INTRODUCTION — Toll-like receptors (TLRs) are cell surface and intracellular molecules on eukaryotic cells that detect and respond to microbial antigens. They derive their name from homology to the Drosophila Toll molecule, an important component of dorsal-ventral patterning and antifungal defense [1,2]. TLRs are part of the innate immune system, which is a phylogenetically ancient system present in invertebrates and conserved through vertebrate evolution. Innate immune responses are initiated rapidly by exposure to microbes and precede the development of adaptive immune responses . Innate immunity is reviewed separately. (See "An overview of the innate immune system".)
ROLE IN INNATE IMMUNITY — TLRs are a type of pattern recognition receptor (PRR), which are receptors specific for molecular components of micro-organisms that are not made by the host. The ligands for TLRs are called pathogen-associated molecular patterns (PAMPs), which are components of pathogenic microbes. In vertebrates, TLRs initiate protective functions that operate independently from adaptive immunity. They also play a critical role in adaptive immune responses by directing the differentiation of naïve T cells into effector T cells. Other examples of PRRs include nucleotide oligomerization domain (NOD)-like receptors (NLRs) and the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs). (See "An overview of the innate immune system", section on 'Pattern recognition receptors'.)
STRUCTURE AND FUNCTION OF TLRs — Different species have different numbers of distinct TLRs. At least 10 have been discovered in humans [1,2,4]. With the exception of TLR1, TLR2, and TLR6, TLRs initiate signaling by homodimerization (eg, two identical TLRs brought together). TLR2 forms heterodimers with TLR1 (TLR2/1) or TLR6 (TLR2/6). Receptor structure/organization, ligands, and signaling are summarized in the table (table 1) and figure (figure 1).
The structure of all of the TLRs is similar. All have an extracellular ligand recognition and binding domain that contains leucine-rich repeats. All have a single transmembrane domain. The cytoplasmic (signaling) domain is homologous to the interleukin-1 (IL-1) receptor and is called a toll/IL-1 receptor or TIR domain.
The final common pathway for TLR signaling involves the transcription factors, nuclear factor (NF) for the kappa (k) light chain enhancer in B cells (NF-kB) and activating protein-1 (AP-1). These transcription factors regulate a multitude of genes, including those encoding important proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), IL-1-beta, IL-6, IL-8, and IL-12. Some TLRs also activate production of type 1 (alpha and beta) interferons by inducing the interferon regulatory factors (IRFs) IRF3, IRF5, and IRF7.
Cellular and tissue distribution — TLRs are differentially expressed on various leukocyte populations [2,5]. Monocytes and macrophages express all TLRs, except TLR3. Myeloid dendritic cells (MDCs) express TLR2/1, TLR3, TLR4, TLR5, and TLR8, while plasmacytoid dendritic cells (PDCs) express TLR7 and TLR9. TLR2/6, TLR4, and TLR8 are found on mast cells. B cells express TLR3, TLR7, TLR9, and TLR10, while T cells express TLR3 and TLR9. A variety of tissue types may express one or more TLRs. As an example, the basolateral surfaces of intestinal epithelial cells express TLR5, whereas fibroblasts and keratinocytes express TLR3 on the cell surface .
The cellular localization of TLRs varies. TLR2/1, TLR2/6, TLR4, TLR5, TLR10, and TLR11 are localized to cell surfaces, whereas TLR3, TLR7, TLR8, and TLR9 are localized within endosomes. The cell surface expression of TLRs, such as TLR4, which recognizes lipopolysaccharide (LPS), is hypothesized to allow recognition of extracellular molecules released from pathogens. Endosomal expression of TLR3, TLR7, TLR8, and TLR9 allows recognition of microbial nucleic acids following their uptake and degradation in phagolysosomes. TLR3, TLR7, TLR8, and TLR9 interact with an endoplasmic reticulum (ER) membrane protein called UNC93B (UNC93 homolog B), which is essential for proper signaling . Additionally, endosomal expression of TLR3, TLR7, TLR8, and TLR9 may prevent activation by host nucleic acids and the development of autoimmunity.
Signaling — All TLRs, except TLR3, utilize the cytoplasmic proximal signaling intermediate myeloid differentiation primary response protein 88 (MyD88), either by itself (TLR5, TLR7, TLR8, and TLR9) or together with the adapter molecule toll/interleukin-1 (IL-1) receptor (TIR) domain-containing adapter protein (TIRAP/MyD88 adapter-like [MAL], TLR2/1, TLR2/6, and TLR4) (figure 1) [1,2,8]. There is also a MyD88-independent pathway via the molecule TIR domain-containing adapter-inducing interferon-beta (IFN-beta) (TRIF), which is used by TLR3 and TLR4 (TLR4 also utilizes the intermediate TRIF-related adapter molecule [TRAM]).
The MyD88 adapter connects through interleukin-1 (IL-1) receptor-associated kinases 1 and 4 (IRAK1, IRAK4), tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and transforming growth factor (TGF)-beta-activated protein kinase 1 (TAK1) to the mitogen-activated protein kinases (MAPKs). This complex kinase cascade culminates in activation of the family of heterodimeric transcription factors known as activating protein-1 (AP-1). These transcription factors regulate a broad array of genes influencing cell survival, activation, and proliferation.
The TAK1 adapter also leads to activation of the inhibitor of NF-kB kinase (IKK) and resultant activation of NF-kB. This is another family of heterodimeric transcription factors regulating many genes with important roles in immune responses, as well as in the physiology of a broad array of other cell types. These include cytokines, chemokines and growth factors, lymphocyte receptors, adhesion molecules, acute-phase reactants, and other transcription factors. Ligation of TLR3 leads to activation of TRAF3 followed by tank-binding kinase 1 (TBK1) and IKK, which in turn activate interferon regulatory factor 3 (IRF3), leading to production of type 1 interferons. TLR7, TLR8, and TLR9 signal via a MyD88-dependent pathway that utilizes IRAK1/4, IKK-alpha, TRAF3/6, and intracellular osteopontin (iOPN), which activates IRF7, leading to production of interferon-alpha (IFN-alpha) . In addition, TLR7-induced production of IFN-alpha has been shown to be dependent upon activation of IRF5 .
