Imperative role of adaptor proteins in macrophage toll-like receptor signaling pathways

Macrophages are indispensable cells that serve dual functions of maintaining tissue homeostasis and regulating inflammation, while also providing vital protection against pathogens [ 1 ]. The functional versatility and differentiation of macrophages are essential elements of their lineage diversification and adaptability [ 2 ]. With tissue-specific roles that are critical to overall tissue health, macrophages play an essential role in promoting the organism's well-being [ 1 ]. Macrophages possess a crucial attribute of plasticity, enabling them to transition from a quiescent M0 state into diverse activated states in response to dynamic environmental conditions [ 2 ]. According to the changes in the tissue environment, macrophages (activated) are polarized into two different subtypes: M1 macrophage and M2 macrophage [ 3 ]. Lipopolysaccharide (LPS) can stimulate classical activation of macrophages, while IL-4 can lead to alternative activation [ 4 ]. The foremost and extensively studied subtype is the classically activated macrophages, which function as effector cells in Th1 cellular immune responses [ 5 ]. Both M1 and M2 macrophages play pivotal roles in orchestrating inflammatory responses, with M1 macrophages primarily governing the pro-inflammatory response and M2 macrophages predominantly regulating the anti-inflammatory response [ 6 ]. Analogous to the color allotment of a chameleon, macrophage plasticity makes it difficult to assign a specific biochemical marker to each population or activity state [ 7 ]. Furthermore, the increasing body of evidence indicates that macrophage diversity may orchestrate tissue development and function, controlling organ functionality during development, homeostasis and ageing [ 8 ]. However, macrophage heterogeneity holds enormous potential for understanding diseases and can be leveraged as biomarkers or surrogate markers of protection after drug treatment or vaccination [ 7 ]. Two different classes of macrophages are speculated to have a similar set of genes required for protein synthesis, phagocytic activities, gene regulations and for basic macrophage functioning [ 9 ]. Anti-inflammatory reprogramming probably acts as a precursor in M1 to M2 macrophage transitions. M2 macrophages, being less harmful to microbes and vulnerable post-mitotic host cells like neurons, are commonly associated with anti-inflammatory or reparative roles [ 9 ]. The process of macrophage polarization is precisely regulated by critical signaling events [ 10 , 11 ]. Macrophages undergo classical activation in response to an injury or infection, which occurs due to microbial products or pro-inflammatory cytokines, including bacterial LPS, interferon-γ (IFN-γ), or tumor necrosis factor-α (TNF-α) [ 12 ]. Studies have shown the prominent involvement of transcription factor such as NF-κB that leads to the activation of various markers of inflammation [ 13 ]. Considering the markers for inflammatory diseases, receptors such as TLRs have been observed to be associated with various diseases and infectious conditions [ 14 ]. M1 macrophages, primarily involved in tissue injury and microbicidal activity are associated with markers namely CD86, CD40, MHCII [ 15 ]. On the other hand, M2 macrophages involved in anti-inflammatory, wound healing is recognized by the presence of CD163, CD36, CD206, CD209 and so on [ 15 , 16 ]. For their peculiar functions and plasticity, macrophages play both protective and pathogenic roles in a wide range of autoimmune, inflammatory as well as cancer diseases [ 17 ]. Adaptor proteins typically possess a modular architecture characterized by the inclusion of one or more dedicated domains, which facilitate their interaction with various other proteins [ 18 , 19 ]. These domains play a significant role in facilitating interactions with phosphorylated tyrosine residues found on the target proteins [ 18 ]. Adaptor molecules assume a pivotal role in diverse receptor-mediated signaling pathways, functioning as vital linkages that connect receptors with other molecules, thereby facilitating essential cellular communication [ 18 ]. Macrophages undergo classical activation upon encountering two essential signals; the initial signal involves the mandatory cytokine IFN-γ, which primes macrophages for activation, but by itself, does not trigger their activation [ 20 ]. The second signal encompasses either TNF or a stimulus that induces TNF production [ 21 ]. Although exogenous TNF can fulfill the role of the second signal, the biologically significant second signal predominantly arises from the ligation of Toll-like receptors (TLRs), leading to endogenous TNF production by the macrophage itself [ 21 ]. TLR2 and TLR4 are key players in recognizing specific molecular patterns from invading pathogens via PRRs and regulate the balance between Th1 and Th2 response, essential for host survival against major infectious diseases like tuberculosis, trypanosomiasis, malaria and filariasis [ 22 , 23 ]. So far, in the efforts to prevent and treat inflammatory diseases, diverse TLR antagonists/inhibitors have proven