resemblance suggests that PANoptosis occurs during infectious diseases and contributes to host defense or severe inflammation in conditions like sepsis.

TLR8 and TLR2 signaling crosstalk affects cell activation. During Borrelia burgdorferi infection, TLR8 and TLR2 cooperatively activate NF-κB-mediated cytokine production in human monocytes, whereas TLR8 alone is solely responsible for the transcription of IFN-β. The activation of TLR2, TLR4, or TLR5 suppresses TLR8-IRF5 signaling and IFN-β expression partly through regulating Interleukin 1 Receptor Associated Kinase 1 (IRAK1) [65, 66, 67, 68]. During Mycobacterium tuberculosis (Mtb) infection, however, TLR4 and TLR8 form heterodimers in endosomes to increase the activation of TLR8, which thereby activates Th1 responses against Mtb [69]. The loss and instability of TLR8 contribute to TLR7 activation and spontaneous autoimmune disorders [70, 71]. TLR8 can inhibit TLR7 and TLR9 functions, and TLR9 can suppress TLR7 function in B cells, but not vice versa [63, 71, 72]. These inhibitory effects seem not to rely on direct competition for downstream signaling proteins but not on the direct or indirect physical interactions between TLR8 and TLR7/9 (probably by forming heterodimers) [63, 73].

TLR tolerance refers to a modified responsiveness (unresponsiveness or low responsiveness) of cells following repeated or chronic activation of TLRs. The phenomenon of LPS-induced tolerance, involving TLR4 and endotoxin, was the first to be described and was observed primarily in monocytes, macrophages, and dendritic cells. LPS tolerance drives comprehensive transcriptional reprogramming, shifting the inflammatory response toward the expression of anti-inflammatory and pro-resolution factors while maintaining innate immune protection. Further studies revealed gene-specific regulatory mechanisms and epigenetic changes involved in TLR-tolerance development [74]. It is worth noticing that TLR tolerance is a reversible phenomenon, and these changes can be reversed over time or in response to competing signals [74, 75, 76].

Heterologous tolerance, or cross-tolerance, which refers to the reduced responsiveness to TLR stimulation after initial exposure to a specific ligand, may not be as effective as auto tolerance induced by repeated stimulation of the same TLR type. For instance, cells initially treated with synthetic lipopeptide TLR2 agonist MALP-2 fail to respond to subsequent stimulation with TLR4 ligand LPS [77]. This MALP-2-induced cross-tolerance to LPS is not attributed to the decrease in TLR4 surface expression. Instead, it is believed that TLR2-induced tolerance specifically affects the signaling of TLR4 and TLR7 ligands, while leaving TLR3 and TLR5 signaling unaffected. This effect is achieved through the inhibition of paracrine type I interferon amplification, leading to the suppression of IL-12 production [78]. Similarly, treatment with a low concentration (1 μg/ml) of Pam3CSK4 induced a state of tolerance, leading to reduced production of TNF-α and IL-6 upon secondary stimulation with LPS [79]. On the other hand, TLR9 triggers the production of IL-12 family members in response to intact Gram-positive bacteria. However, in the absence of TLR2 signaling, this response becomes exaggerated in microglia [80]. Interestingly, when cells are treated with a combination of TLR2, TLR4, and TLR9 ligands, auto tolerance is induced for each TLR toward these ligands, while cross-tolerance is specifically induced by lipoteichoic acid and LPS, but not by CpG (TLR9 ligand). Besides LPS, TLR7/8 agonist (R848) has also been shown to induce homologous and heterologous tolerance to various TLR ligands in macrophages [81]. These findings suggest that TLR cross-tolerance is mediated by distinct mechanisms depending on the specific ligands involved [82].

2.7 TLRs and NLRs signaling crosstalk

NOD-like receptors (NLRs) work distinctly from TLR that function as cytoplasmic receptors to recognize PAMPs and DAMPs. Most NLRs play important roles during inflammatory diseases by driving inflammation, while some NLRs serve as negative regulators of inflammation. The best-characterized members of the NOD subfamily are Nucleotide Binding Oligomerization Domain Containing 1 (NOD1) and NOD2, which detect distinct subunits of bacterial peptidoglycans. The activation of NOD1 was found to inhibit the activation of TLR1/2, leading to a decrease in the secretion of IL-6 and IL-10 and a reduction in the percentage of CD11b⁺ F4/80⁺ macrophages. These findings suggest that NOD1 exhibits antagonistic effects of TLR1/2 response in macrophages [83]. While there is evidence supporting a cooperative interaction between TLR2/4 and NOD2 in cytokine production [84, 85, 86], NOD2 has the ability to inhibit the induction of inflammatory cytokines mediated by TLR2/4 and promote immune tolerance and homeostasis [87, 88, 89]. In mice, intact NOD2 signaling inhibits the NF-κB activation driven by TLR2, primarily through the inhibition of c-Rel [89].

