TIR domains are essential components of the innate immune system (36). The proximal events of TLR-mediated intracellular signaling are initiated by the interaction of TIR-domain of TLRs with TIR-domain-containing cytosolic adaptors and MyD88 is a central adaptor protein for TLRs. With the exception of TLR3, all TLRs mediate the downstream signaling pathway via MyD88 (37). The association of TLRs with MyD88 recruits the members of the interleukin-1 receptor associated kinase (IRAK) family, forming MyD88-IRAK-4 complex. This recruits IRAK-1 and IRAK-2, leading to the phosphorylation of IRAKs and interaction with tumor necrosis factor receptor associated factor 6 (TRAF6). TRAF6 induces the activation of transforming growth factor-β activated kinase 1 (TAK-1), thereby I-κB (IκB) and mitogen-activated protein kinase (MAPK). The activation of IκB and MAPK results in nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1)-mediated gene transcription (38, 39). IRAK activation also stimulates interferon-regulatory factor (IRF) such as IRF7 (40–42) and activates the gene transcription of type I IFN (43). As a result, pro-inflammatory cytokines including tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, IL-12, and interferon (IFN)-α are produced (44).

TRIF was identified as MyD88-independent pathway (alternative pathway). TRIF is recruited to TLR3 and TLR4. TRAF activation recruits TRAF6 and TRAF3. TRAF6 recruits receptor interacting protein 1 (RIP1). The subsequent activation of TGF-β-activated kinase 1 (TAK1)/TAK1-binding proteins (TABs) leads to the activation of NFκB and IFN-β promoter (45) to express pro-inflammatory cytokines and type I interferons. TRIF also activates TANK-binding kinase 1 (TBK1) and inhibitor of NF-κB kinase (IKK). Subsequently interferon regulatory factor 3 (IRF3) is activated and negatively regulates the activation of NF-κB and IFN-β promoter (46).

Inside-out signal is initiated by non-integrin receptors such as G protein-coupled receptors (GPCRs), selectins, and chemokine receptors (49, 53, 54). Their signals are transmitted to activate integrins. Integrin αLβ2 was extensively studied on inside-out signal in the setting of T cells (55, 56). The activation of T cell receptor (TCR) and tyrosine kinase Lck leads to the phosphorylation of ZAP-70 kinase. This triggers the phosphorylation of LAT adaptor protein and the activation of phospholipase Cg1. This ultimately activates the small G protein ras-related protein-1 (Rap1). Rap1 binds to Rap-1 interacting adaptor molecule (RIAM). These events trigger the binding of talin and kindlin to β2 subunit, which induces the conformational change of αLβ2 into its active form (57). Although the binding of talin alone to integrin can activate it, its potency is extremely weak, supporting the critical role of kindlin in this process (58). The activation of αLβ2 results in its binding with ligands including intercellular adhesion molecule-1 (ICAM-1). What kind of molecules each integrin uses for inside-out signal and whether the same integrin uses different combination of molecules depending on cell type would be an important research area.

Upon the inside-out activation, an integrin binds to a specific ligand. However, for the integrin to tightly bind to its ligand to mediate cell adhesion and migration, its cytoplasmic domains must be anchored to the cytoskeleton (59, 60). When the integrin binds to its ligand, it triggers the assembly of large protein complexes known as focal adhesions by incorporating a variety of molecules including cytoskeletal proteins and signaling molecules. Linking the integrin to the actin cytoskeleton promotes firm cell adhesion, cell spreading, migration and proliferation (57). Talin and kindlin serve as seed proteins to recruit proteins and initiate focal adhesion assembly (61). In case of αLβ2, the binding of ICAM-1 induces the activation/deactivation of kinases and phosphatases, leading to the cytoskeletal remodeling for the fine-tuning of effector functions such as T cell migration (62). Interestingly, this outside-in signal can be modified by the heterotrimeric guanine nucleotide-binding protein (G protein) Gα13. GPCRs activate Gα13, triggering its interaction with β integrin to regulate the outside-in signal (53).

