Below the surface: The inner lives of TLR4 and TLR9

Abstract TLRs are a class of pattern recognition receptors (PRRs) that detect invading microbes by recognizing pathogen‐associated molecular patterns (PAMPs). Upon PAMP engagement, TLRs activate a signaling cascade that leads to the production of inflammatory mediators. The localization of TLRs, either on the plasma membrane or in the endolysosomal compartment, has been considered to be a fundamental aspect to determine to which ligands the receptors bind, and which transduction pathways are induced. However, new observations have challenged this view by identifying complex trafficking events that occur upon TLR‐ligand binding. These findings have highlighted the central role that endocytosis and receptor trafficking play in the regulation of the innate immune response. Here, we review the TLR4 and TLR9 transduction pathways and the importance of their different subcellular localization during the inflammatory response. Finally, we discuss the implications of TLR9 subcellular localization in autoimmunity.

some receptors are able to initiate signaling cascades from either the plasma membrane or endosomes.
Here, we provide an overview of the transduction pathways triggered by intracellular TLRs, with a particular focus on the signaling cascades elicited by TLR9 and the intracellular pathways of TLR4. We also discuss how TLR9 signaling may be involved in autoimmunity.

INTRACELLULAR TLRS
TLRs are glycoproteins that consist of 3 domains: a transmembrane domain, an amino-terminal ectodomain, and a cytoplasmic carboxy-terminal Toll IL1-1R homology (TIR) domain. 10,11 To activate downstream signaling pathways, TLRs recruit a variety of adaptor proteins, including the TIR-containing adaptor protein (TIRAP), MyD88, the TIR domain-containing adaptor inducing IFN-(TRIF), and the TRIF-related adaptor molecule (TRAM). 12 Intracellular TLRs (TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13) are expressed in the endoplasmic reticulum (ER), endosomes, multivesicular bodies, and lysosomes; their localization to endosomes and lysosomes, where self-DNA is rarely present, is important to prevent autoimmunity and inappropriate immune responses. Intracellular TLRs recognize either nucleic acids (TLR3, TLR7, TLR8, TLR9, and TLR13) or microbial components (TLR11-TLR12), both derived from the hydrolytic degradation of microorganisms in the endolysosomal compartment. 13 The ligand of TLR3 is double-stranded RNA, such as that of HSV-1, which causes encephalitis, 14 small interfering RNAs, 15 and self RNAs from damaged cells (e.g., RNA damaged by ultraviolet B irradiation). 16 Similarly, TLR7 in plasmacytoid dendritic cells (pDCs) recognizes viral single-stranded RNA, whereas it binds to the RNA of streptococcus B bacteria in conventional dendritic cells (cDCs). 17 In addition, human TLR8 recognizes viral and bacterial RNA and is preferentially activated by ssRNA rich in AU. 18,19 On the other hand, TLR9 primarily binds unmethylated CpG DNA motifs, which are common in bacterial and viral DNA; it can also recognize hemozoin, an iron-porphyrinproteinoid complex derived from the degradation of hemoglobin by malaria parasites. 20 Parroche et al., however, proposed that hemozoin is itself immunologically inert and that its inflammatory activity is due to the presence of parasite DNA in the hemozoin crystal. 21 Another nucleic acid-sensing TLR, TLR13, senses bacterial 23S rRNA 22 and vesicular stomatitis virus. 23 Among the TLRs that recognize microbial components, TLR11 binds to an unknown proteinaceous component of uropathogenic Escherichia coli (UPEC) 24 and a profilin-like molecule derived from Toxoplasma gondii. 25 TLR12 shares many similarities with TLR11: they both recognize Toxoplasma gondii, can form homo-and heterodimers, and can cooperate to recognize their ligands in cDCs and macrophages. 26 Upon ligand binding, intracellular TLRs initiate various signaling pathways. TLR3 induces the expression of inflammatory cytokines and type I IFNs by activating TRIF-dependent signaling through a high-affinity interaction between its TIR domain and the TRIF domain. Notably, this binding is completely TRAM independent. 27 On the other hand, TLR7 and TLR9 activate the transcription factor IRF7 through the MyD88-dependent signaling pathway. 28,29 TLR3, 7, and 9 become active and trigger downstream signaling following internalization of their ectodomains into endosomes, where they undergo proteolytic cleavage. This process requires endosomal proteases and is an additional regulatory mechanism that avoids recognition of self-molecules by strengthening the compartmentalization of intracellular TLRs.
The trafficking of intracellular TLRs from the ER to endolysosomes must be strictly controlled to ensure correct signaling cascades.
Indeed, intracellular TLRs require the multimembrane protein unc-93 homolog B1 (UNC93B1) to exit the ER and enter the secretory pathway. 30 UNC93B1 controls the packaging of TLRs into coat protein complex II (COPII) vesicles, which then shuttle the TLRs from the ER to the Golgi. 31 The role that UNC93B1 plays in the trafficking of TLRs is different for each receptor. 32 Several chaperone proteins, such as glycoprotein 96 and the protein associated with TLR4 A (PRAT4A), also interact with TLRs and are important for shuttling from the ER. 33,34 Notably, nucleic acids may enter the cell through different types of endosomes and the specific site of signaling defines the final outcome of the pathway.

