Critical Roles of the WASP N-Terminal Domain and Btk in LPS-Induced Inflammatory Response in Macrophages

While Wiskott-Aldrich syndrome protein (WASP) plays critical roles in TCR signaling as an adaptor molecule, how it transduces innate immune signals remains to be elucidated. To investigate the roles of WASP in innate immune cells, we established bone marrow-derived macrophage (BMDM) cell lines from WASP15 transgenic (Tg) mice overexpressing the WASP N-terminal region (exons 1–5). Upon LPS stimulation, WASP15 Tg BMDM cell lines produce lower levels of inflammatory cytokines, such as TNF-α, IL-6, and IL-12p40 than the wild-type BMDM cell line. In addition, the production of nitric oxide by WASP15 Tg BMDM cells in response to LPS and IFN-γ was significantly impaired. Furthermore, we uncovered that the WASP N-terminal domain associates with the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk). Overexpression of the WASP N-terminal domain diminishes the extent of tyrosine phosphorylation of endogenous WASP in WASP15 Tg BMDM cells, possibly by interfering with the specific binding between endogenous WASP and Btk during LPS signaling. These observations strongly suggest that the interaction between WASP N-terminal domain and Btk plays important roles in the LPS signaling cascade in innate immunity

The regulation of IL-10 expression

Interleukin (IL)-10 is an important immunoregulatory cytokine and an understanding of how IL-10 expression is controlled is critical in the design of immune intervention strategies. IL-10 is produced by almost all cell types within the innate (including macrophages, monocytes, dendritic cells (DCs), mast cells, neutrophils, eosinophils and natural killer cells) and adaptive (including CD4(+) T cells, CD8(+) T cells and B cells) immune systems. The mechanisms of IL-10 regulation operate at several stages including chromatin remodelling at the Il10 locus, transcriptional regulation of Il10 expression and post-transcriptional regulation of Il10 mRNA. In addition, whereas some aspects of Il10 gene regulation are conserved between different immune cell types, several are cell type- or stimulus-specific. Here, we outline the complexity of IL-10 production by discussing what is known about its regulation in macrophages, monocytes, DCs and CD4(+) T helper cells

Regulatory T cell-derived extracellular vesicles modify dendritic cell function

Regulatory T cells (Treg) are a subpopulation of T cells that maintain tolerance to self and limit other immune responses. They achieve this through different mechanisms including the release of extracellular vesicles (EVs) such as exosomes as shown by us, and others. One of the ways that Treg derived EVs inhibit target cells such as effector T cells is via the transfer of miRNA. Another key target for the immunoregulatory function of Tregs is the dendritic cells (DCs). In this study we demonstrate directly, and for the first time, that miRNAs are transferred from Tregs to DCs via Treg derived EVs. In particular two miRNAs, namely miR-150-5p and miR-142-3p, were increased in DCs following their interaction with Tregs and Treg derived exosomes. One of the consequences for DCs following the acquisition of miRNAs contained in Treg derived EVs was the induction of a tolerogenic phenotype in these cells, with increased IL-10 and decreased IL-6 production being observed following LPS stimulation. Altogether our findings provide data to support the idea that intercellular transfer of miRNAs via EVs may be a novel mechanism by which Tregs regulate DC function and could represent a mechanism to inhibit immune reactions in tissues

MITOCHONDRIAL MICROSATELLITE MARKERS FOR THE AUSTRALIAN ECTOMYCORRHIZAL FUNGUS LACCARIA SP A (HYDNANGIACEAE)

UNLABELLED: PREMISE OF THE STUDY: Microsatellite loci were developed for the ectomycorrhizal fungus Laccaria sp. A to investigate the population genetic structure of this fungal symbiont across its fragmented distribution in southeastern Australia. • METHODS AND RESULTS: A partial genome sequence from an individual collection of Laccaria sp. A was obtained by 454 genome sequencing. Eight microsatellite markers were selected from 66 loci identified in the genome. The selected markers were highly polymorphic (4-19 alleles per locus, average 13 alleles) and amplified reproducibly from collections made across the distribution of this species. Five of these markers also amplified reproducibly in the sister species Laccaria sp. E (1). All eight of the selected microsatellite loci were from the mitochondrial genome. • CONCLUSIONS: The highly polymorphic markers described here will enable population structure of Laccaria sp. A to be determined, contributing to research on mycorrhizal fungi from a novel distribution