Regulation — Both the cell surface expression of TLRs and the intracellular consequences of TLR ligation are regulated by a variety of factors. These include exposure to microbial products, other TLR ligands, and cytokines. Suppressor of cytokine signaling (SOCS) proteins and ubiquitin-mediated degradation of signaling proteins are involved in negative regulation of TLR signaling [11,12]. Within the gastrointestinal tract, the factors that maintain tolerance to commensal host flora while detecting/containing pathogenic bacteria with appropriate inflammatory responses are incompletely understood. The detection of common PAMPs in pathogenic and nonpathogenic bacteria would be anticipated to activate the same inflammatory response. Nevertheless, the detection of commensal bacteria within the intestines can induce tolerance. TLR signaling can contribute to intestinal homeostasis by regulating intestinal epithelial cell proliferation and epithelial integrity . Expression and localization of TLRs in the intestinal epithelium may directly relate to their role in maintaining homeostasis versus inducing inflammation. For example, within the intestinal epithelium, TLR9 activation through the apical membrane induces tolerance, whereas TLR9 activation via the basolateral membrane induces an inflammatory response through the canonical NF-kB pathway. Differential spatial expression of pattern recognition receptors (PRRs) in epithelia may constitute a critical mechanism of distinguishing nonpathologic from pathologic bacteria. Regulation of TLR function is highly complex and still incompletely understood and beyond the scope of this review [14-17].
SPECIFIC TLRs — The ligands, cellular expression, signaling molecules utilized, and resultant cellular effects of ligation for each of the TLRs are summarized in the table (table 1). Additional information is presented below.
●TLR2/1 – The heterodimer of TLR2 with TLR1 binds to cell wall components of bacteria and mycobacteria. TLR2/1 binds triacyl lipopeptides/lipoproteins and some diacyl lipopeptides [18-20]. It also interacts with porins of Neisseria . TLR2/1 is expressed on myeloid dendritic cells (MDCs) and signals via the canonical myeloid differentiation primary response protein 88 (MyD88) pathway with the toll/interleukin-1 (IL-1) receptor (TIR) domain-containing adapter protein (TIRAP). TLR2/1 also exists in a soluble form that can inhibit signaling via the membrane-bound receptor .
●TLR2/6 – The heterodimer of TLR2 with TLR6 displays a preference for recognizing diacyl lipopeptides, although it can recognize some triacyl lipopeptides [18-20]. TLR2/6 is expressed on mast cells, and it also signals via the canonical MyD88 pathway with the TIRAP.
●TLR3 – TLR3 binds double-stranded RNA (dsRNA), which is produced in the replication of many viruses . TLR3 may also bind to synthetic dsRNA, such as polyinosinic-polycytidylic acid (polyI:C) . TLR3 is found within endolysosomes (membrane-bound intracellular compartments formed by the fusion of vesicles of internalized viral debris and lysosomes containing degradative enzymes) of MDCs, B cells, T cells, and natural killer (NK) cells . TLR3 signals via a MyD88-independent pathway (TIR domain-containing adaptor-inducing interferon [TRIF]) and induces production of type 1 interferon-beta (IFN-beta) in addition to inflammatory cytokines.
●TLR4 – The principal ligand for TLR4 is the lipopolysaccharide (LPS) of Gram-negative bacteria . The interaction of LPS with TLR4 occurs in a complex with LPS-binding protein (LBP), CD14, and a molecule called myeloid differentiation factor-2 (MD-2) (figure 1). Additional ligands include the fusion protein of respiratory syncytial virus (RSV), heat shock proteins of organisms, such as Chlamydia pneumoniae, and components of the extracellular matrix, such as fibronectin, hyaluronic acid, and heparan sulfate [27-29]. TLR4 is expressed on MDCs and mast cells and can signal via MyD88 with TIRAP. It may also induce type 1 IFN through the MyD88-independent pathway via TRIF-related adaptor molecule (TRAM).
●TLR5 – TLR5 recognizes bacterial flagellin . It is expressed on MDCs and transduces signals via MyD88.
●TLR7 and TLR8 – TLR7 and TLR8 are very similar in their ligand binding and signaling [31-33]. Both bind to single-stranded RNA (ssRNA) from viruses, such as influenza and human immunodeficiency virus-I (HIV-I). They also recognize imidazoquinolines (synthetic heterocyclic organic molecules), such as imiquimod, and guanosine analogs, such as loxoribine. TLR7 is expressed on MDCs, plasmacytoid dendritic cells (PDCs), and B lymphocytes. TLR8 is expressed on MDCs and mast cells. Both signal directly via MyD88.
●TLR9 – TLR9 interacts with unmethylated cytosine guanine dinucleotide (CpG) motifs in microbial DNA . TLR9 is expressed within endolysosomes of PDCs, B cells, and T cells and signals directly via MyD88. Activation of PDCs (also known as IFN-producing cells) through TLR7 and TLR9 leads to production of large quantities of IFN-alpha, which plays a critical role in antiviral innate immunity. Additionally, PDCs, by virtue of their production of IFN-alpha and IL-12, are potent inducers of differentiation of naïve T cells into T helper type 1 (Th1) effector T cells.