TLR4
Among all PRRs, TLR4 is the best characterized, as it was the first to be discovered in mammalian innate immune cells. 35 Despite TLR4 mainly residing in the plasma membrane, it can also be considered as an intracellular TLR, because it can be internalized and stimulate intracellular pathways. 36 Moreover, although still controversial, it has been proposed that TLR2 also activates NF-kB from endosomes in human monocytes 37 and induces the production of type I IFN in mouse Ly6C high inflammatory monocytes in response to viral ligands. 38 Thus, the endocytic machinery assumes a pivotal role in the regulation of pathways elicited by TLR4 and perhaps TLR2.
The main ligand of TLR4 is LPS, the major component of the outer membrane of Gram-negative bacteria. LPS is composed of lipids and carbohydrates, with a high level of structural complexity, and consists of 3 different components: the O antigen, an O-polysaccharide chain of variable length; the core oligosaccharide; and lipid A, which contributes to most of the immunostimulatory activity of the molecule. 39 The O antigen is specific for each bacterial strain and affects colony morphology; microbial variants with full-length O-polysaccharide chains form smooth colonies, whereas those lacking or carrying reduced chains form rough colonies. 40 Despite TLR4 being the central mediator of innate and adaptive immune responses induced by LPS, endotoxin recognition also requires other surface molecules. Indeed, TLR4 forms the LPS multi-receptor complex with LPS binding protein (LPB), glycosylphosphatidylinositol (GPI)-anchored protein CD14, and myeloid differentiation 2 (MD-2). 12 LPB is a soluble protein that binds large LPS aggregates on the bacterial cell wall, 41 leading to LPS disaggregation and the presentation of monomers to CD14. 42 Upon LPS stimulation, CD14 promotes re-localization of the TLR4-MD-2 complex to lipid rafts, which are enriched in PIP2 (phosphatidylinositol 4,5-bisphosphate). 43

T IR
A P TI RA P F I G U R E 1 TLR4 plasma membrane and endosome signaling. A) LPB protein extracts LPS from the bacterial cell wall and transfers it to CD14. In the presence of LPS, CD14 allows the translocation of the TLR4-MD-2 complex to lipid rafts, where it dimerizes. Then the formation of the "myddosome" complex (containing TIRAP, MyD88, and IRAKs) occurs. IRAKs recruit TRAF6, which interacts with TAB1/2/3 and TAK1 for the activation of NF-B and AP-1. CD14 binds directly to LPS and induces a signal that leads to the activation of NFAT transcription factors. B) The LPS receptor complex is internalized through a CD14-dependent mechanism, involving ITAM-bearing molecules, Syk tyrosine kinase, and PLC 2. Calcium mobilization from the extracellular space via TRPM7 is also required, at least in part. In the endosome, TRAM-TRIF adaptor molecules bind to TRAF3, which interacts with TANK to recruit IKKs and TBK1, which activate IRF3 both the plasma membrane and the endosome; on the plasma membrane, PIP2 binds to TIRAP and mediates the activation of the Myd88 pathway 45 (Fig. 1A), whereas TLR4 activates the TRAM-TRIF pathway upon internalization into the endosome (Fig. 1B). 36 The coordinated actions of all the proteins of the LPS multi-receptor complex, combined with the ability of CD14 and MD-2 to sense and bind LPS, even at picomolar concentrations, ensures the detection of bacteria with high sensitivity. 46 Recent data have shown that not only TLR4 but also CD14 can sense LPS. Indeed, the surface molecule CD14 alone is able to activate a signaling cascade in response to LPS, leading to activation of the NFAT in DCs (Fig. 1A). 47 Moreover, several studies have demonstrated that intracellular LPS activates the formation of a caspase-11dependent noncanonic inflammasome. 48