β1 integrin receptors regulate numerous functions, including cell adhesion, migration, differentiation, growth, and survival. β1 integrin subfamily consists of 12 α-chains that non-covalently bind to β1 chain (CD29) (49, 63). They can be categorized by their binding characteristics such as Arg-Gly-Asp (RGD)-binding integrins (αvβ1, α8β1, and α5β1), Leu-Asp-Val (LDV)-binding integrins (α4β1 and α9β1), collagen-binding integrins through triple helical GFOGER sequence in major collagens (α1β1, α2β1, α10β1, and α11β1), and laminin-binding integrins which includes both non-α Inserted (I) domain-containing integrins (α3β1, α6β1 and α7β1) and αI domain-containing integrins (α10β1, α2β1, and α1β1) (64). The key downstream signaling molecules of integrins include focal adhesion kinase (FAK), AKT, MAPK, Src-family protein tyrosine kinases, and integrin-linked kinase (ILK) (65). Integrins regulate intracellular signal transduction cascades that control differentiation, proliferation, and survival. Upon binding to fibronectin, collagen, and laminin, β1 integrin induces cell adhesion and migration that is extensively observed in pancreatic cancer models. Blockade or knockdown of β1 on cancer cells resulted better prognosis by reducing tumor growth and metastasis (66), which makes β1 integrin as an attractive therapeutic target. β1 integrins, in particular α9β1 has been reported to induce Th17 cell promoting cytokines in dendritic cells and macrophages in synergy with TLR2 and TLR4 through ERK pathway, that developed functional Th17 cells and arthritis (67). In addition to that, upon engaging with extracellular matrix (ECM) or other ligands, they initiate signaling pathways that can either reinforce or inhibit the activity of other receptors through negative or positive feedback loops. Interactions of α3β1 and α4β1 with TLRs have reported in several studies, which will be discussed in the following sections.

β2 integrins consist of four members- αLβ2 (CD11a/CD18, lymphocyte function-associated antigen-1), αMβ2 (CD11b/CD18, macrophage-1 antigen, complement receptor 3), αXβ2 (CD11c/CD18, p150.95, complement receptor 4), and αDβ2 (CD11d/CD18). αLβ2 is ubiquitously expressed on all leukocytes, while αMβ2, αXβ2, and αDβ2 are mainly expressed on myeloid cells at different levels (86). αLβ2 binds to intercellular adhesion molecule (ICAM)-1~5 that can be found on the surface of other cells. αMβ2 has broad versatility, having over 40 known binding partners, such as ICAMs, iC3b, fibrinogen, RAGE (receptor for advanced glycation end products), and CD40L (87). αMβ2 and αXβ2 share several ligands as including iC3b, ICAM-1 and fibrinogen, but their binding sites on the same ligand are not exactly the same (88). αDβ2 also binds to multiple ligands, encompassing extracellular matrix-associated proteins like fibronectin, fibrinogen, vitronectin, and plasminogen as well as ICAM-1 (89). Reactive oxygen species (ROS) produced by the ligation of TLR2 and TLR5 induced rapid β2-integrin activation on myelomonocytes, and promoted leukocyte adhesion, suggesting that TLRs collaborate with one another (90). CD18 (β2) knockout (KO) macrophages and DCs produced higher level of IL-12p40 and IL-6 in response to TLR2, TLR4 and TLR9 stimulation, and higher level of type I interferon in response to TLR4 stimulation (91), suggesting that β2 integrins modulate TLR response. Further investigation of β2 ablation showed NF-κB and p38 MAPK pathway activations were involved in these processes (91, 92). Among β2 integrins, the interplay between αMβ2 and TLRs is well studied, which will be discussed further.

αMβ2 is highly expressed on macrophages, DCs, monocytes, granulocytes, and mature or activated NK cells (93). It regulates TLR signaling positively or negatively, depending on cell types and inflammatory status.