●TLR10 – TLR10 is a modulatory PRR with largely inhibitory functions [35,36]. TLR10 is believed to exert inhibitory functions through competition with ligand binding to TLRs. Furthermore, TLR10 may compete for heterodimer formation with TLR2. TLR10 activation also leads to increased expression of IL-1 receptor antagonist (IL-1RA), which decreases inflammation. Recently, TLR10 was found to be a receptor for HIV gp41 binding, leading to activation of NF-kB and IL-8 production in macrophages, monocytes, and mammary epithelial cells, suggesting it may be an innate sensor of HIV infection. Binding of HIV gp41 to TLR10 leads to upregulation of expression of TLR10. However, increased expression of TLR10 may result in enhanced HIV infection by allowing entry into monocytes, macrophages, and epithelial cells .
CELLULAR RESPONSES INDUCED BY TLRs — The response to various combinations of TLRs depends on the cell expressing that TLR, the relative amounts and types of TLRs present, as well as other stimuli and the developmental program of the target cell.
●TLR ligation of mononuclear cells may potentiate the microbicidal activity of phagocytic cells. As an example, TLR2/1 or TLR2/6 ligation by cell wall components of Mycobacterium tuberculosis induces nitric oxide production in mice and other bactericidal mechanisms in humans . The same ligand/receptor combination may also lead to apoptosis in macrophages . This mechanism could potentially limit the spread of microbes that are replicating intracellularly.
●TLR activation of epithelial cells can induce production of antimicrobial peptides (defensins) that provide protection against a wide variety of pathogens. TLR activation in intestinal epithelial cells can contribute to intestinal homeostasis by regulating intestinal cell proliferation and epithelial integrity. Depending on the cellular localization of TLRs in intestinal epithelia (ie, apical membrane), TLR activation may induce tolerance, whereas activation of TLRs within the basolateral membrane induces an inflammatory response to invasive bacteria [40-42].
●Activation of TLRs on dendritic cells leads to antigen presentation, production of IL-12, and upregulation of costimulatory molecules (CD80, CD86), which directs the differentiation of naïve T cells into antigen-specific Th1 effector T cells . (See "The adaptive cellular immune response: T cells and cytokines".)
●TLR activation (TLR3, TLR7, TLR8, TLR9) results in production of type 1 interferons, which plays a crucial role in antiviral innate immunity. Different TLR ligands may lead to somewhat different cytokine responses or may induce type 1 interferon and lead to activation of qualitatively different types of immune responses (eg, T helper type 1 [Th1] versus T helper type 2 [Th2] T cell differentiation) . The response to various combinations of TLRs also depends on the relative amounts and types of TLRs on the cell surface, as well as other stimuli and the developmental program of the target cell.
Because pathogens coevolved with their hosts, we (the host) have acquired sophisticated mechanisms for resisting infection, including TLRs. In turn, pathogens have developed strategies to circumvent many of these mechanisms. For example, vaccinia virus encodes molecules with homology to toll/interleukin-1 (IL-1) receptor (TIR) domains and can inhibit signaling via TLR3 and TLR4 [44,45].
TLR SIGNALING DEFECTS IN PRIMARY IMMUNODEFICIENCY — Defects have been identified in molecules that transduce signals from TLRs and lead to primary immunodeficiency (table 2).
Specific disorders — The degree of immunologic impairment is related to where in the TLR signaling pathway the defect occurs (figure 2). Defects that occur proximally in the TLR pathway, such as deficiency in myeloid differentiation primary response protein 88 (MyD88) or interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK4) lead to susceptibility to a limited number of pathogens (typically pyogenic bacteria), since these defects only impair the function of TLRs and IL-1R.
In contrast, distal defects involving transcription factors (ie, nuclear factor [NF]-kB complex) lead to broader infectious susceptibilities (eg, bacteria, viruses, fungi, and opportunistic organisms), as well as developmental abnormalities (ie, ectodermal dysplasia). This is because of the diverse immune and developmental mechanisms that signal through NF-kB, including pathways in innate immunity (ie, TLRs), adaptive immunity (ie, T cell receptor, B cell receptor, and CD40), and ectodermal development (ie, ectodysplasin A receptor).
MyD88/IRAK4/IRAK1 deficiency — All TLRs, except TLR3, use the cytoplasmic proximal signaling intermediate myeloid differentiation primary response protein 88 (MyD88), either by itself or together with the adapter molecule toll/IL-1 receptor domain-containing adapter protein (TIRAP). MyD88 deficiency was initially described in nine children suffering from recurrent and severe pyogenic bacterial infections . These children were susceptible to invasive infections with Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa, but had normal resistance to other common bacteria, viruses, fungi, and parasites. Three children died before the age of 1 year, while another six were alive at ages 3 to 16 years at the time of publication. Susceptibility to infection improved with age in affected patients, although MyD88 levels did not change, suggesting that maturation of the adaptive immune system may compensate for defective TLR function. The defect displayed autosomal recessive inheritance. MyD88 deficiency is clinically indistinguishable from IL-1 receptor-associated kinase 4 (IRAK4) deficiency, since IRAK4 associates with MyD88 in signal transduction .
Deficiency of IRAK4 (MIM 607676) impairs signaling from the TLRs that associate only with the MyD88 adapter (figure 3). These include TLR2/1, TLR2/6, TLR5, TLR7, TLR8, and TLR9. TLR4 signals through MyD88, but can also make use of the toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-beta (IFN-beta) (TRIF)-related adapter molecule (TRAM)/TRIF pathway. TLR3 is unaffected. Signaling by the IL-1 receptor and T cell receptor activation are also impaired . Peripheral blood mononuclear cells from patients with IRAK4 deficiency or MyD88 deficiency do not produce proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), IL-1, and IL-6 when stimulated via IL-1 or IL-18 receptors or TLRs. The lack of responsiveness to IL-1 results in impaired fever responses to bacterial infection. The lack of significant fever and the unexpectedly low level of C-reactive protein (CRP) in the setting of a serious bacterial infection can be a clue to the possibility of IRAK4 deficiency or MyD88 deficiency.