TLRPLASMA MEMBRANE AND ENDOSOMAL SIGNALING
TLR4 activates 2 signaling pathways. From the plasma membrane, the receptor induces the TIRAP-MyD88 pathway, which activates NF-B and AP-1. From the endosome, TLR4 initiates the TRAM-TRIF pathway, leading to the activation of IRF3, the production of type I IFNs, and a late wave of NF-B activation. 36 As recently reviewed by Brubaker et al., 12 upon TLR4 activation, TIRAP facilitates the interaction of MyD88 with TLR4 via its TIR domain, leading to the formation of the so-called "myddosome," a large molecular platform composed of MyD88, TIRAP, and IRAK proteins. 52,53 IRAK4 activates both IRAK1 and IRAK2, which, in turn, recruit TRAF6. TRAF6 interacts with TAB1, TAB2, TAB3, and TAK1, regulating the activation of NF-B and AP-1 via IKKs and MAPK, respectively (Fig. 1A). 12 After the first wave of NF-B and AP-1 activation, the bipartite sorting signal of the adaptor protein TRAM controls trafficking of the entire LPS receptor complex to the endosomal compartment. 36 During internalization, the TIRAP-MyD88 complex is released from the invaginating plasma membrane, allowing TRAM-TRIF to engage the TIR domain of TLR4. 36 The first step for TRIF-dependent IRF3 activation entails the recruitment of TRAF3 to TRIF. In turn, TRAF3, by interacting with TANK, recruits TBK1 and IKK-; this complex then activates IRF3 and induces the production of type I IFNs (Fig. 1B). 54,55 It has become clear over the last 10 yr that both plasma-membrane and endosome signaling of TLR4 are required for the full response to LPS, highlighting the importance of both the internalization process and the molecules involved. In addition to the 2 main signaling pathways of TLR4, TLR4 intracellular signaling boosts micropinocytosis and antigen presentation 56,57 and, recently, it has also been shown to be involved in the recognition and uptake of apoptotic cells. 58

