Alberto Visintin

TLR9 signals after translocating from the ER to CpG DNA in the lysosome

Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA–containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.

LPS-TLR4 Signaling to IRF-3/7 and NF-κB Involves the Toll Adapters TRAM and TRIF

Toll–IL-1–resistance (TIR) domain–containing adaptor-inducing IFN-β (TRIF)–related adaptor molecule (TRAM) is the fourth TIR domain–containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-κB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-α/β, regulated on activation, normal T cell expressed and secreted (RANTES), and γ interferon–inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor–like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.

Regulation of Toll-Like Receptors in Human Monocytes and Dendritic Cells

A number of pathogens induce immature dendritic cells (iDC) to migrate to lymphoid organs where, as mature DC (mDC), they serve as efficient APC. We hypothesized that pathogen recognition by iDC is mediated by Toll-like receptors (TLRs), and asked which TLRs are expressed during the progression of monocytes to mDC. We first measured mRNA levels for TLRs 1–5 and MD2 (a protein required for TLR4 function) by Northern analysis. For most TLRs, message expression decreased severalfold as monocytes differentiated into iDC, but opposing this trend, TLR3 and MD2 showed marked increases during iDC formation. When iDC were induced to mature with LPS or TNF-α, expression of most TLRs transiently increased and then nearly disappeared. Stimulation of iDC, but not mDC, with LPS resulted in the activation of IL-1 receptor-associated kinase, an early component in the TLR signaling pathway, strongly suggesting that LPS signals through a TLR. Surface expression of TLRs 1 and 4, as measured by mAb binding, was very low, corresponding to a few thousand molecules per cell in monocytes, and a few hundred or less in iDC. We conclude that TLRs are expressed in iDC and are involved in responses to at least one pathogen-derived substance, LPS. If TLR4 is solely responsible for LPS signaling in humans, as it is in mice, then its extremely low surface expression implies that it is a very efficient signal transducer in iDC.

Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein

Aeroallergy results from maladaptive immune responses to ubiquitous, otherwise innocuous environmental proteins. Although the proteins targeted by aeroallergic responses represent a tiny fraction of the airborne proteins humans are exposed to, allergenicity is a quite public phenomenon—the same proteins typically behave as aeroallergens across the human population. Why particular proteins tend to act as allergens in susceptible hosts is a fundamental mechanistic question that remains largely unanswered. The main house-dust-mite allergen, Der p 2, has structural homology with MD-2 (also known as LY96), the lipopolysaccharide (LPS)-binding component of the Toll-like receptor (TLR) 4 signalling complex. Here we show that Der p 2 also has functional homology, facilitating signalling through direct interactions with the TLR4 complex, and reconstituting LPS-driven TLR4 signalling in the absence of MD-2. Mirroring this, airway sensitization and challenge with Der p 2 led to experimental allergic asthma in wild type and MD-2-deficient, but not TLR4-deficient, mice. Our results indicate that Der p 2 tends to be targeted by adaptive immune responses because of its auto-adjuvant properties. The fact that other members of the MD-2-like lipid-binding family are allergens, and that most defined major allergens are thought to be lipid-binding proteins, suggests that intrinsic adjuvant activity by such proteins and their accompanying lipid cargo may have some generality as a mechanism underlying the phenomenon of allergenicity.

Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9

Hemozoin (HZ) is an insoluble crystal formed in the food vacuole of malaria parasites. HZ has been reported to induce inflammation by directly engaging Toll-like receptor (TLR) 9, an endosomal receptor. “Synthetic” HZ (β-hematin), typically generated from partially purified extracts of bovine hemin, is structurally identical to natural HZ. When HPLC-purified hemin was used to synthesize the crystal, β-hematin had no inflammatory activity. In contrast, natural HZ from Plasmodium falciparum cultures was a potent TLR9 inducer. Natural HZ bound recombinant TLR9 ectodomain, but not TLR2. Both TLR9 stimulation and TLR9 binding of HZ were abolished by nuclease treatment. PCR analysis demonstrated that natural HZ is coated with malarial but not human DNA. Purified malarial DNA activated TLR9 but only when DNA was targeted directly to the endosome with a transfection reagent. Stimulatory quantities of natural HZ contain <1 μg of malarial DNA; its potency in activating immune responses was even greater than transfecting malarial DNA. Thus, although the malarial genome is extremely AT-rich, its DNA is highly proinflammatory, with the potential to induce cytokinemia and fever during disease. However, its activity depends on being bound to HZ, which we propose amplifies the biological responses to malaria DNA by targeting it to a TLR9+ intracellular compartment.

Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor homolog RP105.

Activation of Toll-like receptor (TLR) signaling by microbial signatures is critical to the induction of immune responses. Such responses demand tight regulation. RP105 is a TLR homolog thought to be mostly B cell specific, lacking a signaling domain. We report here that RP105 expression was wide, directly mirroring that of TLR4 on antigen-presenting cells. Moreover, RP105 was a specific inhibitor of TLR4 signaling in HEK 293 cells, a function conferred by its extracellular domain. Notably, RP105 and its helper molecule, MD-1, interacted directly with the TLR4 signaling complex, inhibiting its ability to bind microbial ligand. Finally, RP105 regulated TLR4 signaling in dendritic cells as well as endotoxin responses in vivo. Thus, our results identify RP105 as a physiological negative regulator of TLR4 responses.

Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization

Atopic diseases such as allergy and asthma are characterized by increases in Th2 cells and serum IgE antibodies. The binding of allergens to IgE on mast cells triggers the release of several mediators, of which histamine is the most prevalent. Here we show that histamine, together with a maturation signal, acts directly upon immature dendritic cells (iDCs), profoundly altering their T cell polarizing capacity. We demonstrate that iDCs express two active histamine receptors, H1 and H2. Histamine did not significantly affect the LPS-driven maturation of iDCs with regard to phenotypic changes or capacity to prime naive T cells, but it dramatically altered the repertoire of cytokines and chemokines secreted by mature DCs. In particular, histamine, acting upon the H2 receptor for a short period of time, increased IL-10 production and reduced IL-12 secretion. As a result, histamine-matured DCs polarized naive CD4(+) T cells toward a Th2 phenotype, as compared with DCs that had matured in the absence of histamine. We propose that the Th2 cells favor IgE production, leading to increased histamine secretion by mast cells, thus creating a positive feedback loop that could contribute to the severity of atopic diseases.

Lysines 128 and 132 Enable Lipopolysaccharide Binding to MD-2, Leading to Toll-like Receptor-4 Aggregation and Signal Transduction

Three cell-surface proteins have been recognized as components of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS): CD14, Toll-like receptor-4 (TLR4), and MD-2. Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation. These studies clarify how MD-2 functions: we found that MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. MD-2 must be bound to TLR4 on the cell surface before binding can occur. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction. TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and identify MD-2 as an attractive target for pharmacological intervention in endotoxin-mediated diseases.

Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolysaccharide (LPS) in mammals, requires the binding of MD-2 to its extracellular domain for maximal responsiveness. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2), we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disulfide-linked oligomers formed from stable dimeric subunits and that concentrations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporter cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum/cis Golgi with different-sized oligomers of MD-2, and excess MD-2 is secreted into the medium. We conclude that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4 and that could play a role in regulating responses to LPS and other pathogen-derived substances in vivo.

The Apoptotic Response to Pneumolysin Is Toll-Like Receptor 4 Dependent and Protects against Pneumococcal Disease

ABSTRACT Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.