effective, progressing from preclinical testing to clinical trials [ 24 ]. Immunogenetic variability significantly influences a host's susceptibility or resistance to infectious diseases. Principal polymorphic sites within TLRs, predominantly single nucleotide polymorphisms (SNPs), include l602S (TLR1), R677W (TLR2), P554S (TLR3), D399G (TLR4), F616L (TLR5), S249P (TLR6), Q11L (TLR7), M1V (TLR8), G1174A (TLR9) and G1021T (TLR10) [ 25 ]. Studies demonstrate the anti-inflammatory potential of 3a-u, a synthesized compound from aryl 7-chloroquinolinyl hydrazone derivatives act as TLR4 antagonists on macrophage membranes, modulating the TLR4-NFkB pathway [ 26 ]. Consequently, the development of classically activated macrophages is triggered by the presence of IFN-γ and exposure to a microorganism or microbial component, such as LPS [ 27 , 28 ]. In the murine system, the distinct characteristic of classically activated macrophages is their prominent generation of nitric oxide (NO), enabling facile recognition [ 27 ]. Adaptor proteins are essential immunological molecules that do not possess inherent enzymatic and transcriptional activity [ 29 ]. The including cytokines, hormones and growth factors, involves the coordinated actions of multiple molecular components [ 29 ]. Notably, the activation of receptor tyrosine kinases, which plays a central role in these pathways, is triggered by ligand-induced oligomerization and subsequent autophosphorylation [ 29 ]. This event initiates a downstream signaling cascade that involves various kinases, transcription factors and adaptor molecules, leading to the regulation of diverse cellular processes and functions [ 29 ]. These mechanisms are well-documented in the scientific literature and are critical for understanding the complex interplay between extracellular signals and intracellular responses in health and disease [ 29 ]. A diverse range of origins and functions characterize macrophages, an immune cell population with significant heterogeneity [ 8 , 30 ]. Categorizing macrophages based on their activation state can be effectively accomplished through the concept of macrophage polarization [ 31 ]. In this review, we have summarized the macrophage adaptor proteins which play an imperative role in orchestrating the immune response by activating the Toll-like receptors signaling pathway ( Figure 1 & Table 1 ). The extensive list has been categorized into one major part which includes the adaptor molecules that facilitate the immune response along with the enigmatic role of some macrophage adaptors involved in both pro-inflammatory and anti-inflammatory roles in some cases, respectively. Adaptor proteins that act as facilitators mainly induce activation and stimulate the production of pro-inflammatory cytokines. Role of macrophage adaptor proteins in toll-like receptors signaling pathway. Role of macrophage Adaptor proteins in Toll-Like Receptors signaling pathways. Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein(TIRAP) has been found to function downstream of TLR4 signaling pathways [ 32 , 33 ]. TIRAP is a pivotal mediator of varied immune responses and functions as a key intracellular signaling molecule [ 34 ]. In relation to TLR-mediated innate immune signaling, TIRAP's function as an adaptor molecule has been extensively explored [ 35 ]. Since its discovery in 2001, early studies focused on TIRAP's role in coupling MyD88 with TLRs, which leads to the activation of MyD88-dependent TLR signaling [ 34 , 36 ]. Further studies revealed that TIRAP also transduces signaling events by interacting with non-TLR signaling mediators [ 34 ]. The broad range of intracellular signaling mediators with which TIRAP interacts underscores its central role in multiple immune responses [ 34 , 36 ]. Mice lacking TIRAP demonstrate normal responsiveness to TLR5, TLR7 and TLR9 ligands, as well as to IL-1 and IL-18 [ 37 ]. However, they exhibit deficiencies in cytokine production and activation of NF-κB and MAPKs in response to TLR4 ligand [ 37 ]. Additionally, these mice have impaired responses to ligands for TLR2, TLR1 and TLR6, suggesting that TIRAP plays a distinctive role in the signaling of various TLR members [ 37 ]. The specificity in downstream signaling pathways of individual TLRs is potentially due to the involvement of TIRAP [ 37 ]. Upon binding to their corresponding ligands (PAMP), TLRs prompt the recruitment of MyD88 to the intracellular domain of the receptor, apart from TLR2 and TLR4, for which MyD88 is indirectly recruited via TIRAP [ 38 , 39 ]. This recruitment process leads to the formation of a significant complex called the “myddosome” [ 26 ]. TIRAP is essential for recruiting MyD88 to TLR2 and TLR4 and is anchored to the membrane through a phosphatidylinositol-4,5-bisphoaphate-binding domain [ 40 , 41 ]. TIRAP's centralized role in profuse immunological responses can be elucidated by its interaction with numerous intracellular signaling mediators [ 34 ]. Several transcription factors such as NF-κB and AP-1 get activated as a result of downstream signaling events [ 38 , 42 , 43 ]. The pro-inflammatory cytokine production by immune cells and control of host cell survival mainly