ENDOCYTOSIS OF TLR4
After the first wave of NF-B activation, the LPS receptor complex is internalized and redirected to the endosome. A series of studies have underlined the central role of CD14 in this process and have demonstrated that the production of type I IFN depends on CD14, highlighting the essential role of CD14 in the induction of the type I IFN-mediated response against Gram-negative bacteria. In particular, it has been shown that the TLR4-CD14-TRAM-TRIF pathway is required for the induction of IFN-production in NK cells during Gramnegative bacterial infections. 59 Jiang et al. demonstrated that CD14 is absolutely required for both activation of the TRAM-TRIF pathway and the production of type I IFN in response to smooth and rough LPS, despite its being dispensable for the detection of high doses of LPS by the complex. 60 Two studies have described how CD14 orchestrates endosomal relocalization of the LPS complex: CD14-dependent TLR4 endocytosis, called "inflammatory endocytosis," is mediated by the activation of the tyrosine kinase Syk and phospholipase C 2, of which the activation is regulated by ITAM and the adaptors DAP12 and Fc R (Fig. 1B). 56,61 A recent study has proposed that the chanzyme TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is involved in LPS-induced TLR4 endocytosis in macrophages by mediating calcium influx (Fig. 1B). 59 Indeed, the authors showed that both genetic deletion of trpm7 and pharmacologic inhibition of the channel abolish, at least partially, the calcium influx in response to LPS, preventing TLR4 internalization. 62 However, TRPM7 may control the recycling of TLR4 rather than its internalization. 63 Further research is needed to clarify the mechanism by which TRPM7 regulates TLR4 endocytosis.
Recently, a study has clarified how TLR4 is selected as cargo for endocytosis. 64 Starting from the observation that the endocytosis of CD14 occurs constitutively in resting cells, the authors hypothesized that the tail of TLR4 is dispensable for the initiation of TLR4 internalization. As a TLR4 mutant lacking intracellular domain did not abrogate the process, the authors inferred that the cargo-selection agent resided in the extracellular portion and hypothesized the involvement of the interaction between TLR4 and MD-2. Indeed, they discovered that both direct binding of MD-2 to the TLR4 ectodomain and MD-2-dependent TLR4 dimerization promote TLR4 endocytosis. 61 Thus, MD-2 plays a key role in TLR4 signaling by coordinating both signal transduction and endocytosis.
Depending on the cell type, the endocytosis of TLR4 involves different players. For example, a specific role for CD11b in promoting the endocytosis of TLR4 has been found in DCs but not in macrophages, as the absence of the integrin affects the process only in DCs. 65 Notably, CD11b is required for the correct internalization of TLR4 only in cells with low levels of CD14. 69 Indeed, the treatment of CD11b-deficient DCs with CpG DNA leads to higher levels of expression of CD14 that compensate the TLR4 internalization defect of the cells. However, CpG treatment does not rectify the defect that the cells have in the TRIF/IRF3 pathway, showing that CD11b plays another role in addition to the modulation of TLR4 trafficking. 65 The endocytosis of TLR4 is negatively regulated by the metallopeptidase CD13. CD13 is up-regulated in the presence of LPS and inhibits TRIF signaling in DCs, as shown by higher levels of TLR internalization in CD13-deficient cells. 66 How CD13 negatively regulates TLR4 trafficking is not yet clear, but neither the inhibition of MD-2 nor the inhibition of CD14 seem to be involved. 66 Perkins et al. described a new negative-feedback loop driven by the PGE2-EP4 axis that specifically inhibits TLR4-mediated TRIFdependent type I IFN production by regulating TLR4 trafficking.
Specifically, PGE2 is rapidly secreted and acts in an autocrineparacrine regulatory loop in response to bacterial LPS. 67 Finally, it is worth noting that pathogenic and commensal bacteria prevent TLR4 endocytosis by producing dephosphorylated LPS to evade detection and CD14-mediated transport to the endosome. 64

IT IS ALL ABOUT TRAFFICKING: THE PATH OF TLR9 INTO THE ENDOLYSOSOMAL SYSTEM
The complexity of the endosomal system fine-tunes the immune response by ensuring the correct compartmentalization of intracellular TLRs and their ligands.
In resting cells, TLR9 is localized to the ER 30 Several checkpoints control TLR9 shuttling through vesicles and involve several membrane and adaptor proteins, 70 actin-nucleation factors, cytoskeletal remodeling proteins, 71 lysosome-or vesicleassociated membrane proteins (LAMPs and VAMPs), 72 and folding chaperones. For example, UNC93B1 facilitates TLR9 trafficking from the ER to the Golgi 32 and then controls the loading of TLR9 into COP II + vesicles, which deliver the receptor to the plasma membrane. 31 At the cell membrane, UNC93B1 recruits the adaptor protein AP-2 via its C-terminal YxxΦ motif and mediates clathrin-dependent internalization of TLR9, leading to localization of the receptor to early endosomal compartments. 68,69 The early endosomes that contain TLR9 and its ligand are still poorly characterized. The brain and DC-associated LAMP-like molecule (BAD-LAMP) is a member of the lysosome-associated membrane glycoproteins and controls, together with UNC93B1, the trafficking of TLR9. 72 It is expressed by pDCs, which produce the largest amount of type I IFNs in response to viral infections. 73 Fig. 2). 71