The clinical and laboratory features of IRAK4 deficiency are indistinguishable from MyD88 deficiency, since IRAK4 associates with MyD88 in signal transduction (figure 3). Patients with IRAK4 deficiency have recurrent severe infections (cellulitis, arthritis, meningitis, osteomyelitis, organ abscesses, and sepsis), mainly caused by S. aureus, S. pneumoniae, and P. aeruginosa [47,49,50]. One series described 48 patients who experienced recurrent bacterial infections, mostly of the upper respiratory tract and skin . None had severe viral, fungal, or parasitic infections. Initial invasive infections occurred before the age of two years in 88 percent. Invasive pneumococcal infections caused the most morbidity and mortality and were the cause of death in 33 percent. In patients with either IRAK4 deficiency or MyD88 defects, susceptibility to infections seems to improve with age, regardless of therapy [47,52]. In the largest series, no infectious deaths were reported in patients over the age of 8 years and no invasive infections after the age of 14 years .
Serum immunoglobulins are normal, specific antibody levels against polysaccharide antigens may be low or normal, and B cell subsets and function in vitro are normal. T cell responses to mitogens and recall antigens are also normal. Peripheral eosinophilia and elevated serum immunoglobulin E (IgE) may be observed.
The diagnosis can be made by measuring cytokine production by leukocytes that have been activated by various TLR ligands (TLR function assay, available in at least one commercial laboratory) . Absence of functional MyD88 or IRAK4 results in an absence of proinflammatory production in affected patients. The absence of TLR-induced cytokine production in these patients must be then followed by gene sequencing to identify the precise genetic defect. Targeted gene panels are now commercially available and are cost effective.
Meticulous skin care to minimize bacterial colonization of the skin is also an essential element of patient management, since these patients remain susceptible to skin infections. Antibiotic or other therapy is dictated by specific pathogen sensitivity and the focus of infection. Antibiotic prophylaxis and/or immune globulin replacement therapy may be considered. Most MyD88- or IRAK4-deficient patients are maintained on antibiotic prophylaxis with amoxicillin or sulfamethoxazole-trimethoprim indefinitely.
For the roughly 50 percent of MyD88- or IRAK4-deficient patients that do not have adequate antibody responses to polysaccharide pneumococcal vaccine (ie, Pneumovax), immune globulin replacement therapy is helpful, at least until adolescence when invasive infection risks diminish . (See "Immune globulin therapy in primary immunodeficiency".)
IRAK1 deficiency in a male due to a 112kb deletion, which deleted both MECP2 and IRAK1 on the X chromosome, has been reported . Unfortunately, no information on the infectious consequences of the loss of IRAK1 is available because the patient died of progressive neurologic and respiratory insufficiency, presumably from absence of MECP2. Analysis of transformed fibroblasts from this patient demonstrated absent responses to stimulation of TLR ligands, whereas signaling downstream of IL-1R was relatively unimpaired, suggesting that IRAK1 plays an essential role in signaling downstream of TLRs but a redundant role downstream of IL-1R in fibroblasts. In contrast, TLR signaling in the patient's peripheral blood mononuclear cells was unimpaired, indicating that IRAK1 plays a redundant role that may be compensated by IRAK2.
UNC93B1 deficiency, TLR3 mutations, TRIF deficiency, TRAF3 deficiency, and TBK1 deficiency — UNC93B1 (UNC93 homolog B1) is an endoplasmic reticulum (ER) protein that is involved in the transport of the nucleotide-sensing TLR7 and TLR9 from the ER to endolysosomes. Signaling through TLR3, TLR7, TLR8, and TLR9 normally induces production of type 1 interferons following binding to viral RNA (figure 3). Type 1 interferons are critical to controlling certain viruses during the earliest stages of infections.
HSV-1 encephalitis — Deficiency of UNC93B1 or TLR3 (MIM 610551 and MIM 613002) leads to susceptibility to herpes simplex virus type 1 (HSV-1) encephalitis due to decreased production of interferons in the central nervous system [55-58]. Both autosomal dominant and autosomal recessive forms of toll/interleukin-1 (IL-1) receptor domain-containing adaptor-inducing interferon (TRIF) deficiency have been shown to cause susceptibility to HSV-1 encephalitis . Autosomal dominant deficiency of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), which leads to reduced but not absent TRAF3 function, is another cause of susceptibility to HSV-1 encephalitis . Finally, heterozygous mutations in tank-binding kinase 1 (TBK1) also result in susceptibility to HSV-1 encephalitis . This group of patients with defective TLR3 signaling demonstrates the essential role of TLR3 in the central nervous system in the prevention of HSV-1 encephalitis. (See "Herpes simplex virus type 1 encephalitis", section on 'Host susceptibility'.)
Severe influenza — Deficiencies in TLR3, interferon regulatory factor 7 (IRF7), and IRF9 have been identified in children with life-threatening influenza pneumonia [62-64]. Additionally, a heterozygous missense mutation in TLR3 (F303S) was associated with influenza-associated encephalopathy, likely due to haploinsufficiency of TLR3 function .
