Jochen Mattner

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.

Lysosomal Glycosphingolipid Recognition by NKT Cells

NKT cells represent a distinct lineage of T cells that coexpress a conserved αβ T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized both by mouse and human NKT cells. Impaired generation of lysosomal iGb3 in mice lacking β-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. We suggest that expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity.

Liver Autoimmunity Triggered by Microbial Activation of Natural Killer T Cells

Summary Humans with primary biliary cirrhosis (PBC), a disease characterized by the destruction of small bile ducts, exhibit signature autoantibodies against mitochondrial Pyruvate Dehydrogenase Complex E2 (PDC-E2) that crossreact onto the homologous enzyme of Novosphingobium aromaticivorans, an ubiquitous alphaproteobacterium. Here, we show that infection of mice with N. aromaticivorans induced signature antibodies against microbial PDC-E2 and its mitochondrial counterpart but also triggered chronic T cell-mediated autoimmunity against small bile ducts. Disease induction required NKT cells, which specifically respond to N. aromaticivorans cell wall α-glycuronosylceramides presented by CD1d molecules. Combined with the natural liver tropism of NKT cells, the accumulation of N. aromaticivorans in the liver likely explains the liver specificity of destructive responses. Once established, liver disease could be adoptively transferred by T cells independently of NKT cells and microbes, illustrating the importance of early microbial activation of NKT cells in the initiation of autonomous, organ-specific autoimmunity.

The role of type I interferons in non‐viral infections

Summary:  For a long time, the family of type I interferons (IFN‐α/β) has received little attention outside the fields of virology and tumor immunology. In recent years, IFN‐α/β regained the interest of immunologists, due to the phenotypic and functional characterization of IFN‐α/β‐producing cells, the definition of novel immunomodulatory functions and signaling pathways of IFN‐α/β, and the observation that IFN‐α/β not only exerts antiviral effects but is also relevant for the pathogenesis or control of certain bacterial and protozoan infections. This review summarizes the current knowledge on the production and function of IFN‐α/β during non‐viral infections in vitro and in vivo.

Cutting Edge: Impaired Glycosphingolipid Trafficking and NKT Cell Development in Mice Lacking Niemann-Pick Type C1 Protein

Niemann-Pick Type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked Vα14-Jα18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent α-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of Vα14-Jα18 NKT cells.

Protection against Progressive Leishmaniasis by IFN-β

Type I IFNs (IFN-αβ) exert potent antiviral and immunoregulatory activities during viral infections, but their role in bacterial or protozoan infections is poorly understood. In this study, we demonstrate that the application of low, but not of high doses of IFN-β protects 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral disease after infection with a high (106) or low (104) number of Leishmania major parasites, respectively. IFN-β treatment of BALB/c mice restored the NK cell cytotoxic activity, increased the lymphocyte proliferation, and augmented the production of IFN-γ and IL-12 in the draining lymph node. Low, but not high doses of IFN-β caused enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the levels of suppressor of cytokine signaling-1, and up-regulated the expression of inducible NO synthase in vivo. The IFN-β-induced increase of IFN-γ production was dependent on STAT4. Protection by IFN-β strictly required the presence of inducible NO synthase. In the absence of STAT4 or IL-12, IFN-β led to an amelioration of the cutaneous and visceral disease, but was unable to prevent its progression. These results identify IFN-β as a novel cytokine with a strong, dose-dependent protective effect against progressive cutaneous leishmaniasis that results from IL-12- and STAT4-dependent as well as -independent events.

Cytokines, Signaling Pathways, and Effector Molecules Required for the Control of Leishmania (Viannia) braziliensis in Mice

ABSTRACT Cutaneous leishmaniasis is caused by protozoan parasites of the genus Leishmania. The mechanisms of pathogen control have been established primarily in the mouse model of Leishmania major infection, but they might not hold true for other Leishmania species associated with cutaneous disease. Here, we analyzed the role of cytokines, signaling components, and effector molecules in the control of New World cutaneous leishmaniasis due to L. braziliensis. Unlike L. major, L. braziliensis caused small, nonulcerative, and self-healing skin swelling in C57BL/6 mice, as well as BALB/c mice. In contrast to the results obtained for L. mexicana, mice deficient for interleukin-12 or its key signaling molecule, signal transducer and activator of transcription 4, rapidly succumbed to severe visceral leishmaniasis. Infection of tumor necrosis factor knockout mice with L. braziliensis led to progressive, nonhealing skin lesions with erosions and hemorrhagic ulcerations, but in contrast to the results with L. major, only 20 to 30% of the mice developed fatal visceral disease. As seen with L. major, mice with a deleted inducible nitric oxide synthase gene (iNOS−/−) were unable to contain L. braziliensis in the skin, whereas the control of the parasite in the spleen remained unimpaired. Unlike what happens in L. major infections, NADPH oxidase had no impact on the course of disease in L. braziliensis-infected mice. These results not only define essential components of a protective immune response to L. braziliensis but also illustrate that the requirements for the control of cutaneous leishmaniasis vary between different parasite species.