The game changer: AP-3
The signaling cascade triggered by TLR9 depends on intracellular trafficking of the receptor. APs select the cargo in the vesicles and, specifically, AP-3 determines whether TLR9 is addressed to IRF-SE to promote type I IFN production. 70 Indeed, AP-3 is required for the formation of lysosome-related organelles (LROs), 78 Finally, several studies have suggested that AP-3 is regulated by the phosphoinositide 3-phosphate 5-kinase (PIKfive), a kinase that controls the status of the phosphorylated derivatives of phosphatidylinositol (PI), key components of cell membranes. 82 It has been shown that PIKfive and phosphorylated PIs regulate TLR signaling by orchestrating their intracellular pathways. 83,84 Specifically, in NF-B endosomes, PIKive converts PI(3)P to PI(3,5)P 2 , 85 which recruits and interacts with AP-3. 86 Thus, PIKfive ensures the correct trafficking of TLR9 and CpG to type I IFNs-SE 87 by guaranteeing both the recruitment of AP-3 and the generation of LROs. 88 Moreover, an additional role of PIKfive in pDCs has been suggested, as its inhibition suppresses both IRF7 and NF-B pathways in pDCs, whereas it abrogates only type I IFN production in cDCs. 88 The role of AP-3 in generating LROs thus appears to be clear, although the signaling cascade that is triggered from the LROs is still a matter of debate.

IT IS ALL ABOUT TRAFFICKING: CPG IS LOOKING FOR A RECEPTOR
The trafficking of TLR9 to endosomal compartments is of utmost importance for the initiation of signaling cascades (see Fig. 2). However, CpG also requires controlled shuttling to endolysosomes to encounter TLR9 and activate the pathways. Indeed, upon CpG stimulation of human DCs, the DNA undergoes rapid clathrin-dependent and caveolin-independent internalization into vesicles that localize in juxtanuclear areas. 68 Then, TLR9 is actively shuttled to CpG-rich compartments because of the recruitment of MyD88 in the vesicles. Two studies have also shown that CpG trafficking affects the efficiency of TLR9 signaling, as the abrogation of CpG trafficking to the LAMP + late compartment impairs TLR9 pathways. 87,88 Some of the molecules involved in the shuttling of CpG to the endolysosomal system are discussed below.

Granulin
CpG interacts with a co-receptor that delivers it to the endolysosomes: granulin. 89 M2b phenotype, leading to increased production of pro-inflammatory cytokines, such as TNF-, IL-6, and IL-1 . 96 As TLR9 pathway activation in macrophages induces M1 polarizing signaling, 98 it is likely that granulin-mediated M2b polarization involves an additional receptor or an alternative mechanism. In addition, Chen et al. focused on macrophages, excluding pDCs and B cells from the scenario of activated lymphocyte-derived DNA-induced lupus nephritis. 96 Hence, the role of granulin in autoimmunity appears to be poorly characterized, in particular regarding type I IFN production upon TLR9 engagement in pDCs. Overall, these results highlight the unclear role of granulin in both the TLR9 pathway and autoimmune diseases.

HMGB1
Another co-factor that facilitates DNA sensing is high-mobility group box 1 (HMGB1). HMGB1 is a multifunctional protein that resides in the nucleus and regulates chromatin structure, 99 The authors suggested that HMBG1 may catalyze the TLR9-mediated response to DNA, as it enhances the stimulatory effect of CpG on pDCs by increasing the production of both IFN-and TNF. 107 In addition, the authors demonstrated that, in pDCs, the HMGB1-DNA complex binds to the receptor for advanced glycation end-products (RAGE), which in turn interacts with TLR9, increasing the production of type I IFN production by pDCs via the internalization of DNA. 107 The cru- in BMDCs and BMDMs and plays an essential role in enhancing the release of pro-inflammatory cytokines. 108 The authors showed that the augmented response was not due to increased internalization of HMGB1-CpG, but to a more rapid interaction between TLR9 and CpG.
Indeed, HMGB1 already co-localizes with TLR9 in early vesicles in BMDMs prior to CpG stimulation and accelerates TLR9 redistribution to early endosomes in response to CpG-ODN. 108