depends on TLR-mediated signaling pathway activation [ 34 ]. By undergoing this process, the cell is transformed into a state that aids in reducing the effects of infection [ 34 ]. While regulated activation of TLR is necessary for the host's defense mechanisms, excessive amplification of pro-inflammatory cytokines can have a harmful impact on the host's immune system [ 34 , 44 ]. Tyrosine phosphorylation of TIRAP in the TIR domain is pivotal for its activation [ 45 , 46 ]. In TLR 2/4 signaling, Y86, Y106 and Y159 and Y187 of TIRAP undergo phosphorylation by BTK and PKCδ, which are essential for activating downstream MAPKs [ 45 ]. The tyrosine phosphorylation at the Y86 site leads to conformational changes that draw TIRAP closer to the p38 MAPK kinase domain's active site and it also plays a vital role at the interface site [ 45 , 46 ]. The process gets reversed when the Y86 site is dephosphorylated [ 45 ]. The TIRAP intracellular signaling initiation is intricate and TIR domain is required for numerous protein-protein interactions [ 35 ]. Other interaction partners that have been reported for TIRAP include RAGE, PKCδ, CLIP170, Triad3A, SOCS1, c-JUN etc. [ 34 , 35 ]. The role of TIRAP of mediating inflammation has been reported in various studies. When Mal(-/-) mice (TIRAP KO) were infected with Bordetella pertussis, which causes infectious diseases of the respiratory system, exhibited higher bacterial load in the lungs and lower macrophage infiltration and survival. The pro-inflammatory cytokine production and bacterial killing ability of macrophages from Mal(-/-) mice was decreased [ 47 ]. Another study focusing on sepsis showed that blocking the interaction of TIRAP with PKCδ by dorzolamide in LPS-treated macrophages attenuates the expression of pro-inflammatory cytokines, mediated via suppression of p38 MAPK activation [ 48 ]. Disruption of TIRAP interaction with c-Jun by gefitinib was proposed as a potential treatment of sepsis patients. TIRAP forms complexes with c-Jun upon macrophage treatment with LPS, leading to the activation of AP-1 and increased pro-inflammatory cytokines, an effect that was abolished by gefitinib [ 49 ]. Toll-interleukin-1-receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF) is the principle adaptor molecule to produce IFN-β in direct response to dsRNA along with its analog poly(I:C), which are both recognized by TLR3 [ 50 ]. TLR3 utilizes TRIF to activate IRF3 via TBK1 and it also participates in the activation of the NF-κB pathway by recruiting RIP1 [ 40 ]. Additionally, TRIF has been found to facilitate apoptosis through RIP1 [ 40 ]. TRIF, also known as TICAM-1, plays an essential role in the dimerization of transcription factor IRF-3, late NF-κB activation and LPS-induced phosphorylation, further leading to immune response activation and IFN-β production [ 51 ]. IRF-3 activation mainly depends on the phosphorylation of some specific serine residues by IKKs, IKK-ϵ and TBK [ 52–55 ]. TRIF activates NF-κB by direct binding to TRAF6 which further recruits IKK complex [ 56 , 57 ]. TRIF requires TRAM while interacting with TLR4 but TLR3 signaling via TRIF occurs independently of TRAM [ 58 ]. TRIF generally has three main domains in mammals; one highly conserved TIR domain and two proline-rich domains on the N- and C-terminal regions [ 59 ]. Signal transduction involves a unique contribution from each domain [ 56 ]. IRF3 activation heavily relies on the presence of a binding site TRAF proteins located in the N-terminal domain [ 56 , 59 ]. NF-κB activation and apoptosis depends on RIP1 interaction motif (RHIM) present on C-terminal domain [ 60–62 ]. In mammals, TLR4, TLR5, TLR1/TLR2 and TLR2/TLR6 rely on bridging adaptor TIRAP to facilitate recruitment of MyD88[ 40 ]. However, TLR9, TLR11, TLR13, TLR7/TLR8 and TLR2/TLR10 directly recruit MyD88 [ 40 , 63 ]. TLR3 is the only TLR that does not recruit MyD88 as a signaling adaptor [ 40 , 63 ]. TLR3 recruits TRIF directly, but TRIF is indirectly recruited to TLR4 through the bridging adaptor TRAM [ 40 , 63–65 ]. The TIR domain is inherent to TRIF and is tasked with the duty of engaging with the TIR domain present within TLR3, in addition to the TRAM adaptor that acts as a bridge for TLR4 [ 66 , 67 ]. TLR4 serves as an antecedent for both the TRIF and MyD88 dependent pathways, as it is the only receptor that utilizes TRIF, TRAM, MyD88 and TIRAP [ 37 , 53 , 68–70 ]. Direct interaction of TLR3 and TRIF involves two tyrosine residues in the cytoplasmic domain of TLR3 upon phosphorylation by epidermal growth factors ErbB1 and BTK [ 71 , 72 ]. Simultaneously, MyD88 and TRIF-dependent pathways distinctly constitute TLR signaling which are activated through the usage of the adaptor [ 73 ]. CD14, a GPI-anchored protein, governs the internalization of TLR4 induced by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and consequently activates the TRIF-dependent pathway through the engagement of TRAM and TRIF adaptor proteins in early-endosomes after receptor endocytosis [ 74 ]. The first signaling cascade involves TIRAP and MyD88 adaptor proteins and is induced in the plasma membrane [ 74 ]. Studies reveal that macrophages from TRIF -/- mice have