NLRX1 is a unique member of the NLR family which is localized in the mitochondria. Xia et al. demonstrated that NLRX1 acts as a negative regulator of TLR-mediated NF-κB signaling [90]. Upon LPS stimulation, NLRX1 is rapidly ubiquitinated, disassociates from TRAF6, and then binds to the IKK complex, leading to inhibition of IKKα and IKKβ phosphorylation and NF-κB activation [90, 91]. The NLRX1-dependent suppression has also been tested with a number of NLRX1 binding molecules, e.g. punicic acid (PUA) in both in vitro as well as an in vivo DSS model of colitis [92]. Similarly, additional NLRs, including NLRC3, NLRC5, NLRC6, and NLRP12 have been shown to play a role in regulating TLR-induced canonical and noncanonical NF-κB activation and MAPK signaling pathways [93, 94, 95, 96, 97, 98, 99].

2.8 TLR and CLR crosstalk

C-type lectin receptors (CLRs) are intracellular receptors expressed on antigen-presenting cells (APCs) and function as PAMP recognition and antigen-uptaking, playing a crucial role in the immune system. When activated, CLRs recognize specific carbohydrate structures on pathogens and initiate immune responses. In the context of APCs, such as dendritic cells (DCs), triggering the dendritic cell immunoreceptor (DCIR), a specific CLR, does not affect the upregulation of neither TLR4- nor TLR8-mediated co-stimulatory molecules CD80 and CD86. Interestingly, DCIR activation inhibits the production of IL-12 and TNF-α during TLR8 activation. This suggests that DCIR can modulate specific cytokine responses induced by TLR8 signaling. However, the production of cytokines induced by TLR2, TLR3, and TLR4 is not affected by DCIR triggering [100]. Mannose Receptor, an important CLR that interacts with a number of products generated by a variety of helminths, is able to downregulate TLR4-mediated IL-12 production in DCs to favor Th2 cell responses. The engagement of macrophage galactose-type lectin (MGL) enhances TLR-induced IL-10 expression, which in turn promotes the generation and activation of regulatory T cells. This regulatory T cell response contributes to immune tolerance and the control of immune reactions, helping to maintain a balanced immune system.

2.9 Crosstalk between complement receptor and TLRs

The complement system and TLRs are two crucial components of the innate defense system that rapidly respond to infection. In recent years, there has been increasing evidence indicating the existence of crosstalk between the complement system and TLR signaling pathways [101]. The crosstalk between C5a receptor (C5aR) and TLR4 signaling leads to the downregulation of primarily proinflammatory mediators, such as IL12B, IL2RA, and jagged canonical notch ligand 1 (JAG1), and the upregulation of anti-inflammatory factors, including Sphingosine kinase 1 (SPHK1), adrenoceptor beta 2 (ADRB2), and four and a half LIM domains 2 (FHL2), in monocyte-derived dendritic cells (moDCs). This crosstalk is mediated, at least in part, by the transcription factors Forkhead Box O1 (FOXO1), FOXO3, and serum/glucocorticoid regulated kinase 1 (SGK1) [102, 103]. C5a-C5aR1 pathway activated by Porphyromonas gingivalis infection can induce ubiquitination and proteasomal degradation of the TLR2 adaptor myeloid differentiation primary response 88 (MyD88), leading to suppression of the antimicrobial effect of neutrophils [104]. While C5aR1-TLR2 crosstalk also dampens intracellular killing of P. gingivalis in macrophages with a mechanism that is different to what happens in neutrophils. C5aR1-TLR2 crosstalk induces massive Cyclic adenosine monophosphate (cAMP) production to activate protein kinase A (PKA), which inactivates GSK-3β and inhibits the expression of inducible nitric oxide synthase (iNOS). The consequence of this causes the reduction of nitric oxide-dependent killing of P. gingivalis in macrophage [105].