THE CONTROVERSIAL ROLE OF TLR9 PROTEOLYTIC CLEAVAGE EVENTS
An additional mechanism that limits TLR9 activation involves a multistep proteolytic cleavage that is required for MyD88 recruitment and the triggering of both signaling cascades. 109 The cleavage of TLR9 occurs in endolysosomal compartments as an evolutionary strategy to prevent aberrant self-recognition, such that the 150 kDa full-length receptor on the plasma membrane, which is potentially in contact with self-DNA, remains nonfunctional.
In the endolysosomes of macrophages, lysosomal cathepsins and endopeptidases, which function only at acidic pH, cleave the TLR9 ectodomain between LRR14 and 15 into an 80 kDa protein. [109][110][111] Additional proteolytic events that involve asparagine endopeptidase occur in both myeloid and plasmacy-  117 These data suggest that TLR9 471-1032 is generated from full-length TLR9 in the endosome in the presence of its ligand; if these conditions are not met, the active form is not properly glycosylated and may act as a negative regulator. 117

DOWNSTREAM TLRENGAGEMENT
Signal transduction begins once TLR9 and its ligand enter the endolysosomal system. TLR9 engagement leads to the recruitment of different players, depending on the cell type. In cDCs, macrophages, and pDCs, TLR9 activates the signaling cascade that culminates with the production of pro-inflammatory cytokines, such as TNF-, IL-6, and IL-12. Instead, the receptor initiates the pathway that leads to type I IFN release primarily, but not exclusively, in pDCs. These 2 signaling cascades are discussed in detail below.

TIRAP: An adaptor only for the TLRs on the plasma membrane?
Several studies have investigated whether intracellular TLRs require sorting adaptor molecules, such as TIRAP, to signal. The sorting capacity of TIRAP relies on its amino-terminal localization domain, which was initially believed to strictly localize TIRAP at the plasma membrane, in association with PI(4,5)P2. 84 lations that generate the bifurcated pathway. 70 Intriguingly, upon HSV stimulation, TIRAP knockout pDCs were unable to produce IFN-but not IL-12p40. 119 These results suggest that TIRAP is essential for the signaling that begins from late endosomal compartments. 70 Finally, the authors confirmed that TIRAP can bind to multiple lipids 122 and showed that its interaction with 3 ′ PIs and phosphatidylserine (PS) in the endosome is sufficient to promote the TLR9 signaling that leads to type I IFN production. 119 Recently, Ve et al. proposed a sequential and cooperative model for the assembly of TIR-signaling complexes. Their structural and kinetic data demonstrate that sequential monomer addition, rather than dimerization and trimerization, is more favorable, providing a more sensitive response. 123

The production of pro-inflammatory cytokines
Once the ligand binds to the leucine-rich repeats in the ectodomain of TLR9, the receptor undergoes a conformational change that allows the formation of homodimers and association of the TIR domains. 125 Depending on the cell type and stimulus, the juxtaposed TIR domains recruit TIRAP 119,120  Simultaneously, IRF1 and IRF5 are directly activated by MyD88. 137,138 Finally, the activated IRFs, NF-B, and AP-1 induce the expression of pro-inflammatory cytokines (TNF-, IL-6, and IL-12).

The production of type I IFN
Several studies have shown that only pDCs produce type I IFN following TLR9 engagement; however, cDCs and macrophages can also release IFNs upon TLR9 challenge. 139,140 Here, we describe the signaling pathway in pDCs, the major producers of type I IFN.

pDCs and type I IFN production
Type I IFN production by pDCs is essential to protect the host against viral infections. 141 Whether the signal from the IRF-SE occurs sequentially or simultaneously to that triggered from the NF-B-SE is still under discussion. Once AP-3 interacts with TLR9 and shuttles from the NF-B-SE to the LROs, the pathway forks. 70 At this point, TIRAP acts as a sorting adaptor and is required for the formation of the myddosome, a multiprotein complex. MyD88 recruits IRAK4, 141,142 which then interacts with TRAF6, TRAF3, 143,144 and IRAK 1. 145 Once this multiprotein complex has formed, IRF7 association with MyD88 and TRAF6 promotes IFN-production. 28,29,146 In addition, IKKenhances IFN-release via IRF7 phosphorylation. 147 Hence, IRF7 disassociation from the complex and its translocation to the nucleus induces type I IFN transcription.
An additional player in the pathway is osteopontin (OPN), which contributes to the induction of IFN-production, specifically in pDCs, because they express intracellular OPN, as opposed to cDCs, which do not. Although the precise mechanism by which OPN supports type I IFN release is still unknown, it is considered to be a functional member of the multiprotein complex. Indeed, upon TLR9 engagement, it localizes near TLR9 and MyD88, favoring the IRF7 pathway. 148 The importance of OPN in TLR9 signaling has also been confirmed by the fact that OPN-deficient animals produce reduced levels of IFN-when challenged with inactivated HSV. 148 Another pathway that supports type I IFN release is phosphoinositol 3-OH kinase (PI3K)-mTOR signaling. The pharmacologic inhibi-tion of the kinase or mTOR reduces the interaction between TLR9 and MyD88 and impairs the production of type I IFN. 149 The mechanism by which PI3K promotes IRF7 activation and translocation into the nucleus in human pDCs has not yet been fully dissected, 143 but it is likely that PI3K acts together with other regulatory elements of the pathway.
Finally, type I IFN is a positive regulator of its own pathway; it enhances the expression of TLR9 and MyD88, further increasing its production. 72