diminished expression of IFN-β which is accordant with results observed in TLR -/- mice [ 75 , 76 ]. Research finding of another study showed that polyguanine treatment of LPS-induced macrophages downregulates the expression of TLR4 and TRIF, decreases the pro-inflammatory cytokine production and M1 polarization, while increases M2 macrophage polarization [ 77 ]. Moreover, macrophage stimulation with oxidized low-density lipoprotein (ox-LDL) induces the inflammatory response via TRIF pathway, which upregulates ERK and SOCS1-STAT3-NF-κB signaling pathways and elevates the levels of IL-6 and TNF-α. Silencing of TRIF rescued this phenotype [ 78 ]. Toll-interleukin-1-receptor(TIR)-domain-containing adapter-inducing interferon-β (TRIF)-related adaptor molecule (TRAM) is the fourth essential TIR-domain-containing adaptor protein which actively participates in Toll receptor signaling [ 58 ]. TRIF is recruited by TRAM which is myristoylated to membranes and regulated by PKCϵ [ 40 ]. Two different signaling pathways are elucidated by TLR4 administered by the TIRAP-MyD88 and TRIF-TRAM pairs of adaptor proteins, which further evoke the pro-inflammatory cytokines production along with type-I IFNs [ 79 ]. TRAM leads to activation of IRF-3, IRF-7 and NF-κB signaling similar to TRIF [ 58 ]. TLR3 and TLR4 lead to the activation of these pathways [ 58 ]. Studies reveal the downstream functioning of TRIF in both TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas TRAM is restricted to TLR4 [ 58 ]. During innate immune responses to LPS, MyD88-independent pathway regulation occurs by both TRIF and TRAM through LPS-TLR4 signaling [ 58 ]. Evolutionary divergence of TLR3 and TLR4 from other TLRs is mainly involved in the activation of gene expression and enhanced immune responses by combined activation of NF-κB and IRF-3 [ 58 ]. TRAM, also known as TICAM-2, acts as a bridging adaptor between TRIF and TLR4 [ 80 ] and plays an essential role in the dimerization of transcription factor IRF-3, late NF-κB activation and LPS-induced phosphorylation, further leading to immune response activation and IFN-β production [ 51 ]. TIRAP and TRAM show similar structural features, but TRAM exhibits greater sequence homology with TRIF in its TIR domain [ 68 ]. TRAM possesses a cysteine residue in contrast to other TLR adaptors, at position 117 instead of the conserved proline [ 81 ]. TRAM requires this cysteine residue to form homodimers and for TRIF heterodimerization [ 59 , 81 ]. The major function of the TRAM/TRIF pathway is to enhance the expression of innate interferons transcriptionally, while the TIRAP/MyD88 pathway is implicated in the escalation of inflammatory cytokines like TNF-α [ 82 ]. Despite that, signals that rely on TRAM and TRIF have been observed to contribute to the expression of LPS-induced TNF-α [ 53 ]. When macrophages are stimulated with LPS, a deficiency in either TRAM or TRIF leads to a significant reduction in the amount of TNF-α that is secreted [ 53 , 74 ]. The proposed mechanism of TLR4 activation involves the induction of MyD88-dependent signaling at the plasma membrane followed by its internalization, which subsequently stimulates early endosomal MyD88-independent (TRIF-TRAM) signaling [ 83 ]. Myeloid differentiating factor 88 (MyD88) is a characteristic cytosolic adaptor molecule central to signaling in both innate and adaptive immune responses [ 84 , 85 ]. It is known to carry the signals downstream from the membrane receptors such as TLRs and IL-1 receptor families [ 32 ]. MyD88 is composed of three distinct domains viz. death domain (DD), an intermediate segment domain (INT) and Toll/Interleukin-1 receptor domain (TIR). The intermediate domain forms a bridge between the death and TIR domains [ 86 ]. The N-terminal DD consists of 54 to 109 aa, the INT spans from 110 to 155 aa and the TIR is longest from 155 to 296 aa [ 87 ]. MyD88 utilizes its C-terminal TIR domain to interact with the upstream receptor's TIR domain [ 88 ]. Death domain located at N-terminal is crucial for interaction with IRAK4 and for downstream signaling [ 89 ]. Studies have reported that the absence of INT is strongly linked with the dampening of MyD88 transduced signaling [ 90 ]. MyD88 is recruited differentially under the influence of different types of TLRs. TLR1/2, TLR2/6, TLR4 and TLR5 require the adaptor protein TIRAP which facilitates MyD88 recruitment to the receptor [ 40 , 63 ], while it is directly recruited to TLR7, TLR8 and TLR9 respectively [ 40 , 63–65 ]. TLR3 is an exception that does not recruit MyD88 as a signaling adaptor [ 40 , 63–65 ]. MyD88 is also reported to influence the activation of the signaling cascades involving transcription factors such as NF-κB, AP-1, IRF1, IRF5 and IRF7 [ 40 ]. The activation of different transcription factors is responsible for the production of various pro-inflammatory mediators [ 32 , 91 , 92 ]. Since MyD88 is observed to be majorly involved in TLR-mediated signaling, various studies have shown that its absence helps in abrogating the development of chronic inflammatory conditions [ 93 , 94 ]. In two distinct studies, Muzio et al. and Wesche et al. found that MyD88 is a central adaptor