Severe COVID-19 — Type 1 interferon production plays an essential role in host defense against coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is an RNA virus that is likely detected intracellularly by TLR3, 7, and 8, as well as RIG-I and MDA5. SARS-CoV-2 encodes numerous genes that can inhibit type 1 interferon production by blocking innate sensing (nsp16, 14), interferon production (M protein, N protein, nsp3, ORF 3b,4a, 4b, 5, 6), and interferon signaling (nsp1, ORF4b, 6), which contributes greatly to its virulence. Defects in genes involved type 1 interferon production, including TLR3, IRF7, UNC93B1, TICAM1, TBK1, and interferon alpha and beta receptor subunit 1 (IFNAR1) were found in 3.5 percent of patients hospitalized with life-threatening COVID-19 (figure 3) . Ten of 23 patient for whom blood samples were available from early in their hospitalization showed very low IFN-alpha levels compared with other patients with milder COVID-19 disease. In a parallel study by the same group, the presence of autoantibodies against specific type 1 interferons, which resulted in undetectable serum levels, were found in 10.2 percent of patients with life-threatening COVID-19 pneumonia compared with 0.33 percent of healthy individuals . Thus, defects in the pathways generating type 1 interferons, inhibition of type 1 interferon production, and autoantibodies that neutralize interferons predispose to severe COVID-19.
NEMO defects — NF-kB essential modifier (NEMO), also called the inhibitor of NF-kB kinase (IKK)-gamma, is encoded by a gene on the X chromosome (IKBKG). NEMO is required for the proper function of IKK, which inactivates the inhibitor of NF-kB (I-kappa [k]-B) proteins (figure 3). In the absence of IKK activity, I-kB remains bound to NF-kB, holding it in the cytoplasm in an inactive form and preventing its translocation to the nucleus to exert its transcriptional regulatory activities. Thus, in the absence of NEMO function, NF-kB signaling is blocked, shutting down many pathways, including innate immune functions (ie, TLRs, nucleotide-binding oligomerization domain [NOD]-like receptors [NLRs], retinoic acid-inducible gene-I [RIG-I]-like receptors [RLRs]), adaptive immune functions (T cell receptor, B cell receptor, CD40), and developmental pathways (ie, ectodysplasin A receptor in ectoderm and receptor activator of NF-kB [RANK] in osteoclasts) (figure 2). Complete absence of NEMO function is lethal in male embryos, and partial deficiency in females leads to a condition called incontinentia pigmenti . (See "Incontinentia pigmenti".)
Mutations that permit partial function of NEMO (ie, hypomorphic mutations) cause immunodeficiency of varying severity in males, ranging from recurrent sinopulmonary infections to extreme susceptibility to all classes of pathogens, including mycobacteria and opportunistic organisms. Because NF-kB also transduces signals from receptor systems important for the development of skin and skin appendages, the majority of patients with hypomorphic mutations also have ectodermal dysplasia, a disorder known as ectodermal dysplasia with immunodeficiency (ED-ID) (figure 2), although it can be present in very subtle ways in some patients. Ectodermal dysplasia is characterized by conical or absent teeth; fine, sparse hair; and hypohidrosis due to decreased sweat glands. NEMO mutations are discussed separately. (See "Mendelian susceptibility to mycobacterial diseases: Specific defects", section on 'NEMO deficiency'.)
I-kappa-B defects — Multiple patients have been found to have mutations in the alpha subunit of the I-kB complex, an autosomal dominant form of ED-ID (figure 3). The phenotype is very similar to NEMO deficiency, leading to variable susceptibilities to infectious agents with (or without) ectodermal dysplasia. Testing of TLR function in patients suspected of having impaired NF-kB function can be informative. The degree of impairment in TLR function can range from insignificant to severely impaired, depending on the mutation in IKBKG or NFKBIA . (See "Combined immunodeficiencies: An overview".)
DOCK8 deficiency — Dedicator of cytokinesis 8 (DOCK8) deficiency is a combined immunodeficiency characterized by markedly elevated levels of immunoglobulin E (IgE), hypereosinophilia, atopic disorders, recurrent sinopulmonary infections, cutaneous candidiasis, and viral infections of the skin . These patients have T cell lymphopenia and impaired serologic memory. DOCK8 is a guanine nucleotide exchange factor and an adapter protein important in TLR9 signaling and T cell receptor-driven Wiskott-Aldrich syndrome protein (WASp) activation and cytoskeleton reorganization . DOCK8 deficiency results in impaired TLR9-induced B cell proliferation and immunoglobulin production, leading to poorly sustained protective antibody responses (figure 4). Patients also have a significant reduction in the number of circulating plasmacytoid dendritic cells (PDCs). The PDCs of DOCK8-deficient patients appear to produce less IFN-alpha in response to TLR9 activation, perhaps contributing to their susceptibility to viral infections [71,72]. (See "Combined immunodeficiencies: Specific defects", section on 'DOCK8 deficiency'.)
HOIL-1/HOIP deficiency — Heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1) is a component of the linear ubiquitination chain assembly complex (LUBAC) that is essential for stabilization of the LUBAC and NF-kB activation downstream of the IL-1 receptor. HOIL-1 deficiency is an autosomal recessive disorder that causes impaired IL-1 activation of NF-kB in patient fibroblasts. Conversely, monocytes from these patients are hyper-responsive to IL-1 (figure 5). This condition results in a unique association of immunodeficiency, autoinflammation, periodic fever, and muscular amylopectinosis. Homozygous mutations in HOIL-1 interacting protein (HOIP), the gene encoding the catalytic component of LUBAC, leads to similar cellular impairments and a clinical condition nearly identical to HOIL-1 deficiency [73,74]. (See "Autoinflammatory diseases mediated by NFkB and/or aberrant TNF activity", section on 'LUBAC deficiency'.)
Common variable immunodeficiency — Defects in the functioning of TLR7 and TLR9, normally prominent on B cells and dendritic cells, have been identified in patients with common variable immunodeficiency. These findings may be important in ultimately understanding the impaired B cell maturation that characterizes this disorder. TLR9-induced activation of STAT3 in B cells plays an important role in B cell maturation into memory B cells. A recent report that examined seven patients with CVID demonstrated reduced TLR9-induced activation of STAT3, which may underlie impaired antibody responses in some CVID patients [75-77]. (See "Pathogenesis of common variable immunodeficiency".)
