Myeloid-Specific Deletion of Mcl-1 Yields Severely Neutropenic Mice That Survive and Breed in Homozygous Form

Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2Cre/CreMcl1flox/flox (Mcl1ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1flox/flox (Mcl1ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease

Specific pathways mediating inflammasome activation by Candida parapsilosis

Candida albicans and C. parapsilosis are human pathogens causing severe infections. The NLRP3 inflammasome plays a crucial role in host defence against C. albicans, but it has been previously unknown whether C. parapsilosis activates this complex. Here we show that C. parapsilosis induces caspase-1 activation and interleukin-1beta (IL-1beta) secretion in THP-1, as well as primary, human macrophages. IL-1beta secretion was dependent on NLRP3, K+-efflux, TLR4, IRAK, Syk, caspase-1, caspase-8 and NADPH-oxidase. Importantly, while C. albicans induced robust IL-1beta release after 4 h, C. parapsilosis was not able to stimulate the production of IL-1beta after this short incubation period. We also found that C. parapsilosis was phagocytosed to a lesser extent, and induced significantly lower ROS production and lysosomal cathepsin B release compared to C. albicans, suggesting that the low extent of inflammasome activation by C. parapsilosis may result from a delay in the so-called "signal 2". In conclusion, this is the first study to examine the molecular pathways responsible for the IL-1beta production in response to a non-albicans Candida species, and these results enhance our understanding about the immune response against C. parapsilosis

New insights on Hygrophorus penarioides and H. penarius (Agaricales, Hygrophoraceae) from Hungary

The ectomycorrhiza forming basidiomycete Hygrophorus penarius sensu lato was recognized as two species, H. penarioides and H. penarius in 2007 based on molecular and morphological evidence. However, this was based on few collections of restricted geographical origin and no studies have performed molecular analysis on additional specimens to date. To this end we sequenced the nrDNA ITS regions of 29 H. penarius s. 1. collections from Hungary and Slovakia and analysed with collections from previous studies. Bayesian and Maximum Likelihood phylogenetic analyses confirmed the separation of H. penarioides and H. penarius in two Glades with the majority of the specimens belonging to H. penarioides and only three to H. penarius. Despite the strong support of the lineages, there was no coherent difference in basidiome and spore traits. We also provide the first evidence of an intraspecific nrDNA sequence variation in the ITS2 region of H. penarioides together with morphological variability that has not been reported to date. On the basis of our result, we conclude that the Quercus associated H. penarioides is far more common in Hungary than the Fagus associated H. penarius and we suggest that identification of the two species in the absence of DNA sequence analysis should be handled with caution, especially when herbarium specimens are examined

Dark septate endophytic pleosporalean genera from semiarid areas

Dark septate endophytes (DSE) are distributed worldwide as root-colonising fungi, and frequent in envi - ronments with strong abiotic stress. DSE is not a taxon, but constitutes numerous fungal taxa belonging to several orders of Ascomycota . In this study we investigate three unidentified DSE lineages belonging to Pleosporales that were found previously in semiarid sandy grasslands. For molecular phylogenetic studies seven loci (ITS, partial 18S nrRNA, 28S nrRNA, actin, calmodulin, transcription-elongation factor 1-α and ß-tubulin genes) were amplified and sequenced. Based on morphology and the resulting molecular phylogeny these isolates were found to represent three novel genera within the Pleosporales , namely Aquilomyces , Flavomyces and Darksidea , with eight novel spe - cies. Molecular data revealed that monotypic Aquilomyces belongs to Morosphaeriaceae , monotypic Flavomyces represents an incertae sedis lineage related to Massarinaceae , and Darksidea , with six new species, is allied to the Lentitheciaceae . During this study we tested numerous conditions to induce sporulation, and managed for the first time to induce several DSE to form their sexual morphs

Investigation of OCH1 in the virulence of Candida parapsilosis using a new neonatal mouse model

Candida parapsilosis is an opportunistic human fungal pathogen that poses a serious threat to low birth weight neonates, particularly at intensive care units. In premature infants, the distinct immune responses to Candida infections are not well understood. Although several in vivo models exist to study systemic candidiasis, only a few are available to investigate dissemination in newborns. In addition, the majority of related studies apply intraperitoneal infection rather than intravenous inoculation of murine infants that may be less efficient when studying systemic invasion. In this study, we describe a novel and conveniently applicable intravenous neonatal mouse model to monitor systemic C. parapsilosis infection. Using the currently developed model, we aimed to analyze the pathogenic properties of different C. parapsilosis strains. We infected 2 days-old BALB/c mouse pups via the external facial vein with different doses of C. parapsilosis strains. Homogenous dissemination of yeast cells was found in the spleen, kidney, liver and brain of infected newborn mice. Colonization of harvested organs was also confirmed by histological examinations. Fungal burdens in newborn mice showed a difference for two isolates of C. parapsilosis. C. parapsilosis CLIB infection resulted in higher colonization of the spleen, kidney and liver of neonatal mice compared to the C. parapsilosis GA1 strain at day 2 after the infection. In a comprehensive study with the adult mice infection, we also presented the attenuated virulence of a C. parapsilosis cell wall mutant (OCH1) in this model. Significantly less och1 Delta/Delta null mutant cells were recovered from the spleen, kidney and liver of newborn mice compared to the wild type strain. When investigating the cytokine response of neonatal mice to C. parapsilosis infection, we found elevated TNF alpha, KC, and IL-1 beta expression levels in all organs examined when compared to the uninfected control. Furthermore, all three measured cytokines showed a significantly elevated expression when newborn mice were infected with och1 Delta/Delta cells compared to the wild type strain. This result further supported the inclusion of OCH1 in C. parapsilosis pathogenicity. To our current knowledge, this is the first study that uses a mice neonatal intravenous infection model to investigate C. parapsilosis infection