Complement receptor 3 (CR3; CD11b/CD18) plays a controversial role in regulating TLR response by either promoting TLR response through membrane-bound phosphatidylinositol-(4,5)-bisphosphate (PIP2) to recruit Mal/MyD88 for initiation of MyD88-dependent signaling or suppressing TLR response through activating Syk and promoting the degradation of MyD88 and TIR domain-containing adaptor molecule 1 (TRIF) via the E3 ubiquitin ligase Cbl-b [106, 107, 108]. Activation of C5aR1 and CR3 signaling by P. gingivalis selectively downregulates TLR2-induced IL-12 production through activation of the phosphoinositide 3-kinase (PI3K) and ERK1/2 pathways in macrophages. In addition, gC1qR, a complement receptor for C1q, also suppresses TLR-induced IL-12 but no other inflammatory genes in human monocytes [105]. The selective inhibition of IL-12 production by complement receptor causes less IFN-γ production, ultimately leading to enhanced survival of the pathogen during infection [106].

2.10 GPCR and TLR signaling crosstalk

Recent studies have shed light on the intricate interplay between G-protein coupled receptor (GPCR) and TLR signaling pathways, revealing a fascinating crosstalk that significantly influences immune responses and cellular functions. Protease activated receptors (PARs) are a family of GPCRs that mediate serine proteases triggered cellular effects. Activation of PAR2 by PAR2-AP reduced TLR3-mediated STAT1 activation and TLR3/IRF3-induced IFN-β expression [109]. However, using a global PAR1 deficient mouse model, it has been revealed that PAR1 promoted polyinosinic-polycytidylic acid (poly I:C)-triggered CXCL10 expression while suppressed CXCL1 induction [110]. In addition, PAR1 activation was found to suppress TLR4-mediated NF-κB activation in murine embryonic fibroblasts [111].

A2A adenosine receptor (A2AR), another member of the GPCR family, plays a critical role in a physiological immunosuppressive pathway. By utilizing A2AR-deficient mice, Lukashev et al. uncovered the role of A2AR in inhibiting NF-κB activity and the transcription of proinflammatory cytokines induced by TLR in vivo [112]. Chemokine receptors, belonging to the large family of GPCRs, play a crucial role in facilitating cell migration through binding to their specific chemokine ligands. These receptors can be categorized into four main subtypes: CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors. Among CCR8 has been implicated in the progression of various diseases such as sepsis, type I diabetes, and experimental autoimmune encephalomyelitis [113]. CCL1 per se, as a ligand of CCR8, can induce TNF-α and IL-6 production in macrophages; however, CCL1 also exerts a suppressive effect on LPS-mediated cytokine production [113].

2.11 Crosstalk between immunosuppressive mediators and TLRs

TLR signaling pathways were also being regulated via immunosuppressive mediators. IL-10, known for its potent inhibitory effects on innate immune cells, is the most effective regulator of TLR-induced inflammatory cytokine production [114, 115]. Some pathogens take advantage of the regulatory effects of IL-10 to dampen immune responses and evade host immune defenses. This manipulation allows the pathogens to establish infectious niches within the host, leading to immune evasion and disease progression. These studies offer mechanistic insights into the immunoregulation of IL-10. IL-10 distinctly affects LPS-induced IP-10 and IL-12B expression—by inhibiting type I interferon production and restraining RNA polymerase II recruitment [116, 117]. Conversely, IL-10-responsive microRNA, miR-146b, emerges through an IL-10-mediated loop, adeptly targeting various TLR4 signaling components [118]. It achieves this not only through direct inhibition of cytokine transcription but also by destabilizing the coding RNA [116, 117] and upregulation of miRNA by and IL-10-mediated STAT3-dependent loop [118]. Furthermore, it has been observed that IL-10 activates the PI3K-Akt-GSK signaling pathway, leading to the suppression of inflammatory gene expression [119]. These findings collectively highlight the multifaceted role of IL-10 in quelling immune responses by intervening at various points within TLR4 signaling pathways.