TLR4 KO neutrophils reduced αMβ2 activation, but not αLβ2 or αXβ2, suggesting that TLR4 would selectively facilitate the activation of αMβ2 on neutrophils. TLR4-mediated αMβ2 induction involved the activation of transcription factors NF-κB and c-Jun (94). αMβ2 can affect several TLRs. Upon in vivo challenge with TLR ligand stimulations (LPS, poly(I:C), and CpG) pro-inflammatory cytokines (TNF, IL-6, IL-10, and IFN-β) were greatly increased in the serum of CD11b (αM) KO mice (39). Higher level of pro-inflammatory cytokines in the serum was observed in CD11b KO mice during methicillin-resistant Staphylococcus aureus (MRSA) (95) and Escherichia coli (E.coli)<i> (96) </i>infection. Bacterial loads were higher in CD11b KO mice following MRSA and E. coli infections. In contrast, CD11b KO mice demonstrated better clearance of L. monocytogenes following its infection, despite higher serum TNF and IL-6 levels were detected (95). The difference in the phenotype may be because TNF induces apoptosis of certain bacteria (97). In case of MRSA and E.coli infection, TLR4 ligation activated αMβ2 on macrophages by inside-out signaling through PI3K and RapL pathway, which negatively looped back TLR4 signaling ( Figure 3 ) (96). Outside-in signaling activated Src-Syk and promoted degradation of MyD88 and TRIF ( Figure 3 ). This feedback loop in macrophages may control balance of both TLR4 and αMβ2 signaling pathways since their uncontrolled activation can cause harmful pathogenesis. Of note, syk is typically associated with other receptors such as C-type lectin receptors (CLRs). To make complicated further, resident macrophages or bone marrow derived macrophages from CD11b KO mice showed similar level of pro-inflammatory cytokines and activation status upon LPS stimulation, thus suggesting that the interplay of αMβ2 with TLR4 was not involved in steady-state macrophages (96). Thus, the interplay between TLRs and αMβ2 may be dictated by cell types and their cellular state. In fact, the lack of αMβ2 in DCs resulted in decreased pro-inflammatory cytokines and reduced MyD88-dependent phosphorylation of p38, Erk1/2, JNK, and IκBα in response to LPS stimulation (96). Upon stimulation with LPS, αMβ2 was clustered in DCs and co-localized with CD14, which has been shown important for TLR4 endocytosis, suggesting that αMβ2 was a part of TLR4 endocytosis. Furthermore, CD11b KO in DCs impaired RANTES production in LPS induced TRIF–mediated signaling in the endosome (44). Unlike TLR4, αMβ2 in DCs negatively regulated TLR9 signaling by selectively reducing IL-12p70 production, which was possibly regulated by upregulated miR-146. The consequence of IL-12p70 production affected poor cross-priming of DCs to cytotoxic T lymphocyte (CTL) response (98). TLR3 and αMβ2 interplay has been reported on NK cells. KO and neutralization of αMβ2 enhanced cytotoxic function of NK cells in response to TLR3 stimulation and limited acute liver infection (93). αMβ2 deficiency impaired the activation of MAPK/JNK pathway, suggesting that it inhibited TLR3 mediated activation of NK cells (99). Inside-out activation of αMβ2 by TLR2 in association with CD14 was reported in monocytes during the infection of Porphyromonas gingivalis, a pathogen implicated in chronic periodontitis and atherosclerosis. The activation of αMβ2 induced adhesion and recruitment of monocytes to the site of the infection (100). This recruited inflammatory monocytes can be beneficial to control infection, but uncontrolled accumulation results a tissue destruction. Although current data are all based on either αMβ2 or TLR KO system or depletion by neutralizing antibodies, the studies suggested a possible indirect interplay between αMβ2 and TLR2 (100).

While most studies examined the interaction between TLRs and αMβ2 by inhibiting or deleting αMβ2, some studied by activating it. αMβ2 activation by leukadherin-1 (LA1), its allosteric agonist, protected mice from pathological injuries and reduced the mortality induced by LPS (101). αMβ2 activation by LA-1 inhibited M1 macrophage response to LPS both in vivo and in vitro. Although it is not clear whether LA-1 facilitated a direct interaction between αMβ2 and TLR4 on macrophages, it induced an endocytosis of both αMβ2 and TLR4 and prevented LPS binding to TLR4 (101). While it prevented an excessive activation of TLR4 signaling pathway and pro-inflammatory response in macrophages, LA-1 pretreatment induced pro-inflammatory cytokines in DCs, suggesting that the effect of LA-1 could be cell type-dependent (101). It is worth noting that the expression levels of αMβ2 on macrophages and DCs are different (102), which may be in part responsible for the different effect of LA-1 on these two cell types.