molecule to IL-1R signaling transduction and showed to facilitate signaling switch from IL-1R to activate NF-κB transcription factor [ 93 , 94 ]. Another study by Sun and Ding showed the involvement of MyD88 in transducing the signals from IFN-γ in macrophages and reported that MyD88 -/- condition decreased IFN-γ-inducible protein 10 (IP-10) and TNF-α production [ 95 ]. In MyD88 -/- macrophages, it has been shown that LPS-induced NF-κB signaling is impaired, while this effect was not evident in TRIF -/- macrophages [ 80 ]. In the hippocampi of SE mice, it was observed that inhibition or gene knockout of MyD88 resulted in the reduction of M1 MG/MΦ and gliosis while causing an increase in M2 MG/MΦ [ 96 ]. In another research using infectious models in MyD88 and TRIF KO mice, treated with TLR agonists, it was demonstrated that MyD88 pathway is important to induce TLR-mediated trained immunity in macrophages [ 97 ]. Macrophage specific MyD88 KO mice were protected against high-fat diet (HFD)-induced hepatic injury, lipid accumulation and fibrosis. The protective effect against liver damage is associated with decreased infiltration of macrophages in liver tissues and decreased inflammatory responses [ 98 ]. Atherosclerosis is another macrophage-mediated inflammatory disease. Investigations reported that lnc-MRGPRF-6:1 is upregulated in M1 macrophages of coronary atherosclerotic disease (CAD) patients and positively correlates with the pro-inflammatory cytokine production via TLR4-MyD88-MAPK signaling pathway [ 99 ]. Src kinase-interacting membrane protein (SCIMP) belongs to the family of pTRAP. pTRAP are kinase recruits and modulate pro-inflammatory response [ 100 ]. SCIMPs are generally composed of 150 residues, which include an extracellular domain, transmembrane domain which contains palmitoylation site, and a proline-rich cytoplasmic tail for scaffolding several signaling factors such as Lyn kinase, Grb2, CSK, SLP65/76 [ 101 ]. SCIMP has multiple phosphorylation sites (Y58, Y96 and Y120) at its cytoplasmic tail region for the recruitment of different effectors (Grb2, CSK, SLP65/76 ) to produce downstream signaling under LPS stimulation in macrophages [ 102 ]. More recently, Syk recruitment by SCIMP has been discovered, showing that TLR4 dependent Syk activation is mediated by SCIMP [ 103 ]. SCIMPs are generally expressed in B-cells, dendritic cells and macrophages [ 104 ]. SCIMP plays an important role in antigen presentation on B cells via modulating MHC-II signaling [ 105–107 ]. SCIMP, an immune-specific transmembrane adaptor, engages with TLR4 employing a TIR-non-TIR association to generate a pro-inflammatory response in macrophages [ 108 , 109 ]. TLR4 phosphorylation through tyrosine is crucial in signal initiation which has been implicated by various tyrosine kinases namely the Src family of tyrosine kinases (Lyn) [ 110 ], BTK [ 111 ] and SYK [ 112 ]. SCIMP acts as a scaffold for the Src family kinase, Lyn, to promote phosphorylation and activation of TLR4 [ 103 ]. This allows SCIMP to selectively induce the generation of pro-inflammatory cytokines such as IL-6, IL-12 etc. downstream of TLR4 [ 109 ]. Phosphorylation of TLR-4 leads to the recruitment of MAPK Erk1/2 to the plasma membrane ruffles and macro-pinosomes that activates c-Fos, NF-κB initiating TLR mediated pro-inflammatory responses [ 113 ]. In macrophages, knockout of SCIMP inhibits the signaling mediated by various TLRs namely TLR 2, 3, 4 and 9 [ 114 ]. Indeed, SCIMP has been proposed as a universal Toll-like receptor adaptor in macrophages [ 109 ]. The adaptor protein growth factor receptor-bound protein 2 (Grb2) may recruit other proteins into the signaling pathways of macrophages [ 115 ]. Grb-2 has two SH3 domains and one SH2 domain, with the SH2 domain participating in tyrosine kinase pathways and the SH3 domain recognizing proline-rich signaling molecule sequences [ 115 , 116 ]. The SH3 domain of Grb2 can bind to BCAP which can inhibit TLR-mediated inflammatory signaling [ 117 ]. Grb2 exists in a monomeric-dimeric equilibrium. The monomeric Grb2 associates, via the N-terminal SH3 domain, with the mammalian homologue of the drosophila protein son-of-sevenless (SOS) and activates the MAPK signaling, while the dimeric inhibits this process. Phosphorylation of tyrosine 160 on Grb2 leads to dimer dissociation and this phosphorylated form is highly detected in various malignant types of cancer, pointing the role of monomeric-dimeric Grb2 in controlling MAPK pathway and tumour growth [ 118 ]. Grb2 has been shown to have a role in several disorders including the development of atherosclerotic lesions, since Grb2+/apoE/mice were found to be resistant to the disease [ 119 ]. Erbin interaction with Grb2 results in the activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways that reduce the proliferation of acute leukemia cells and increase acute myeloid leukemia cell differentiation [ 120 ]. Grb2 contribution to carcinogenesis appears to be associated with its regulatory capacity in controlling multiple miRNA synthesis and oncogene expression [ 121 ]. In the context of inflammatory driven diseases, after interacting with SAMSN1,Grb2 activates