IL-4 and IL-13, both significant immunoregulatory cytokines, prompt alternative macrophage polarization. IL-4 exhibits inhibitory effects on IFN-β and IFN-responsive gene expression upon TLR7 and TLR9 stimulation in conventional dendritic cells (cDCs), achieved by curbing IFN-dependent and NF-κB-dependent signaling pathways [120]. These inhibitory actions of IL-4, along with the suppression of proinflammatory cytokine production, operate through the STAT6 pathway. Recent research emphasizes the critical role of IL-4-activated STAT6 transcription factor in directly repressing the LPS-induced inflammatory program of macrophages. This repression results in reduced lineage-determining transcription factors, p300, and RNA polymerase II binding, coupled with diminished enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. Notably, the repressed enhancers influenced by STAT6 significantly overlap with the NF-κB p65 cistrome, resulting in reduced responsiveness to LPS post-IL-4 stimulation. This cascade ultimately leads to decreased inflammasome activation, IL-1β production, and pyroptosis [121].

In addition to the interleukin signaling pathways that suppress TLR activation, two orphan receptors, SIGIRR and ST2, also play significant roles in modulating immune responses. SIGIRR, also known as TIR8, possesses a unique architecture with a single extracellular immunoglobulin domain, a transmembrane domain, a cytoplasmic TIR domain, and an unusually long tail [122, 123, 124]. It selectively inhibits NF-κB activation induced by TLRs and IL1R1, which are TIR domain-containing receptors. Interestingly, SIGIRR also modulates the TRIF-dependent pathway and inhibits TLR3 signaling. Its inhibitory mechanism involves blocking interactions between TRAM and TLR4, as well as TRIF and TRAM. ST2, or IL1RL1, is another orphan receptor with a cytoplasmic TIR domain. It inhibits NF-κB activation in response to IL-1R1 and TLR stimulation as well. ST2 interacts with Mal and MyD88, suggesting its inhibitory roles are specific to the MyD88-dependent pathway of TLRs. Thus, overexpression of ST2 specifically prevents NF-κB activation induced by TLR4, but not TLR3, which utilizes a TRIF-dependent downstream pathway. Notably, ST2 can form homodimers with TLR4, MyD88, and TRAM, while its interactions with Mal and TRIF disrupt Mal homodimerization and TRAM-TRIF interaction, respectively. Although the physiological relevance of ST2’s interaction with TRAM and TRIF may require further investigation, computational analysis predicts these interactions.

Transcription factor aryl hydrocarbon receptor (AhR) is a cytosolic sensor. AhR has been suggested as an immunosuppressive effector on various types of immune cells, playing a crucial role in modulating immune responses [125, 126]. Under TLR activation, AhR is induced in both macrophages [127] and DCs [128]. In AhR-deficient macrophages, the production of IL-6 and TNF-α in response to LPS was notably increased compared to wild-type (WT) cells. Interestingly, activation of AhR increased the expression of IL-1β but decreased the expression of IL-12A in TLR-activated MoDCs through a NF-κB RelB-dependent manner.

Tryptanthrin, a natural alkaloid, exhibits anti-inflammatory effects by modulating the immune response. In PMA-differentiated THP-1 cells, tryptanthrin reduces the phosphorylation of STAT1 in response to TLR3 ligand poly I:C. This results in the suppression of interferon-stimulated gene expression and the inhibition of IFN-β induction [129].

2.12 Crosstalk between nuclear receptor and TLRs

Ligand-activated nuclear receptor (NR) transcription factors, such as the glucocorticoid receptor (GR), peroxisome proliferator-activated receptor gamma (PPARγ), and liver X receptor (LXR), exhibit robust inhibitory effects on TLR-induced inflammatory gene expression. In a recent study, it was discovered that Nuclear Receptor Related 1 (NURR1) was found to decrease the production of TNF-α by interacting with NF-κB p65 and preventing its translocation during LPS stimulation. The interaction between NURR1 and NF-κB p65 contributes to the regulation of the TLR4 NF-κB signaling process [130]. Furthermore, treatment with NR ligands also inhibited the association of AP-1 and NF-κB subunits, repressing both basal and TLR-modulated HIV-1 replication in macrophages [131]. Crosstalk between GR and TLR has been well summarized in previous reviews briefly; while glucocorticoids have long been recognized as negative regulators of NF-κB and AP-1 transcriptional activity, they also play a broader role in modulating immune and inflammatory responses through a mechanism involving competition with the transcription factor IRF3 for binding to the Rel domain of the p65 subunit of NF-κB [132, 133, 134]. Similarly, PPAR agonists serve as potential therapeutic targets in neuroinflammation, CNS disorders, and cancer due to their ability to inhibit TLR-induced proinflammatory cytokine production in different cell types. They achieve anti-inflammatory effects by targeting MyD88, NF-κB, MAP kinase pathways, and IRF3, thereby offering a promising approach for managing these diseases and reducing inflammation-associated pathology [135, 136]. LXRs function as transcriptional regulators of lipid homeostasis and possess strong anti-inflammatory properties. Upon activation, LXRs effectively suppress signaling from TLRs 2, 4, and 9 to downstream NF-κB and MAPK. This inhibition is achieved through Abca1-dependent modifications in membrane lipid organization, which disrupt the recruitment of MyD88 and TRAF6, essential components of TLR signaling pathways [91, 137], leading to the suppression of proinflammatory cytokine production and promotion of cholesterol efflux, and preventing foam cell formation and plaque progression during atherosclerosis progression.

