Alessandra Mazzoni

Leucine-rich repeats and pathogen recognition in Toll-like receptors

Toll-like receptors (TLRs) are the major cell-surface initiators of inflammatory responses to pathogens. They bind a wide variety of pathogenic substances through their ectodomains (ECDs). Here, we ask: what is the structural basis for this interaction? Toll-like receptor ECDs comprise 19-25 tandem copies of a motif known as the leucine-rich repeat (LRR). No X-ray structure of a TLR-ECD is currently available but there are several high-resolution LRR-containing proteins that can be used to model TLRs. We suggest that the basic framework of TLRs is a horseshoe-shaped solenoid that contains an extensive beta-sheet on its concave surface, and numerous ligand-binding insertions. Together, these insertions and the beta-sheet could provide a binding surface that is 10-fold greater in area than binding surfaces in antibodies and T-cell receptors.

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.

Myeloid Suppressor Lines Inhibit T Cell Responses by an NO-Dependent Mechanism

CD11b+Gr-1+ myeloid suppressor cells (MSC) accumulate in lymphoid organs under conditions of intense immune stress where they inhibit T and B cell function. We recently described the generation of immortalized MSC lines that provide a homogeneous source of suppressor cells for dissecting the mechanism of suppression. In this study we show that the MSC lines potently block in vitro proliferation of T cells stimulated with either mitogen or antigenic peptide, with as few as 3% of MSC cells causing complete suppression. Inhibition of mitogenic and peptide-specific responses is not associated with a loss in IL-2 production or inability to up-modulate the early activation markers, CD69 and CD25, but results in direct impairment of the three IL-2R signaling pathways, as demonstrated by the lack of Janus kinase 3, STAT5, extracellular signal-regulated kinase, and Akt phosphorylation in response to IL-2. Suppression is mediated by and requires NO, which is secreted by MSC in response to signals from activated T cells, including IFN-γ and a contact-dependent stimulus. Experiments with inducible NO synthase knockout mice demonstrated that the inhibition of T cell proliferation by CD11b+Gr-1+ cells in the spleens of immunosuppressed mice is also dependent upon NO, indicating that the MSC lines accurately represent their normal counterparts. The distinctive capacity of MSC to generate suppressive signals when encountering activated T cells defines a specialized subset of myeloid cells that most likely serve a regulatory function during times of heightened immune activity.

L-arginine metabolism in myeloid cells controls T-lymphocyte functions

Although current attention has focused on regulatory T lymphocytes as suppressors of autoimmune responses, powerful immunosuppression is also mediated by a subset of myeloid cells that enter the lymphoid organs and peripheral tissues during times of immune stress. If these myeloid suppressor cells (MSCs) receive signals from activated T lymphocytes in the lymphoid organs, they block T-cell proliferation. MSCs use two enzymes involved in arginine metabolism to control T-cell responses: inducible nitric oxide synthase (NOS2), which generates nitric oxide (NO) and arginase 1 (Arg1), which depletes the milieu of arginine. Th1 cytokines induce NOS2, whereas Th2 cytokines upregulate Arg1. Induction of either enzyme alone results in a reversible block in T-cell proliferation. When both enzymes are induced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T lymphocytes to undergo apoptosis. Thus, NOS2 and Arg1 might act separately or synergistically in vivo to control specific types of T-cell responses, and selective antagonists of these enzymes might prove beneficial in fighting diseases in which T-cell responses are inappropriately suppressed. This Opinion is the second in a series on the regulation of the immune system by metabolic pathways.

IL-4-Induced Arginase 1 Suppresses Alloreactive T Cells in Tumor-Bearing Mice

We previously demonstrated that a specialized subset of immature myeloid cells migrate to lymphoid organs as a result of tumor growth or immune stress, where they suppress B and T cell responses to Ags. Although NO was required for suppression of mitogen activation of T cells by myeloid suppressor cells (MSC), it was not required for suppression of allogenic responses. In this study, we describe a novel mechanism used by MSC to block T cell proliferation and CTL generation in response to alloantigen, which is mediated by the enzyme arginase 1 (Arg1). We show that Arg1 increases superoxide production in myeloid cells through a pathway that likely utilizes the reductase domain of inducible NO synthase (iNOS), and that superoxide is required for Arg1-dependent suppression of T cell function. Arg1 is induced by IL-4 in freshly isolated MSC or cloned MSC lines, and is therefore up-regulated by activated Th2, but not Th1, cells. In contrast, iNOS is induced by IFN-γ and Th1 cells. Because Arg1 and iNOS share l-arginine as a common substrate, our results indicate that l-arginine metabolism in myeloid cells is a potential target for selective intervention in reversing myeloid-induced dysfunction in tumor-bearing hosts.

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.

TLR9 is localized in the endoplasmic reticulum prior to stimulation.

In mammals, 10 TLRs recognize conserved pathogen-associated molecular patterns, resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN), containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in lysosome-associated membrane protein type 1-positive lysosomes, and we asked which intracellular compartment contains TLR9 before CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum. By contrast, human TLR4 trafficked to the cell surface, indicating that endoplasmic reticulum retention is not a property common to all TLRs. Because TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.

Controlling the Toll road to dendritic cell polarization

The activation of dendritic cells (DC) via Toll‐like receptors (TLRs) plays a decisive role in shaping the outcome of primary immune responses. Following TLR engagement by microbial products, DC migrate from peripheral tissues to lymphoid organs and up‐regulate major histocompatibility complex and costimulatory molecules, acquiring the unique capacity to prime pathogen‐specific, naïve T cells. In addition, DC determine the character of the ensuing immune response by secreting cytokines that drive the development of T cells into T helper cell type 1 (Th1), Th2, or T regulatory effector cells. Three major factors influence the pattern of cytokines released by DC and accordingly, the Th balance: the lineage to which DC belong; the maturation stimulus; and inflammatory mediators present at the site of infection. A major focus of this review is the capacity of DC to integrate these factors and elicit distinct classes of immune responses.

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.

Immortalized myeloid suppressor cells trigger apoptosis in antigen-activated T lymphocytes

We described a generalized suppression of CTL anamnestic responses that occurred in mice bearing large tumor nodules or immunized with powerful recombinant viral immunogens. Immune suppression entirely depended on GM-CSF-driven accumulation of CD11b+/Gr-1+ myeloid suppressor cells (MSC) in secondary lymphoid organs. To further investigate the nature and properties of MSC, we immortalized CD11b+/Gr-1+ cells isolated from the spleens of immunosuppressed mice, using a retrovirus encoding the v-myc and v-raf oncogenes. Immortalized cells expressed monocyte/macrophage markers (CD11b, F4/80, CD86, CD11c), but they differed from previously characterized macrophage lines in their capacities to inhibit T lymphocyte activation. Two MSC lines, MSC-1 and MSC-2, were selected based upon their abilities to inhibit Ag-specific proliferative and functional CTL responses. MSC-1 line was constitutively inhibitory, while suppressive functions of MSC-2 line were stimulated by exposure to the cytokine IL-4. Both MSC lines triggered the apoptotic cascade in Ag-activated T lymphocytes by a mechanism requiring cell-cell contact. Some well-known membrane molecules involved in the activation of apoptotic pathways (e.g., TNF-related apoptosis-inducing ligand, Fas ligand, TNF-α) were ruled out as candidate effectors for the suppression mechanism. The immortalized myeloid lines represent a novel, useful tool to shed light on the molecules involved in the differentiation of myeloid-related suppressors as well as in the inhibitory pathway they use to control T lymphocyte activation.