Heterozygous mutations in NF-kB1 (also known as p105/p50), leading to haploinsufficiency of NF-kB1 function and impaired activation of the canonical NF-kB pathway, have been shown to cause common variable immunodeficiency and hypogammaglobulinemia [78,79]. These patients suffered from recurrent sinopulmonary infections, chronic obstructive pulmonary disease (COPD), bronchiectasis, lymphadenopathy, skin infections, autoimmune cytopenias, inflammatory bowel disease, carcinomas, and adenocarcinomas. In addition, heterozygous mutations in NF-kB2 (also known as p100/p52), causing haploinsufficiency of NF-kB2 function and impaired activation of the noncanonical NF-kB pathway, lead to hypogammaglobulinemia, recurrent infections, autoimmunity, and adrenal insufficiency. Defective activation of TLRs has not been described in NF-kB1 or NF-kB2 haploinsufficiency (figure 2).
When to suspect — Immunodeficiencies resulting from defects with TLR pathways are extremely rare. The clinical scenarios in which a TLR pathway defect should be considered include the following:
●An infant or child who develops invasive pyogenic infections (meningitis, adenitis, abscesses) caused by S. pneumoniae, S. aureus, or P. aeruginosa, particularly if there is minimal or no fever or an unexpectedly small rise in CRP. IRAK4 or MyD88 deficiency should be considered in this situation, after more common forms of immunodeficiency have been excluded (figure 2). (See 'MyD88/IRAK4/IRAK1 deficiency' above.)
●The patient with HSV-1 encephalitis could have a defect within the TLR3 pathway, although assessment of TLR function is not helpful, because TLR function is usually normal in blood cells and evaluation of fibroblasts is required. Instead, gene sequencing through targeted gene panels or whole exome sequencing may be the most efficient approach. (See 'UNC93B1 deficiency, TLR3 mutations, TRIF deficiency, TRAF3 deficiency, and TBK1 deficiency' above.)
●The patient with ectodermal dysplasia and recurrent infections (bacterial, viral, or mycobacterial) most likely has a mutation within NEMO or I-kB-alpha, as this combination of findings is relatively specific for these immunodeficiencies. However, not all patients with mutations in these proteins have ectodermal dysplasia. TLR-induced proinflammatory cytokine production has been shown to be variably reduced in many but not all affected individuals. Genetic detection of NEMO mutations can be confounded by the presence of a pseudogene. Genetic analysis can be obtained through commercial laboratories . (See 'NEMO defects' above and 'I-kappa-B defects' above.)
Initial evaluation — The initial evaluation of all patients suffering from recurrent infections begins with a detailed history of past infections, including frequency and severity, the specific sites of infections, and review of any available culture data to identify causative organisms. Because many immunodeficiencies are recessive traits, the clinician must ask about consanguinity within the family, although a lack of consanguinity does not rule out the possibility of compound heterozygous mutations. A family history of recurrent infection only affecting males focuses the evaluation on X-linked disorders.
Physical exam findings can provide important clues to possible immune disorders (eg, lack of tonsils in X-linked agammaglobulinemia or ectodermal dysplasia in NEMO/I-kB-alpha defects).
Laboratory evaluation should start with a complete blood count and differential to assess numbers of neutrophils and lymphocytes. Immunoglobulin levels should be measured, along with an assessment of specific antibody production (tetanus and pneumococcal titers). Immunophenotyping of lymphocytes, including assessment of memory T lymphocyte and memory B lymphocyte populations should be performed, along with T lymphocyte proliferation in response to mitogens and antigens. Immunologic testing is reviewed in more detail separately. (See "Laboratory evaluation of the immune system".)
If these evaluations fail to identify a potential immunodeficiency, further specialized testing is indicated.
Referral and testing for TLR function — Additional testing is best performed by an experienced immunologist who is familiar with this rare group of immunodeficiencies. Testing of TLR function is available through commercial reference laboratories [81,82]. A variety of in vitro methods may be applied. Most measure inflammatory cytokine production (eg, IL-6, IL-1, or tumor necrosis factor [TNF]) after stimulation with one or more TLR ligands (eg, bacterial lipopolysaccharide [LPS], an agonist of TLR4) .
Tests of TLR function are very sensitive to the condition of the cells. Blood should be sent as soon as possible after collection, especially if it is to be shipped a long distance (ship at ambient temperature, protect from heat or cold). If possible, the patient should be clinically well for at least a few weeks prior to testing and not actively recovering from any serious illness. A shipping control from an unrelated healthy donor should accompany the specimen. Abnormal tests need to be confirmed by repeat testing. Persistently abnormal tests should be followed up by specific genetic tests.
Significance of TLR polymorphisms — A rapidly expanding area of research is the epidemiologic study of the many single nucleotide polymorphisms (SNPs) that have been identified in various TLR genes. Some early reports stated that certain variants of TLR2 and TLR4 were associated with impaired signaling after binding their natural ligands [84,85]. SNPs in TLR1 have been associated with candidemia, and SNPS in TLR4 have been associated with invasive aspergillosis among recipients of hematopoietic cell transplants [86,87]. (See "Epidemiology and clinical manifestations of invasive aspergillosis", section on 'Risk factors'.)
However, subsequent reports have challenged these findings and have stated that some of these polymorphisms do not have functional consequences [83,88,89]. It is not clear which is correct. Perhaps the answer depends on the specific stimulus, the response that is measured, or other variables.
ASSOCIATIONS BETWEEN TLRs AND OTHER DISEASES
Atherosclerosis — A large body of evidence has accumulated that various infectious agents have epidemiologic links with atherosclerotic heart disease . These include Chlamydia pneumoniae, Helicobacter pylori, cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), and hepatitis B and C . Chronic infection and serum lipopolysaccharide (LPS) level have been identified as risk factors in a prospective study of the development of atherosclerotic heart disease .
