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Summary: Mouse p202 containing two hemopoietic expression, interferon inducibility, nuclear localization (HIN) domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found that only HIN1 of p202 binds double-stranded DNA (dsDNA), while HIN2 forms a homotetramer. Crystal structures of HIN1 revealed that dsDNA is bound on face opposite the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with the AIM HIN domain. We propose that this results in a spatial separation of the AIM2 pyrin domains, and indeed p202 prevented the dsDNA-dependent clustering of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility. [Copyright &y& Elsevier]

Abstract: The murine mononuclear phagocyte (MNP) system comprises a diverse population of cells, including monocytes, dendritic cells (DC) and macrophages. Derived from the myeloid haematopoietic lineage, this group of cells express a variety of well characterized surface markers. Expression of the integrin alpha X (Itgax, CD11c) is commonly used to identify classical DC, and similarly expression of colony stimulating factor 1 receptor (Csf1r, CD115) to identify macrophages. We have characterized the expression of these markers using a variety of transgenic mouse models. We confirmed previous observations of Itgax expression in anatomically defined subsets of MNPs in secondary lymphoid organs, including all MNPs identified within the germinal centres. The majority of MNPs in the intestinal lamina propria and lung express Itgax. All mucosal Itgax expressing cells also express Csf1r suggesting Csf1-dependent haematopoietic derivation. This double-positive population included germinal centre MNPs. These data reveal that Itgax expression alone does not specifically define classical DC. These results suggest more cautious interpretation of Itgax-dependent experimentation and direct equation with uniquely DC-mediated activities, particularly in the functioning of non-lymphoid MNPs within the intestinal lamina propria. [Copyright &y& Elsevier]

Abstract: We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor (tlr)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA micro-arrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R. [Copyright &y& Elsevier]

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169+ macrophages in vivo eliminates F4/80+ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells. [Display omitted] • Genuine macrophages are absent from hematopoietic tissue single-cell preparations • Fragmented remnants of macrophages containing cytoplasm adhere to other cells • Macrophage remnants convolute hematopoietic tissue flow cytometry analysis • Remnant-derived macrophage-RNA is misattributed to other cells in RNA-seq data Millard et al. demonstrate intact macrophages are absent in hematopoietic tissue single-cell suspensions as they are disrupted during preparation. Macrophage remnants containing membrane and intracellular contents remain attached to other cells within the suspensions. This results in misassignment of macrophage identity and misattribution of macrophage-expressed genes. [ABSTRACT FROM AUTHOR]

Responses to cytosolic DNA can protect against both infectious organisms and the mutagenic effect of DNA integration. Recognition of invading DNA is likely to be fundamental to eukaryotic cellular life, but has been described only in mammals. Introduction of DNA into chicken macrophages induced type I interferon mRNA via a pathway conserved with mammals, requiring the receptor cGAS and the signalling protein STING. A second pathway of cytosolic DNA recognition in mammalian macrophages, initiated by absent in melanoma 2 (AIM2), results in rapid inflammasome-mediated pyroptotic cell death. AIM2 is restricted to mammals. Nevertheless, chicken macrophages underwent lytic cell death within 15 min of DNA transfection. The mouse AIM2-mediated response requires double stranded DNA, but chicken cell death was maintained with denatured DNA. This appears to be a novel form of rapid necrotic cell death, which we propose is an ancient response rendered redundant in mammalian macrophages by the appearance of the AIM2 inflammasome. The retention of these cytosolic DNA responses through evolution, with both conserved and non-conserved mechanisms, suggests a fundamental importance in cellular defence. [ABSTRACT FROM AUTHOR]

Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors. [ABSTRACT FROM AUTHOR]