2.13 Lipid metabolism-TLR crosstalk: bridging immunity and metabolism

In addition to nuclear receptor LXR, lipid metabolism pathways have been implicated in the regulation of TLR responses. Oxidized PAPC (oxPAPC), derived from phospholipid PAPC and lipoproteins, exerts modulatory effects on TLR4-induced inflammatory responses through a GPCR. At low concentrations, oxPAPC acts as an antagonist to counteract TLR4-induced inflammation, while higher doses of oxPAPC enhance the proinflammatory response. The precise mechanism underlying the anti-inflammatory function of oxPAPC remains unclear, but it is known that oxPAPC inhibits NF-κB transcription factor activity by generating cAMP and by reducing TLR4 sensitivity to LPS through binding to CD14 and LPS binding protein (LBP) [138, 139]. However, it is worth noting that the inhibitory effects of OxPAPC on proinflammatory products, such as TNF-α, were specifically observed in the signaling pathways downstream of TLR2 and TLR4 but no other TLRs.

GPR120 serves as a receptor/sensor for omega-3 fatty acids. When stimulated by omega-3 or specific chemical agonists, GPR120 exerts broad anti-inflammatory effects in various immune cells, including monocytic RAW 264.7 cells and primary intraperitoneal macrophages. Activation of GPR120 by omega-3 fatty acids can inhibit TLR4 signaling, resulting in a decrease in the production of proinflammatory cytokines such as IL-6 and TNF-α [140]. Prostaglandin E2 (PGE2), a lipid mediator, has been found to suppress alveolar macrophages (AMs) immune responses by inhibiting signaling events downstream of PRRs. In a study on rat AMs, it was observed that while PGE2 did not reduce TLR4 mRNA levels, it decreased TLR4 protein levels [141], or through induction of IL-1R–associated kinase-M (IRAKM), which blocks the scavenger receptor-mediated phagocytosis and the TLR-dependent activation of TNF-a [142]. PGE2 also inhibits the production of IL-23 by monocytes stimulated with LPS. The inhibitory effects of PGE2 on IL-23 production were mediated through the cAMP signaling pathway, as the cAMP enhancer forskolin significantly reduced IL-23 and IL-12 production by monocytes [143]. However, in the case of DCs, the addition of PGE2 has been shown to enhance their migratory capacity in TLR ligands (poly(I:C) and/or R848) matured DCs, while maintaining the production of IL-12 by DCs when they encounter T cells [144]. These findings suggest that the effects of PGE2 on TLR signaling can vary depending on the cell type.