AMPKa2, preventing the LPS-induced acute lung damage [ 122 ]. In vitro studies showed that treatment of bone marrow derived macrophages (BMDMs) with ox-LDL induces MAPK activation and foam cell formation and this effect is mediated by Grb2 [ 119 ]. According to a meta-analysis, immune cell infiltration, which is crucial in the pathophysiology of CAVD, may be linked to Grb2 [ 123 ]. Consequently, it has undesirable as well as desirable functions in certain disorders [ 124 ]. Numerous studies have suggested that Grb2 plays a paradoxical role in macrophage polarization [ 124 ]. Knockdown of Grb2 reactivated alveolar macrophages to an alternative phenotype [ 124 ]. Contrary to this, HCMV IE2 alternatively activates macrophages through a Grb2/IL-4-relatedmechanism, enabling the virus's long-term survival in the host [ 125 ]. In another study in metastatic cancer, binding to Grb2 resulted in SOS1 expression, which in turn leads to CCL2-mediated alternatively activated macrophages [ 126 ]. Grb2 also plays role in monocyte/macrophage differentiation, activation and proliferation, by binding to phosphorylated CSF1R and regulating the activation of ERK signaling [ 127 ]. Therefore, Grb2 macrophage activation appears to be context-led and requires needs more study. B-cell adaptor for PI3K (BCAP) was initially discovered as an adaptor protein specifically expressed in B-cells. Ligation with B-cell receptor (BCR) resulted in tyrosine phosphorylation and downstream, recruitment of PI3K p85 and phosphorylation of AKT [ 117 ]. BCAP is known to possess various conserved domains that may potentially contribute in its interactions with different proteins and conduct signaling from both receptor and non-receptor tyrosine kinases [ 128 ]. Structurally the N-terminal TIR domain of BCAP is associated with a transcription factor-Ig (TIG) and 3-a-helix (3a) structural unit, armadillo repeats (ARM) and a C-terminal helical (a/a) that is common phospho-tyrosine site [ 129 ]. BCR-induced tyrosine phosphorylation of BCAP is regulated by activation of two kinases namely Btk and Syk in ITAM mediated manner and this plays a crucial role in B-cell survival and proliferation [ 130 , 131 ]. Recently, BCAP was shown to be a positive regulator of PI3K-AKT signaling in CD4 + T cells [ 132 ]. In macrophages, the gene pi3kap1 is known to encode BCAP [ 133 ]. Researchers found that a conserved sequence YxxM was responsible for PI3K p85 binding and therefore can act as a connecting link for the activation of PI3K via TLR signaling [ 134 ]. Another study reported that the absence of BCAP gave rise to identical colitis susceptibility induced by DSS in macrophages, similar to what was earlier demonstrated in full-body BCAP-deficient mice [ 129 ], thus highlighting that BCAP is critically important for the reduction of vulnerability to colonic inflammation and the associated diseases [ 135 ]. A significant increment in the upregulation of the reparative genes was observed in the WT BMDMs and, as expected, in BCAP -/- BMDMs did not show significant production of Arg1, Klf4 and Mmp9 after 24 and 48 h of LPS removal [ 135 ]. The TIR domain of BCAP was found to be similar to the TIR of adaptors of TLR-mediated signaling such as TIRAP and MyD88 [ 33 , 136 ] and that BCAP was actively involved in repressing the activation of NF-κB reporter that is present in the signaling pathway mediated by TLR4 (CD4/TLR4) [ 129 ]. The signal-transducing adaptor proteins (STAPs) have an important role in the inflammatory response and cancer progression through interaction with tyrosine phosphorylated proteins [ 137 , 138 ]. The STAP-1 and STAP-2 are the members of STAP adaptor proteins [ 137 ]. They are structurally homologous to pleckstrin homology domain at the N-terminal end and Src-homology 2 domain at the C-terminal [ 139 ]. However, in case of STAP-2 the C terminal also has a proline rich domain and YXXQ motif [ 140 ]. Based on yeast hybrid screening, STAP-1 was named as B cell antigen receptor downstream signaling 1 (BRDG1) [ 141 ]. Earlier STAP-1 was identified as a stem cell-specific adaptor protein and a substrate for c-kit [ 142 ]. The SH2 domain can bind with many tyrosine-phosphorylated proteins like c-kit and STAT5 [ 142 ]. Research studies of STAP-1 in macrophage activation are scarce [ 143 ]. Stoecker et al. demonstrated for the first time the expression of STAP-1 in activated macrophages and microglia [ 143 ]. Cells expressing STAP-1 were more cytotoxic and phagocytic with reduced migration, thereby elucidating the role of STAP-1 in pro-inflammatory activation of microglia [ 143 ]. Studies by Yang et al. supported the evidence of macrophage activation by alternative method [ 144 ]. In glioma, microglia and macrophages play an important role in the progression and invasiveness of cancer [ 145 ]. Increased expression of STAP-1 was found in glioma-associated microglia/macrophages (GAM), where it increases the expression of ARG-1 viaSTAT3/IL-6 pathway and inhibits phagocytosis, thus promoting alternative activation of microglia [ 144 ]. In uterine macrophages, higher expression ofCYP26A1 leads to the classically activated macrophage phenotype through STAP-1 and Slc7a2, thus