Material Supplementary 3.DC1http://www.jimmunol.org/content/suppl/2010/02/15/jimmunol.090401

Calcineurin is a conserved Ca2؉ -calmodulin-activated, serine/threonine-specific protein phosphatase that regulates a variety of physiological processes, e.g., cell cycle progression, polarized growth, and adaptation to salt and alkaline pH stresses. In the pathogenic yeast Cryptococcus neoformans, calcineurin is also essential for growth at 37°C and virulence. To investigate whether calcineurin plays a role in the virulence of Candida albicans, the major fungal pathogen of humans, we constructed C. albicans mutants in which both alleles of the CMP1 gene, encoding the calcineurin catalytic subunit, were deleted. The C. albicans ⌬cmp1 mutants displayed hypersensitivity to elevated Na ؉ , Li ؉ , and Mn 2؉ concentrations and to alkaline pH, phenotypes that have been described after calcineurin inactivation in the related yeast Saccharomyces cerevisiae. Unlike S. cerevisiae calcineurin mutants, which exhibit reduced susceptibility to high Ca 2؉ concentrations, growth of C. albicans was inhibited in the presence of 300 mM CaCl 2 after the deletion of CMP1, demonstrating that there are also differences in calcineurin-mediated cellular responses between these two yeast species. In contrast to C. neoformans, inactivation of calcineurin did not cause temperature sensitivity in C. albicans. In addition, hyphal growth, an important virulence attribute of C. albicans, was not impaired in the ⌬cmp1 mutants under a variety of inducing conditions. Nevertheless, the virulence of the mutants was strongly attenuated in a mouse model of systemic candidiasis, demonstrating that calcineurin signaling is essential for virulence in C. albicans.

Background: Dual specificity phosphatases (DUSPs) have emerged as important regulators of MAPK signaling and responses, including immune responses. Results: Dusp16-deficient gene trap mice were generated and show a phenotype of perinatal lethality and altered cytokine responses to TLR stimulation. Conclusion: Dusp16 has non-redundant functions in the innate immune system. Significance: This is the first description of a mouse model of Dusp16 deficiency.

The purpose of this study was to evaluate the effect of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) on BALB/c mice infected s.c. with the intracellular pathogen Leishmania major. Daily i.p. application of 1 microgram rGM-CSF for 21 days following the infection led to an aggravated course of the disease in most animals. In no case was a therapeutic effect observed. In vitro analysis revealed that the parasite burden was approx. 2- to 7-fold higher in the infected lesions, in the lymph nodes draining the infection and in the spleens of rGM-CSF-treated animals than in tissues from nontreated mice. L. major-infected macrophages obtained from chronically infected mice proliferated in the presence of rGM-CSF in vitro without gaining antiparasitic effector function. However, antiparasitic effector function increased and macrophage growth was inhibited in the presence of recombinant interferon-gamma (IFN-gamma). These data indicate that rGM-CSF-induced macrophage proliferation alone is not sufficient to overcome infections with intracellular pathogens like L. major, since simultaneous activation of macrophages by IFN-gamma is required.

The intracellular pathogen Coxiella (C.) burnetii causes Q fever, a usually self-limiting respiratory infection that becomes chronic and severe in some patients. Innate immune recognition of C. burnetii and its role in the decision between resolution and chronicity is not understood well. However, TLR2 is important for the response to C. burnetii in mice, and genetic polymorphisms in Myd88 have been associated with chronic Q fever in humans. Here, we have employed MyD88-deficient mice in infection models with the attenuated C. burnetii Nine Mile phase II strain (NMII). Myd88−/− macrophages failed to restrict the growth of NMII in vitro, and to upregulate production of the cytokines TNF, IL-6, and IL-10. Following intraperitoneal infection, NMII bacterial burden was significantly higher on day 5 and 20 in organs of Myd88−/− mice. After infection via the natural route by intratracheal injection, a higher bacterial load in the lung and increased dissemination of NMII to other organs was observed in MyD88-deficient mice. While wild-type mice essentially cleared NMII on day 27 after intratracheal infection, it was still readily detectable on day 42 in multiple organs in the absence of MyD88. Despite the elevated bacterial load, Myd88−/− mice had less granulomatous inflammation and expressed significantly lower levels of chemoattractants, inflammatory cytokines, and of several IFNγ-induced genes relevant for control of intracellular pathogens. Together, our results show that MyD88-dependent signaling is essential for early control of C. burnetii replication and to prevent systemic spreading. The continued presence of NMII in the organs of Myd88−/− mice constitutes a new mouse model to study determinants of chronicity and resolution in Q fever.

First quantitative phosphoproteome analysis of TDM-activated macrophages reveals new insights in biological processes of macrophages stimulated with the mycobacterial cord factor. Surprisingly, the bioinformatic results revealed Mincle-dependent and-independent phosphorylation, which appear to affect different biological processes. Whereas PI3K/AKT signaling, dependent on Mincle, is involved in TDM-induced cytokine regulation, Mincle-independent phosphorylation and transcriptomic changes were linked to cell cycle regulation. Collectively, the observed reprogramming of macrophages by TDM might be relevant in the mycobacteriamacrophage interaction.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis‐aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti‐microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1−/− mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1−/− mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1−/− mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1‐derived itaconate is a key factor in the macrophage‐mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.

This information is current as GBP1 Antigen Presentation and Expression of Enhanced Inflammation yet Impaired towards γ Macrophage Response to IFN-Mycobacterial Cord Factor Reprograms the Lang