Interferon Regulatory Factor 8 (IRF8) is required for development, maturation and expression of anti-microbial defenses of myeloid cells. BXH2 mice harbor a severely hypomorphic allele at Irf8 ...(Irf8(R294C)) that causes susceptibility to infection with intracellular pathogens including Mycobacterium tuberculosis. We report that BXH2 are completely resistant to the development of cerebral malaria (ECM) following Plasmodium berghei ANKA infection. Comparative transcriptional profiling of brain RNA as well as chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) was used to identify IRF8-regulated genes whose expression is associated with pathological acute neuroinflammation. Genes increased by infection were strongly enriched for IRF8 binding sites, suggesting that IRF8 acts as a transcriptional activator in inflammatory programs. These lists were enriched for myeloid-specific pathways, including interferon responses, antigen presentation and Th1 polarizing cytokines. We show that inactivation of several of these downstream target genes (including the Irf8 transcription partner Irf1) confers protection against ECM. ECM-resistance in Irf8 and Irf1 mutants is associated with impaired myeloid and lymphoid cells function, including production of IL12p40 and IFNγ. We note strong overlap between genes bound and regulated by IRF8 during ECM and genes regulated in the lungs of M. tuberculosis infected mice. This IRF8-dependent network contains several genes recently identified as risk factors in acute and chronic human inflammatory conditions. We report a common core of IRF8-bound genes forming a critical inflammatory host-response network.
Here, we focus on Leishmania extracellular vesicles (EVs) and their DNA content, detailing a protocol for the isolation of these nanoparticles and their subsequent genomic characterization. We ...describe a robust and comprehensive approach for obtaining, storing, and analyzing EVs derived from cultured parasites. We detail a user-friendly bioinformatics pipeline for sequence analysis and visualization of CNV analysis and ploidy changes.
For complete details on the use and execution of this protocol, please refer to Douanne et al. (2022).1
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•Isolation of extracellular vesicles from Leishmania parasites and DNA analysis•Comprehensive guidelines for obtaining, storing, and analyzing EVs•User-friendly bioinformatics pipeline for sequence analysis•Steps for visualization of CNV analysis and ploidy changes
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Here, we focus on Leishmania extracellular vesicles (EVs) and their DNA content, detailing a protocol for the isolation of these nanoparticles and their subsequent genomic characterization. We describe a robust and comprehensive approach for obtaining, storing, and analyzing EVs derived from cultured parasites. We detail a user-friendly bioinformatics pipeline for sequence analysis and visualization of CNV analysis and ploidy changes.
IRF8 (Interferon Regulatory Factor 8) plays an important role in defenses against intracellular pathogens, including several aspects of myeloid cells function. It is required for ontogeny and ...maturation of macrophages and dendritic cells, for activation of anti-microbial defenses, and for production of the Th1-polarizing cytokine interleukin-12 (IL-12) in response to interferon gamma (IFNγ) and protection against infection with Mycobacterium tuberculosis. The transcriptional programs and cellular pathways that are regulated by IRF8 in response to IFNγ and that are important for defenses against M. tuberculosis are poorly understood. These were investigated by transcript profiling and chromatin immunoprecipitation on microarrays (ChIP-chip). Studies in primary macrophages identified 368 genes that are regulated by IRF8 in response to IFNγ/CpG and that behave as stably segregating expression signatures (eQTLs) in F2 mice fixed for a wild-type or mutant allele at IRF8. A total of 319 IRF8 binding sites were identified on promoters genome-wide (ChIP-chip) in macrophages treated with IFNγ/CpG, defining a functional G/AGAAnTGAAA motif. An analysis of the genes bearing a functional IRF8 binding site, and showing regulation by IFNγ/CpG in macrophages and/or in M. tuberculosis-infected lungs, revealed a striking enrichment for the pathways of antigen processing and presentation, including multiple structural and enzymatic components of the Class I and Class II MHC (major histocompatibility complex) antigen presentation machinery. Also significantly enriched as IRF8 targets are the group of endomembrane- and phagosome-associated small GTPases of the IRG (immunity-related GTPases) and GBP (guanylate binding proteins) families. These results identify IRF8 as a key regulator of early response pathways in myeloid cells, including phagosome maturation, antigen processing, and antigen presentation by myeloid cells.
