The stalk domain and the glycosylation status of the activating natural killer cell receptor NKp30 are important for ligand binding.

The natural cytotoxicity receptors are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The human natural cytotoxicity receptor family comprises the three type I membrane proteins NKp30, NKp44, and NKp46. Especially NKp30 is critical for the cytotoxicity of NK cells against different targets including tumor, virus-infected, and immature dendritic cells. Although the crystal structure of NKp30 was recently solved (Li, Y., Wang, Q., and Mariuzza, R. A. (2011) J. Exp. Med. 208, 703-714; Joyce, M. G., Tran, P., Zhuravleva, M. A., Jaw, J., Colonna, M., and Sun, P. D. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 6223-6228), a key question, how NKp30 recognizes several non-related ligands, remains unclear. Therefore, we investigated the parameters that impact ligand recognition of NKp30. Based on various NKp30-hIgG1-Fc fusion proteins, which were optimized for minimal background binding to cellular Fcγ receptors, we identified the flexible stalk region of NKp30 as an important but so far neglected module for ligand recognition and related signaling of the corresponding full-length receptor proteins. Moreover, we found that the ectodomain of NKp30 is N-linked glycosylated at three different sites. Mutational analyses revealed differential binding affinities and signaling capacities of mono-, di-, or triglycosylated NKp30, suggesting that the degree of glycosylation could provide a switch to modulate the ligand binding properties of NKp30 and NK cell cytotoxicity.

Priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota.

Mononuclear phagocytes are an important component of an innate immune system perceived as a system ready to react upon encounter of pathogens. Here, we show that in response to microbial stimulation, mononuclear phagocytes residing in nonmucosal lymphoid organs of germ-free mice failed to induce expression of a set of inflammatory response genes, including those encoding the various type I interferons (IFN-I). Consequently, NK cell priming and antiviral immunity were severely compromised. Whereas pattern recognition receptor signaling and nuclear translocation of the transcription factors NF-κB and IRF3 were normal in mononuclear phagocytes of germ-free mice, binding to their respective cytokine promoters was impaired, which correlated with the absence of activating histone marks. Our data reveal a previously unrecognized role for postnatally colonizing microbiota in the introduction of chromatin level changes in the mononuclear phagocyte system, thereby poising expression of central inflammatory genes to initiate a powerful systemic immune response during viral infection.

RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells.

The mucosal immune system of the intestine is separated from a vast array of microbes by a single layer of epithelial cells. Cues from the commensal microflora are needed to maintain epithelial homeostasis, but the molecular and cellular identities of these cues are unclear. Here we provide evidence that signals from the commensal microflora contribute to the differentiation of a lymphocyte population coexpressing stimulatory natural killer cell receptors and the transcription factor RORgammat that produced interleukin 22 (IL-22). The emergence of these IL-22-producing RORgammathiNKp46+NK1.1(int) cells depended on RORgammat expression, which indicated that these cells may have been derived from lymphoid tissue-inducer cells. IL-22 released by these cells promoted the production of antimicrobial molecules important in the maintenance of mucosal homeostasis.

Lymphadenopathy in a novel mouse model of Bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/beta.

In immunocompetent humans, cat scratch disease (CSD) is elicited by the Gram-negative bacterium Bartonella henselae and is characterized by a benign regional lymphadenopathy, the pathogenesis of which is poorly understood. Here, we describe a novel mouse model of Bartonella-induced CSD-like disease that allowed us to investigate the mechanisms leading to lymphadenopathy in vivo. In wild-type mice, a subcutaneous inoculation of either viable or inactivated B. henselae led to a strong swelling of the draining lymph node, which was long-lasting despite the rapid elimination of the bacteria. Carboxyfluorescein- and bromodesoxyuridine-labeling experiments showed that lymph node enlargement resulted from modified immigration and enhanced proliferation of lymphocytes, preferentially of B cells. A comparative analysis of B. henselae and the rodent pathogen B. grahamii in wild-type versus interferon-alpha/beta-receptor I chain-deficient mice revealed that interferon-alpha/beta is not only differentially induced by these two Bartonella species but also exerts an inhibitory effect on the development of lymphadenopathy both in vitro and in vivo. These data demonstrate that the lymphadenopathy of human CSD can be reproduced and studied in a mouse model and provide the first insights into the underlying immunological mechanisms.