AUTOIMMUNITY: THE CASE OF TLR9
The etiopathogenesis of most autoimmune diseases is still unclear, as several factors may contribute to their onset, such as the presence of autoantibodies, high serum levels of type I IFNs, 150 or increased cell death, which trigger diseases such as RA and system lupus erythematosus (SLE). 145

Psoriasis
Another molecule that promotes aberrant activation of TLR9, thus inducing autoimmune diseases, is the anti-microbial cathelicidin LL37, a hallmark of psoriasis also found in synovial membranes of arthritis patients. 163 Human LL37 is a carboxy-terminal peptide fragment derived from the cathelicidin precursor (human cationic antibacterial protein of 18 kDa orhCAP18) and has many anti-microbial properties. 164 Upon tissue damage, LL37 binds covalently to self-DNA in pDCs and facilitates DNA internalization into the endolysosomal system; once in the endosome, TLR9 may bind to self-DNA, inducing type I IFN production and triggering the onset of psoriasis. 165,166 Also, LL37 in keratinocytes contributes to the exacerbation of psoriasis via the activation of TLR9 and the production of type I IFN. 167,168

Intracellular TLRs and other autoimmune diseases
Aside from the aberrant sensing of self-DNA by TLR9, autoimmune diseases may also result from the misregulation of intracellular Indeed, SMCR8 negatively regulates endosomal TLR signaling, and the entire complex contributes to the vesicle acidification required to degrade TLR ligands and avoid persistent stimulation. 169 A study has also suggested that amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in humans could be caused by C9ORF72 repeat expansion because it generates a loss-of-function SWC complex. 171 Thus, the role of TLR9 in distinct autoimmune disorders is still unclear and further insights are required to shed light on the context-dependent effects of the engagement of this receptor.

CONCLUDING REMARKS
Over the last few years, a more comprehensive picture of the plasma membrane and intracellular signaling cascades, networks, transcriptional regulation, and other processes associated with the TLR response has emerged. In this review, we have discussed up-to-date knowledge of the regulation of the pathways elicited by TLR4 and TLR9 and their roles in host defense and autoimmunity.
Endocytosis and protein trafficking in TLR4 signaling are recently identified regulatory mechanisms of innate immunity and many studies have focused on the identification of the molecules involved in their modulation, leading to the discovery of new players and functions. For example, CD14 and MD-2 are now considered to comprise a novel category of regulators of innate immunity, called transporter associated with the execution of inflammation (TAXI), rather than "classic" chaperone proteins. 172 The trafficking of TLR9 has also emerged as a crucial checkpoint of its pathway, as adaptor proteins, LAMPs, cytoskeleton stabilizers, and PI kinases contribute to guiding TLR9 signal transduction.
However, some of the mechanisms behind TLR9 trafficking are still poorly understood.
Further studies are needed to fully understand the regulation of TLR9 and TLR4 signaling. An in-depth understanding of the regulatory mechanisms would allow, for example, steering the TLR9 pathway toward a specific immune response. Moreover, TAXI and trafficking regulators may become novel targets to prevent overt inflammation and potentiate vaccines and cancer therapies.

DISCLOSURES
The authors declare no conflicts of interest.