Human atherosclerotic plaques contain endothelial cells and macrophages expressing TLR1/2 and TLR4 . Minimally modified low density lipoprotein is an agonist of TLR4 . It binds independently of CD14 and myeloid differentiation factor-2 (MD-2) and can induce production of chemokines, such as interleukin-8 (IL-8). Animal data strongly suggest roles for TLRs (particularly TLR4) in recognizing foreign (microbial) and self (extracellular matrix, heat shock proteins) molecules, activating endothelial cells, and recruiting and activating monocytes and macrophages to form inflammatory foci that could lead to the formation of atheromatous plaques .
Allergy and asthma — Most studies have found that exposure to bacterial products has been associated with protection from the development of allergic disease, and TLRs may be integral to the mechanisms involved. However, subsequent work suggests that this relationship may not be so straightforward.
●An association between exposure to endotoxin, which forms complexes with TLR4, and protection from allergic sensitization was found in multiple studies. As an example, a report of 61 wheezing infants recorded significantly lower levels of endotoxin in the house dust of those with the highest degree of immunoglobulin E (IgE) sensitization to environmental allergens . Another study showed that lipoteichoic acid, another component of bacterial cell walls, interacts with TLR2 to downregulate the receptor of IgE (Fc-epsilon-RI) on mast cells .
●In contrast, a different model showed that activation of TLR4 on the surface of pulmonary epithelial cells seemed to promote the development of allergic airway responses and asthma . In a mouse model, naturally occurring combinations of dust mite allergens and LPS were found to trigger epithelial cells through TLR4, leading to eosinophil recruitment and the production of the T helper type 2 (Th2) cytokines, interleukin-5 (IL-5) and interleukin-13 (IL-13) . Simultaneous inhalation of a TLR4 antagonist completely eliminated the effect. However, the dose of the TLR agonist also plays a role. In another study, low doses of endotoxin acting on TLR4 were observed to enhance Th2 inflammation in a mouse asthma model, whereas high doses induced a T helper type 1 (Th1) response .
●The importance of early exposure to a rich, diverse microbiota in the prevention of atopy is an active area of research. Microbial components (endotoxin) provide robust activation of TLRs, which shape immune responses . Loss of microbiota diversity, on the other hand, may predispose to the development of atopy and asthma. Microbiome diversity can be influenced by infections and the use of antibiotics [100,101]. Mouse models suggest that TLR activation of CD103+ dendritic cells in the gut may play an important role in inducing development of T regulatory cells, leading to induction of tolerance . Furthermore, increased expression of TLR2 and TLR4 on T regulatory cells may be associated with augmented suppressive functions .
●A meta-analysis of TLR2, TLR4, and TLR9 polymorphisms found relatively minor effects on asthma risk with the possible exception of the TLR2 Arg753Gln alteration, which may have a significant association with asthma risk .
Thus, activation of innate immune responses through TLRs appears to have critical impact on the early stages of allergic disease, although the nature of this influence requires further study.
TLR-BASED THERAPIES — There are several new therapies in use or development that utilize emerging knowledge of TLR biology.
TLR7 agonists — TLR7 agonists have been found to be clinically useful in slowing the growth of malignancies and inhibiting viral replication. However, the precise mechanism of action of these agents remains unknown.
Imiquimod — Imiquimod is a synthetic agonist of TLR7 that has been proven very effective as monotherapy for basal cell carcinoma when applied topically. It is also used in the treatment of actinic keratoses and genital warts. (See "Condylomata acuminata (anogenital warts): Treatment of vulvar and vaginal warts", section on 'Imiquimod' and "Treatment and prognosis of basal cell carcinoma at low risk of recurrence" and "Treatment of actinic keratosis".)
Isatoribine — Isatoribine is another synthetic TLR7 agonist. In 12 adults with chronic hepatitis C infection who were otherwise untreated, seven daily injections of isatoribine reduced blood viral load measured by polymerase chain reaction . The therapy was well-tolerated, although the antiviral effect was modest.
TLR9 and CpG DNA — Coadministration of oligodeoxynucleotides (ODNs) containing cytosine guanine dinucleotide (CpG) motifs during immunization enhances T helper type 1 (Th1) immune responses in animal models . These ODNs mimic bacterial DNA sequences and interact with TLR9. (See 'Specific TLRs' above.)
ODNs are also being studied in human trials. Subcutaneous injection of ODNs alone into healthy volunteers leads to readily measurable increases of serum Th1 type cytokine levels (interleukin-12 [IL-12], interferon-alpha [IFN-alpha]), as well as cytokines generally associated with T helper type 2 (Th2) activation (interleukin-6 [IL-6]) .
Vaccination — In a study of an ODN added to a hepatitis B vaccine (Engerix-B), recipients of the vaccine with added ODN had significantly faster and higher hepatitis B surface antibody responses in comparison with controls who received the vaccine alone . The recipients of the vaccine with ODN also had a significantly higher rate of adverse reactions, although these were mostly mild to moderate in severity. TLR4 agonists are used in several vaccines, including those against cervical cancer, shingles, and hepatitis B (brand names Cervarix, Shingrix, and Fendrix, respectively) . During the SARS-CoV-2 pandemic, highly effective TLR-activating mRNA vaccines against COVID-19 were developed, demonstrating the potential of pattern recognition receptors as adjuvants. (see "Hepatitis B virus immunization in adults" and "Hepatitis B virus immunization in infants, children, and adolescents").
Newborns and infants are more susceptible to infection, particularly by intracellular pathogens. The development of vaccines utilizing TLR agonists within nanoparticles shows promise in inducing adult-like immune responses in neonates, perhaps enhancing immune protection in this vulnerable population .