Dendritic cells prime natural killer cells by trans-presenting interleukin 15.

Natural killer (NK) cells are important effector cells in the control of infections. The cellular and molecular signals required for NK cell activation in vivo remain poorly defined. By using a mouse model for the inducible ablation of dendritic cells (DCs), we showed that the in vivo priming of NK cell responses to viral and bacterial pathogens required the presence of CD11c(high) DCs. After peripheral Toll-like receptor (TLR) stimulation, NK cells were recruited to local lymph nodes, and their interaction with DCs resulted in the emergence of effector NK cells in the periphery. NK cell priming was dependent on the recognition of type I IFN signals by DCs and the subsequent production and trans-presentation of IL-15 by DCs to resting NK cells. CD11c(high) DC-derived IL-15 was necessary and sufficient for the priming of NK cells. Our data define a unique in vivo role of DCs for the priming of NK cells, revealing a striking and previously unappreciated homology to T lymphocytes of the adaptive immune system.

In vitro susceptibility of Bartonella species to 17 antimicrobial compounds: comparison of Etest and agar dilution.

OBJECTIVES: In vitro susceptibility testing of 31 Bartonella spp. strains including 21 Bartonella henselae isolates was performed for 17 antimicrobial agents (telithromycin, four macrolides, five fluoroquinolones, five aminoglycosides, doxycycline and rifampicin). METHODS: MICs were determined by agar dilution and Etest using chocolate agar containing 5% defibrinated sheep blood as assay medium. Longer incubation periods of 3-5 days in a humid atmosphere with 5% CO(2) were required until bacterial growth became visible and MICs could be read. RESULTS: The ketolide telithromycin was the most active agent exhibiting the lowest MICs. The Bartonella spp. were also highly susceptible to macrolides, particularly clarithromycin, and to doxycycline and rifampicin, with MICs of

Mycoplasma pneumoniae infection and Tourette's syndrome.

An association between infection and Tourette's syndrome (TS) has been described repeatedly. A role for streptococcal infection (PANDAS) has been established for several years, but the involvement of other infectious agents such as Borrelia Burgdorferi or Mycoplasma pneumoniae has only been described in single case reports. We examined antibody titers against M. pneumoniae and various types of antibodies by immunoblot in patients and in a sex- and age-matched comparison group. Participants comprised 29 TS patients and 29 controls. Antibody titers against M. pneumoniae were determined by microparticle agglutination (MAG) assay and confirmed by immunoblot. Elevated titers were found in significantly more TS patients than controls (17 vs. 1). Additionally, the number of IgA positive patients was significantly higher in the TS group than in the control group (9 vs. 1). A higher proportion of increased serum titers and especially of IgA antibodies suggests a role for M. pneumoniae in a subgroup of patients with TS and supports the finding of case reports implicating an acute or chronic infection with M. pneumoniae as one etiological agent for tics. An autoimmune reaction, however, has to be taken into account. In predisposed persons, infection with various agents including M. pneumoniae should be considered as at least an aggravating factor in TS.

Hemin binding, functional expression, and complementation analysis of Pap 31 from Bartonella henselae.

Growth of Bartonella henselae is strongly heme dependent, and B. henselae is unable to synthesize heme itself. At least five outer membrane-associated proteins from B. henselae bind hemin, including the 31-kDa protein designated Pap31. The gene of this protein was heterologously expressed in Escherichia coli M15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction. Complementation of the hemA-deficient mutant E. coli K-12 EB53 (aroB tsx malT hemA) with pap31 demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis of B. henselae.