Regulation of type 2 nitric oxide synthase by type 1 interferons in macrophages infected with Leishmania major.

We recently reported that the infection of macrophages with Leishmania major led to the release of type 1 interferons (IFN‐α /β ). Moreover, at day 1 of infection of mice with L. major, IFN‐α /β  was required for the expression of type 2 (inducible) NO synthase (NOS2 or iNOS) which, however, was restricted to a few macrophages in the dermis. Here, we further characterized the regulation of NOS2 by IFN‐α /β . Macrophages that were either simultaneously or sequentially exposed to L. major promastigotes and IFN‐α /β  expressed NOS2 and anti‐leishmanial activity. In contrast, when high amounts of IFN‐α /β  were used or when IFN‐α /β  was added to the macrophages 2 h prior to the parasites, almost no induction of NOS2 was observed. After pretreatment with IFN‐α /β , tyrosine phosphorylation and nuclear DNA binding of Stat1α, the degradation of the NF‐κB inhibitor (IκBα and β), and the nuclear translocation of NF‐κB were strongly impaired compared with macrophages exposed to IFN‐α /β  and L. major simultaneously. Thus, IFN‐α /β  exerts agonistic or antagonistic effects on the expression of NOS2 in macrophages infected with a microbial pathogen, depending on the sequence of the stimuli and the amount of IFN‐α /β  added. The limited number of NOS2‐positive macrophages at day 1 of infection in vivo might result from a blockage of non‐infected macrophages by IFN‐α /β  that is released by neighboring infected cells.

Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope.

Clostridium difficile is the cause of emerging nosocomial infections that result in abundant morbidity and mortality worldwide. Thus, the development of a vaccine to kill the bacteria to prevent this disease is highly desirable. Several recently identified bacterial surface glycans, such as PS-I and PS-II, are promising vaccine candidates to preclude C. difficile infection. To circumvent difficulties with the generation of natural PS-I due to its low expression levels in bacterial cultures, improved chemical synthesis protocols for the pentasaccharide repeating unit of PS-I and oligosaccharide substructures were utilized to produce large quantities of well-defined PS-I related glycans. The analysis of stool and serum samples obtained from C. difficile patients using glycan microarrays of synthetic oligosaccharide epitopes revealed humoral immune responses to the PS-I related glycan epitopes. Two different vaccine candidates were evaluated in the mouse model. A synthetic PS-I repeating unit CRM197 conjugate was immunogenic in mice and induced immunoglobulin class switching as well as affinity maturation. Microarray screening employing PS-I repeating unit substructures revealed the disaccharide Rha-(1→3)-Glc as a minimal epitope. A CRM197-Rha-(1→3)-Glc disaccharide conjugate was able to elicit antibodies recognizing the C. difficile PS-I pentasaccharide. We herein demonstrate that glycan microarrays exposing defined oligosaccharide epitopes help to determine the minimal immunogenic epitopes of complex oligosaccharide antigens. The synthetic PS-I pentasaccharide repeating unit as well as the Rha-(1→3)-Glc disaccharide are promising novel vaccine candidates against C. difficile that are currently in preclinical evaluation.

Synthesis and evaluation of stimulatory properties of Sphingomonadaceae glycolipids

Glycosphingolipids (GSLs) from the Sphingomonadaceae family of bacteria have been reported to be potent stimulators of natural killer T cells. These glycolipids include mono-, tri- and tetraglycosylceramides. Here we have prepared the GSL-1 to GSL-4 series of glycolipids and tested their abilities to stimulate natural killer T cells. Among these glycolipids, only GSL-1 (1) is a potent stimulator. Using a series of synthetic diglycosylceramides, we show that oligoglycosylceramides from Sphingomonadaceae are not effectively truncated to GSL-1 in lysosomes in antigen-presenting cells, possibly because the higher-order GSLs are poor substrates for lysosomal acyltransfer enzymes.