Cancer therapy — CpG ODN are also being studied as agents for cancer therapy. These have been administered in combination with rituximab in a phase 1 trial in 20 patients with relapsed non-Hodgkin lymphoma . The therapy was well tolerated. Increased levels of mRNA for interferon-inducible genes were detected in the peripheral blood mononuclear cells of patients after receiving ODN.
ODN have also been studied as an adjuvant for a cancer vaccine. Patients with newly diagnosed metastatic colon cancer and elevated serum carcinoembryonic antigen (CEA) levels were given a regimen combining chemotherapy with a cancer vaccine . The vaccine was comprised of a CEA-derived peptide with one of three adjuvants: ODN, granulocyte colony-stimulating factor (G-CSF), or autologous dendritic cells. The vaccines were of apparently equivalent immunogenicity. Eight of 17 (47 percent) patients developed cytotoxic anti-CEA T cell responses, despite having received concomitant immunosuppressive chemotherapy.
Loading of the TLR7 agonist resiquimod onto nanoparticles shows promise in enhancing the immune response against tumor cells. Subcutaneous injection of resiquimod loaded nanoparticles into tumor-bearing mice have been shown to accumulate into tumor-draining lymph nodes where they can activate dendritic cells, leading to a cytotoxic T cell response .
Allergen immunotherapy — Coupling of an ODN to an allergen has been evaluated as possible immunotherapy for atopic disease [114-116]. This strategy uses TLR9 as a potent inducer of Th1 T cell responses, thereby reducing Th2 or atopic responses:
●In one randomized trial, 19 adults allergic to ragweed received subcutaneous injections of a major ragweed pollen allergen (Amb a 1) coupled to an ODN . The injections were well tolerated, and the investigators demonstrated in vitro a shift of the peripheral blood T cell cytokine secretion profile from Th2 ("allergic") to Th1 ("nonallergic").
●In two separate double-blind, placebo-controlled trials of 57 adults  and 25 adults  with ragweed allergy, subjects were randomly assigned to treatment using similar preparations of ragweed pollen allergen coupled to ODN, given by subcutaneous injection. In both trials, patients reported clinical benefits for up to two successive ragweed pollen seasons following a course of only six injections.
●Toll-like receptors (TLRs) belong to a class of molecules known as pattern recognition receptors (PRRs). The ligands for these receptors are components of pathogenic microbes called pathogen-associated molecular patterns (PAMPs). TLRs are part of the innate immune system, although they also modulate mechanisms that impact the development of adaptive immune responses. (See 'Role in innate immunity' above.)
●TLRs are expressed on leukocytes and various solid tissue cells. The cellular localization of TLRs varies. TLR2/1, TLR2/6, TLR4, TLR5, TLR10, and TLR11 are localized to cell surfaces, whereas TLR3, TLR7, TLR8, and TLR9 are localized within endosomes. (See 'Cellular and tissue distribution' above.)
●The structure of all of the TLRs is similar. Receptor structure, organization, ligands, and signaling are summarized in the table (table 1) and figure (figure 1). The final common pathway for TLR signaling involves transcription factors that regulate a multitude of genes, including those encoding important proinflammatory cytokines and type 1 interferons. (See 'Structure and function of TLRs' above.)
●Defects in proximal signaling molecules within TLR signaling pathways, including myeloid differentiation primary response protein 88 (MyD88) and interleukin-1 receptor-associated kinase 4 (IRAK4), have been implicated in primary immunodeficiency to a narrow spectrum of microbes, resulting in susceptibility to pyogenic bacterial infections. (See 'MyD88/IRAK4/IRAK1 deficiency' above.)
●UNC93B1 (UNC93 homolog B1), TLR3, toll/interleukin-1 (IL-1) receptor (TIR) domain-containing adapter-inducing interferon-beta (IFN-beta) (TRIF), tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), and tank-binding kinase 1 (TBK1) mutations have been identified in patients with selective susceptibility to herpes simplex virus type 1 (HSV-1) encephalitis, severe influenza pneumonia and encephalopathy, and severe coronavirus disease 2019 (COVID-19). (See 'UNC93B1 deficiency, TLR3 mutations, TRIF deficiency, TRAF3 deficiency, and TBK1 deficiency' above.)
●Defects in more distal components of TLR signaling pathways (ie, nuclear factor [NF]-kappa-B essential modulator [NEMO], I-kappa-B-alpha, heme-oxidized IRP2 ubiquitin ligase-1 [HOIL-1], and HOIL-1 interacting protein [HOIP]) are associated with broader infectious susceptibilities (bacteria, mycobacteria, viruses, fungi, opportunistic organisms). Dedicator of cytokinesis 8 (DOCK8) deficiency impairs multiple cellular functions in a variety of cells types, leading to a combined immunodeficiency. (See 'TLR signaling defects in primary immunodeficiency' above.)
●TLR-based therapies in development or in use include the TLR7 agonists imiquimod and isatoribine. In addition, oligodeoxynucleotides (ODN) containing cytosine guanine dinucleotide (CpG) motifs, presumably through binding to TLR9, have been found to augment T helper type 1 (Th1) immune responses and have been applied to vaccination, cancer therapy, and immunotherapy for allergic disease. (See 'TLR-based therapies' above.)
●The use of TLR agonists in several vaccines in clinical use demonstrates their utility in the development of highly effective vaccines in the prevention human diseases. (See 'Vaccination' above.)
ACKNOWLEDGMENTS — The UpToDate editorial staff acknowledges Francisco A Bonilla, MD, PhD, who contributed to earlier versions of this topic review.
The UpToDate editorial staff also acknowledges E Richard Stiehm, MD, who contributed as a Section Editor to earlier versions of this topic review.
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