producing inflammation-mediated embryo implantation, which is reduced upon CYP26A1 knock down [ 146 ]. However, the role of STAP-1 is enigmatic in macrophage polarization, as both anti-inflammatory and pro-inflammatory role has been described in microglia and macrophages respectively. Signal-transducing adaptor protein (STAP-2) is structurally related to STAP-1 [ 140 ] and contains a unique C-terminal and N-terminal regions featuring combinations of PH (Pleckstrin homology) domain, SH2-like domains and proline-rich regions [ 147 ]. Being a member of the STAP family of adaptor proteins, it engages with molecules responsible for cellular signal transduction pathways, including signaling and transcriptional molecules [ 29 ]. Significantly, STAP-2 plays essential involvement in the control of both innate and adaptive immune responses [ 29 ]. STAP-2 functions as a connector molecule controlling multitude of cellular communications [ 148 ]. STAP-2 participates in cytokine signaling pathways by binding to STAT3 along with STAT5 [ 29 ]. STAP-2 exhibits the capacity to regulate multiple molecular events in the context of pathological inflammatory responses [ 149 ]. The potential of STAP-2 to bind and regulate different types of signaling and transcriptional molecules has been highlighted [ 150 ]. In TLR4 signaling, STAP-2 interacts with MyD88 and inhibitor (I)κB kinase α/β (IKK-α/β), leading to the upregulation of inflammatory cytokine production in macrophages [ 29 ]. The impairment of STAP-2 results in diminished cytokine synthesis and decreased NF-κB activation upon exposure to LPS/TLR4, indicating a potential functional role of STAP-2 in these processes [ 151 ]. On the other hand, the overexpression of STAP-2 augments these biological responses induced by LPS/TLR4 [ 151 ]. The SH2-like domain of STAP-2 is found to bind with MyD88 and IκB kinase (IKK)-αβ, but not with IL-1R-associated kinase 1 or TNFR-associated factor 6 [ 151 ]. As a result, a functional complex composed of MyD88-STAP-2-IKK-αβ is formed [ 151 ]. The augmentation of MyD88 and/or IKK-αβ-dependent signals via these interactions results in an increased NF-κB activity [ 151 ]. The protein STAP-2 was discovered to interact with c-fms, indicating a potential functional relationship between the two proteins [ 140 ]. Also, STAP-2 plays a role in the adaptive immune response, modulating TCR signaling after TCR engagement [ 152 ]. Ionized calcium-binding adaptor molecule 1 (IBA1) is a calcium-binding protein that is mainly found in microglia and macrophages [ 153 ]. IBA1 has been determined by x-ray structures to be a small, single-domain protein with two EF-hand motifs [ 153 ]. It participates in the activation of Rac and controls the actin cytoskeleton's reorganization, which results in the production of lamellipodia and membrane ruffles [ 154 ]. IBA1 contributes to membrane ruffling, phagocytosis and actin-bundling in activated microglia [ 155 ]. In an SAE rat investigation, it was discovered that decreasing IBA1 expression in the mesenteric lymph nodes lowered the classical activation of macrophages [ 156 ]. In another study, the classical activation was discovered in the glioma microenvironment of the CD8/+ animals, where CD11b+ and IBA1+ monocytes and macrophages were abundant [ 157 ]. Neoplasm of monocytic/histiocytic and DC origin do express IBA1 in a highly specific manner [ 158 ]. Human psoriasis lesions were also found to be associated with an increase in macrophages with elevated IBA1 further to classical activation [ 159 ]. Also, in canine and feline inflammatory conditions, IBA1 is found to be a helpful marker of cells of the monocyte/macrophage lineage [ 160 ]. IBA1 is also utilized as a classically activated marker in a study on chronic brain hypoperfusion-induced Sprague Dawley (SD) rats, wherein microglial/macrophage classical polarization was measured by an increased ratio of (CD68 + and CD206 + )/IBA-1 immunofluorescence [ 161 ]. The caspase-recruitment domain-containing adaptor protein (CARD9), an inflammation-associated protein, is known to initiate the inflammatory signaling pathway of NF-κB and/or MAPK [ 162 ]. Playing an indispensable part in both innate and adaptive immunity, CARD9 is a signaling protein that contains a caspase recruitment domain [ 162 ]. CARD9 comprises of two essential parts: a carboxy-terminal coiled domain and an amino-terminal. It is closely related to well-distinguished CARMA1 [ 163 ]. CARD9 is expressed in various tissues, including spleen, liver, placenta, lungs, peripheral blood, brain, bone marrow and fetal liver [ 164 ]. Macrophage and dendritic cells express CARD9 at high levels, highlighting its crucial involvement in immune cell activation and inflammatory processes [ 164 , 165 ]. It has been proposed that CARD9 plays a crucial role in the activation of NF-κB through dectin-1-dependent signaling in innate immunity following a fungal infection [ 166 ]. Studies have demonstrated that CARD9 signaling can collaborate with TLR/MyD88 signaling, leading to increased activation of immune cells [ 167 ]. Genetic mutations in the human CARD9 gene can lead to alterations in protein structure, reduced expression, or loss