CCDC88B is a risk factor for several chronic inflammatory diseases in humans and its inactivation causes a migratory defect in DCs in mice. CCDC88B belongs to a family of cytoskeleton-associated ...scaffold proteins that feature protein:protein interaction domains. Here, we identified the Rho/Rac Guanine Nucleotide Exchange Factor 2 (ARHGEF2) and the RAS Protein Activator Like 3 (RASAL3) as CCDC88B physical and functional interactors. Mice defective in Arhgef2 or Rasal3 show dampened neuroinflammation, and display altered cellular response and susceptibility to colitis; ARHGEF2 maps to a human Chromosome 1 locus associated with susceptibility to IBD. Arhgef2 and Rasal3 mutant DCs show altered migration and motility in vitro, causing either reduced (Arhgef2) or enhanced (Rasal3) migratory properties. The CCDC88B/RASAL3/ARHGEF2 complex appears to regulate DCs migration by modulating activation of RHOA, with ARHGEF2 and RASAL3 acting in opposite regulatory fashions, providing a molecular mechanism for the involvement of these proteins in DCs immune functions.
While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the ...inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response.
IRF8 and IRF1 are transcriptional regulators that play critical roles in the development and function of myeloid cells, including activation of macrophages by proinflammatory signals such as ...interferon-γ (IFN-γ). Loss of IRF8 or IRF1 function causes severe susceptibility to infections in mice and in humans. We used chromatin immunoprecipitation sequencing and RNA sequencing in wild type and inIRF8andIRF1mutant primary macrophages to systematically catalog all of the genes bound by (cistromes) and transcriptionally activated by (regulomes) IRF8, IRF1, PU.1, and STAT1, including modulation of epigenetic histone marks. Of the seven binding combinations identified, two (cluster 1 IRF8/IRF1/STAT1/PU.1 and cluster 5 IRF1/STAT1/PU.1) were found to have a major role in controlling macrophage transcriptional programs both at the basal level and after IFN-γ activation. They direct the expression of a set of genes, the IRF8/IRF1 regulome, that play critical roles in host inflammatory and antimicrobial defenses in mouse models of neuroinflammation and of pulmonary tuberculosis, respectively. In addition, this IRF8/IRF1 regulome is enriched for genes mutated in human primary immunodeficiencies and with loci associated with several inflammatory diseases in humans.
Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a ...key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H(+) NK cells from Ly49h-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and Ly49h-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the Ifng gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific cis-regulatory mechanism of Ifnγ transcript expression potentially relevant to NK cell function in health and disease.
The influence of powder characteristics on the moldability of metal injection molding feedstock is relatively well understood, but remains difficult to quantify other than by real-scale injections. ...This study aims to correlate feedstock moldability performances with melt viscosity and dry powder rheology. Four stainless steel powders exhibiting different particle sizes and shapes were tested in dry conditions using an FT4 powder rheometer to obtain thirteen different powder rheology-related metrics. These powders were subsequently mixed with a wax-based binder to formulate four feedstocks that were tested in melt conditions using a rotational rheometer to characterize the injection-related rheology of the feedstock formulations. The moldability of the feedstocks was finally quantified using real-scale injections performed into a spiral mold cavity to measure the injected lengths, and the correlation between the dry and melt rheology metrics and the real-scale injection moldability was evaluated. Results showed that the feedstock melt viscosities and the injected lengths were almost perfectly correlated. Furthermore, two dry powder rheology metrics, namely, the normalized basic flow energy and the conditioned powder bulk density, showed the correlation with the feedstock moldability which is comparable to that obtained with the melt rheology. The results of this study indicate that although the feedstock melt viscosity and the dry powder rheology take fewer variables into account as compared to real-scale injections, these simplified tests (especially dry powder rheology) could be considered as rapid and reliable approaches to assessing the impact of powder characteristics on the moldability of powder-binder feedstocks used in metal injection molding.
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•MIM powders were characterized for the first time by a powder rheometer in this paper.•The effect of size and shape of the powders on their rheology was clearly observable.•The powders were mixed with binders to create LPIM feedstocks.•The feedstocks were characterized by viscosity and moldabilty measurements.•Strong correlation was found between moldability, viscosity, and dry rheology.
Recent advances in genome analysis have provided important insights into the genetic architecture of infectious and inflammatory diseases. The combined analysis of loci detected by genome-wide ...association studies (GWAS) in 22 inflammatory diseases has revealed a shared genetic core and associated biochemical pathways that play a central role in pathological inflammation. Parallel whole-exome sequencing studies have identified 265 genes mutated in primary immunodeficiencies (PID). Here, we examine the overlap between these two data sets, and find that it consists of genes essential for protection against infections and in which persistent activation causes pathological inflammation. Based on this intersection, we propose that, although strong or inactivating mutations (rare variants) in these genes may cause severe disease (PIDs), their more subtle modulation potentially by common regulatory/coding variants may contribute to chronic inflammation.
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