3. Conclusions

Signaling crosstalk occurs consistently in a sophisticated milieu and determines whether a host can successfully tackle an infection, tumorigenesis, and inflammatory response or not. TLRs play vital roles during the whole life of a host. The signaling crosstalk between TLRs and other molecules affect our bodies in tackling sophisticated situations. Such signaling crosstalks include synergistic effect, training/priming, antagonist effect, and tolerance. Mechanistically, these signaling crosstalks influence immune responses by manipulating the downstream signaling pathways and epigenetic status of immune cells. In this chapter, we enumerate multiple examples of signaling crosstalk between TLR and other molecules contributing to host defense or inflammatory diseases. There is still more evidence for signaling crosstalk between TLR and other molecules. In the natural environment, coinfection with viruses and bacteria is a prevalent occurrence, and it is probable that concurrent activation of both RIG-I-like receptors (RLRs), which serve as receptors for viral pathogens, and TLRs takes place. Negishi et al. found that virus-induced activation of cytosolic RLRs led to the selective suppression of transcription of the gene encoding the p40 subunit of interleukin 12 (Il12b) that was effectively induced by the activation of TLRs [12]. The transcription factor IRF3, activated by RLRs, prominently bound to the promoter region of Il12b and interfered with the assembly of a functional transcription-factor complex induced by TLR activation. TLR4 and CD40 costimulation synergistically increased the frequency of IL-10-producing but not proinflammatory cytokine-producing B cells at multiple sclerosis relapse [145]. Platelets-enriched serum suppresses TLR-induced inflammation and has been used to treat joint inflammation such as rheumatoid arthritis [146, 147, 148, 149]. However, the detailed mechanisms of these signaling crosstalks are not fully understood yet. Discovering and depicting the detailed mechanisms of TLR signaling crosstalk with other molecules are important for better understanding the sophisticated situation of disease and for helping to develop new therapeutic strategies to tackle the disease.

Conflict of interest

The authors declare no conflict of interest.

Appendices and nomenclature

A2AR	A2A adenosine receptor
ADRB2	adrenoceptor beta 2
AhR	aryl hydrocarbon receptor
AKT	AKT serine/threonine kinase
APCs	antigen-presenting cells
BCR	B cell receptor
C5aR	C5a receptor
cAMP	cyclic adenosine monophosphate
CCL	C-C Motif Chemokine Ligand
CLRs	C-type lectin receptors
COVID-19	coronavirus disease 2019
CR	complement receptor
CXCL	chemokine (C-X-C Motif) ligand
CXCR	C-X-C motif chemokine receptor
DAMPs	damage-associated molecular
DCIR	dendritic cell immunoreceptor
DCs	dendritic cells
FHL2	four and a half LIM domains 2
FOXO	Forkhead box O1
GPCR	G-protein coupled receptor
GR	glucocorticoid receptor
GSK-3β	glycogen synthase kinase
HCV	hepatitis C virus
Hrs	hours
IFN	interferon
iNOS	nitric oxide synthase
IRAK	interleukin 1 receptor associated kinase
IRAKM	IL-1R-associated kinase-M
IRF	interferon regulatory factor
IRGs	IFN-responsive genes
IκBα	NF-κB inhibitor alpha
JAG1	jagged canonical notch ligand 1
LBP	LPS binding protein
LPS	lipopolysaccharide
LXR	liver X receptor
MGL	macrophage galactose-type lectin
moDCs	monocyte-derived dendritic cells
Mtb	Mycobacterium tuberculosis
MyD88	myeloid differentiation primary response 88
NA	nucleic acid
NF-κB	nuclear factor-kappa B
Nlrp	NLR family pyrin domain containing
NLRs	NOD-like receptors
NOD	nucleotide binding oligomerization domain containing
NR	ligand-activated nuclear receptor
NRF2	NFE2 like BZIP transcription factor 2
NURR1	nuclear receptor related 1
ORN8L	TLR8 oligoribonucleotides ligand
oxPAPC	oxidized PAPC
PAMPs	pathogen-associated molecular patterns
PARs	protease activated receptors
PBMCs	peripheral blood mononuclear cells
pDCs	plasmacytoid dendritic cells
PGE2	prostaglandin E2
PHZ	phenylhydrazine
PI3K	phosphoinositide 3-kinase
PIP2	phosphatidylinositol-(4,5)-bisphosphate
PKA	protein kinase A
poly I:C	polyinosinic-polycytidylic acid
PPARγ	peroxisome proliferator-activated receptor gamma
PRRs	pattern recognition receptors
PUA	punicic acid
RA	rheumatoid arthritis
RF	rheumatoid factor
RLRs	RIG-like receptors
ROS	reactive oxygen species
SGK1	serum/glucocorticoid regulated kinase 1
SLE	systemic lupus erythematosus
SPHK1	sphingosine kinase 1
SSc	systemic sclerosis
STAT	signal transducer and activator of transcription
TLRs	toll-like receptors
TNF	tumor necrosis factor
TNFAIP3	TNF alpha-induced protein 3
TRIF	TIR domain-containing adaptor molecule 1
XBP1	X-box binding protein 1