of function [ 162 ]. These have been connected with inflammatory reactions, heightened vulnerability to fungal infections [ 168 , 169 ], ulcerative colitis [ 170 , 171 ], along with the intensity of pulmonary tuberculosis [ 172 ]. CARD9 polymorphism show a positive correlation with elevated susceptibility to fungal infection and autoimmune diseases [ 173 ]. The essential role of the CARD9 adaptor has been extensively demonstrated in facilitating PRRs to activate NF-κB and MAPK, which in turn initiates a downstream inflammatory cytokine cascade and provides robust protection against microbial invasions, particularly fungal infections [ 162 ] and is responsible for regulating the innate immune signaling cascades [ 166 ]. Studies have found that the TLR3 ligand polyinosinic-polycytidylic acid and the TLR7 ligand loxoribine significantly suppress the production of IL-6, IL-12 and TNF-α in macrophages lacking CARD9 (CARD9 -/- ) [ 166 , 174 ]. CARD9 was found to be indispensable for cytokine secretions through TLR3 and TLR7, but not TLR4, TLR2, TLR5 and TLR9 [ 163 ]. Moreover, the difference in the expression of other adaptors or enzymes that interact with CARD9 could partially explain the varying ability of TLRs to recruit CARD9 [ 163 ]. CARD9-NF-κB signaling pathway is involved in peritoneal macrophages in SAP and blocking the CARD9 expression downregulates transcription activity of NF-κB and reduces the production and release of cytokines, such as TNF-α and IL-6, thereby reducing inflammation [ 175 , 176 ]. According to studies, after establishment of an activated macrophage model, CARD9 shRNA was classically activated, which confirmed that CARD9 shRNA interfered with CARD9 expression, resulting in significant decreases in CD86 + expression on the surface of macrophages, phosphorylation of NF-κB and secretion of the pro-inflammatory cytokines TNF-α and IL-6 by macrophages [ 176 ]. These results confirmed that downregulation of CARD9-NF-κB signaling pathway inhibited classical activation of macrophages [ 176 ]. Althea et al. showed enhanced alternative activation in mice lacking CARD9 [ 177 ]. The recognition of pathogenic fungi is primarily attributed to the families of C-type lectin receptors (CLRs) and TLRs [ 178 ]. Many of the members in these families use CARD9 and MyD88 as signaling adapter proteins to trigger their functions and initiate a defensive response against fungal invasion [ 178 ]. While both CLRs and TLRs activate immune responses against fungi, the observed effects of human deficiencies in either of the shared signaling adaptors indicate that CARD9 has a distinct and superior function in controlling fungal diseases in humans compared with MyD88 [ 179 ]. Individuals with genetic CARD9 deficiencies display a primary immunodeficiency disorder (PID) that leads to a heightened susceptibility to fungal infections [ 179 ].

In the next 5–10 years, the exploration of adaptor proteins in macrophage Toll-like receptor signaling pathways is poised to advance significantly. Future research endeavors may delve deeper into uncovering the intricate mechanisms by which these adaptor proteins regulate immune responses, potentially revealing novel therapeutic targets for infectious and inflammatory diseases. Additionally, the application of advanced imaging techniques and computational modeling may provide unprecedented insights into the spatiotemporal dynamics of adaptor protein interactions within macrophages. Moreover, the integration of omics approaches, such as proteomics and single-cell transcriptomics, holds promise for deciphering the complex signaling networks orchestrated by adaptor proteins. Overall, continued interdisciplinary efforts are warranted to unravel the full spectrum of functions and therapeutic implications of adaptor proteins in macrophage-mediated immune responses.

Inflammation is a tightly regulated process, and among the various immune cells, macrophages play a crucial and central role in the initiation, maintenance and termination of inflammation. They are activated by different stimuli via different receptors and trigger downstream signaling pathways that are facilitated by adaptor proteins. These adaptor proteins serve as mediators between membrane-bound receptors and effector signaling molecules and coordinate cellular responses to external and intrinsic signals, primarily through specific interactions between proteins and phospholipids. They act as a bridge between membrane-bound receptors and effector signaling molecules. Macrophage adaptor molecules play an indispensable role in various neoplasms and chronic inflammatory diseases such as asthma, neurological diseases, rheumatoid arthritis, type 1 diabetes, atherosclerosis, endometriosis and inflammatory bowel disease. The adaptor molecules compiled in this review mainly contain the TIR domain as well as SH2, SH3, PH and CARD and targeting these domains may open up new ways to combat the inflammatory response. Overall, this review provides a detailed insight into the involvement of adaptor proteins in macrophage activation. It represents a valuable resource for future research and may expand our understanding of macrophage biology.