A recent study showed that CD11b deficiency of donor non-classical monocytes increased CXCL2 production and exacerbated primary graft dysfunction in lung transplantation model (103). High mobility group box 1 (HMGB1), a DAMP released from dying cells, activates TLR4 in nonclassical monocytes. It was released from the donor lungs with primary graft dysfunction. Interestingly, HMGB1 stimulation induced lower CXCR2 production by TLR4 single KO or TLR2/TLR4 double KO monocytes, but not TLR2 single KO. It is uncertain whether αMβ2 interacts directly with TLR2 or TLR4, however, αMβ2 agonist LA-1 prevented primary graft dysfunction, suggesting that αMβ2 might facilitate TLR endocytosis.

Although all the evidence supports the presence of crosstalk between αMβ2 and TLRs, several questions still need to be answered. Do αMβ2 and TLRs bind to the same ligands? In fact, several studies reported that αMβ2 binds viral dsRNA (104), and bacterial toxins (105) and LPS (106–108). So far the interaction between ligands and αMβ2 has been shown in vitro. Therefore, it will be critical to determine if reported ligands for αMβ2 are in fact relevant in vivo. If so, it is not known whether both TLRs and αMβ2 bind a ligand at the same time. If they do, which signaling should be activated first? Does ligand binding avidity and affinity affect downstream signal? Or do they limit activation? If they don’t, is there ligand binding competition between TLR and αMβ2? Would it be possible that the rest of β2 integrin members interplay with TLRs? This may be very possible since extracellular part of αXβ2 has over 80% of sequence homology to αMβ2 and about 50% of homology in intracellular tail, for example (109, 110). Furthermore, LPS also binds to caspase-11 intracellularly (111), which makes the crosstalk complicated. β2 integrin members may also collaborate each other to inhibit or induce TLR response, since β2 integrin members are expressed upon activation of cells, especially on myeloid cells. They may function synergistically. Apparently, the proposed crosstalk between αMβ2 and TLRs depend on cell types, but does ligand binding affinity or avidity affect crosstalk? For example, high-affinity ligand binding affects the degree of up- or downstream signal? There might be a rivalry between the ligands. It is interesting to know whether these crosstalks depend on the timing of activation or not. A previous study showed αMβ2 on dendritic cells was activated through inside-out signaling by TLR4 (112) that was necessary for αMβ2-induced phagocytosis but not affected αXβ2, suggesting a bidirectional action between αMβ2 and TLR4.

αV integrin also known as CD51 or MSK8, is a transmembrane protein that is involved in cell adhesion, migration, and signaling (113). αV integrin forms heterodimers with various β integrin subunits such as β1, β3, β5, β6, and β8. Together they designate a various array of receptors to bind to specific ligands in the extracellular matrix (ECM) including fibronectin, vitronectin, fibrinogen, and osteopontin, enabling cells to adhere and respond to their surrounding environment (114, 115).

In addition to adhesion, αV integrin promotes the activation of a multitude of signaling pathways, primarily the FAK pathway (116). The phosphorylation of FAK will in turn recruit Src kinases, phosphoinositide 3-kinase (PI3K) subunit p85, or phospholipase (PL)Cγ and stimulate the signaling cascades of Ras/Erk, PI3k/Akt, and Crk/Dock180/Rac. These pathways contribute to cell survival, proliferation, differentiation, and migration, emphasizing the multifaceted role of integrin αV.

The significance of αV integrin is not only linked with normal physiological functions. Dysregulation of αV integrin function has been associated with a variety of pathological conditions, including cancer, metastasis, angiogenesis, and wound healing (115). Additionally, αV integrin is involved in vascular remodeling and fibrosis (117).