In-speck-ting Inflammasomes by Flow
Inflammasome complex formation in macrophages is triggered by the detection of microbial products and results in proteolytic activation of IL-1β and IL-18. One of the defining features of this formation is the rapid relocation of diffuse apoptosis-associated speck-like protein containing a CARD (ASC) from the cytoplasm and nucleus to the inflammasome site, clustering in what appears as a “speck” in the cell. It has been difficult to quantify inflammasome activity directly without manually counting ASC-containing cells via microscopy. To formulate an alternative quantitative measure of inflammasome activity, Sester et al. (p. 455) developed a flow cytometry assay dubbed “time of flight inflammasome evaluation” (TOFIE) that detects cellular changes that occur during rapid relocalization of ASC. To do this, they treated LPS-primed bone marrow–derived macrophages with nigericin to induce inflammasome activation, followed by a specific ASC immunostaining protocol to enable detection of this protein via flow cytometry. Using a flow cytometer capable of detecting pulse width allowed the investigators to compare the pulse width to pulse area profile (W:A) and pulse width to pulse height ratio in activated versus unstimulated cells. Activated cells exhibited a substantially decreased W:A profile, reflecting the sudden relocalization of diffuse ASC into a punctate speck in the cell. The results of TOFIE were validated by comparison with traditional microscopic techniques. The TOFIE technique was further used to identify inflammasome-competent cells in mixed cell populations in both mice and humans. This protocol enables measurement of inflammasome activity in cells more rapidly than counting ASC specks via microscopy and more quantitatively than indirect measurement of inflammasome activity via other methodologies.
Shedding Stemness with Lupus Locus
Numerous disease susceptibility genes have been linked to the autoimmune disorder systemic lupus erythematosus (SLE), but the contributions of these genes are not well understood. Defects in mesenchymal stem cells (MSCs) are thought to contribute to immune dysregulation in lupus, and the immunosuppressive functions of MSCs are critical to maintaining immune homeostasis. Lu et al. (p. 43) used several genetically engineered mouse strains to characterize the function of Pbx1-d, a dominant negative splice isoform of the pre–B cell leukemia homeobox (Pbx1) gene, which has been shown previously to be the only gene present in the lupus susceptibility locus Sle1a1. Mice carrying the Sle1a1 locus expressed higher levels of Pbx1-d transcripts in MSCs compared with congenic C57BL/6 (B6) mice. Sle1a1 MSCs derived from bone marrow cultures multiplied more rapidly and developed a more “differentiated” phenotype relative to B6 MSCs. Expression analysis confirmed that Sle1a1 MSCs had a significant global decrease in gene expression associated with “stemness” or pluripotent capacity, and a shift toward expression of genes associated with innate immunity and inflammation. Relative to B6 control MSCs, Sle1a1 MSCs were less able to suppress lupus-associated immune responses, including TLR-induced Ab production from B cells or IL-2 secretion by activated CD4+ T cells. Together, these results indicated that Pbx1-d expressed from the Sle1a1 locus can alter MSC function, contributing to lupus immunopathogenesis and perhaps also explaining why allogeneic MSC immunotherapy is unsuccessful in lupus patients.
Doubts about DTR in DCs
The tissue-specific expression of diphtheria toxin receptor (DTR) in mice and subsequent treatment with DT has been used as a strategy to specifically ablate a cell population of interest in immunological studies. Previously, it was thought that extraneous expression of DTR in cells did not adversely affect the immune cell repertoire, but in this issue, van Blijswijk et al. (p. 307) report that DTR expression in dendritic cells (DCs) may have immunological consequences in the absence of DT treatment. The authors crossed mice expressing Cre recombinase under the control of Clec9a, which encodes a molecule expressed on conventional DC (cDC) precursors, to ROSA26-LSL-DTR mice, a strain commonly crossed with Cre transgenic mice to induce tissue-specific expression of DTR. In the resulting Clec9a+/CreROSAiDTR mice, CD8α+ DCs and most CD11b+ DCs could be specifically ablated using DT treatment. However, comparisons of PBS-treated Clec9a+/CreROSAiDTR mice with genetically matched wild-type controls revealed that mice expressing DTR exhibited reduced DC frequency in skin-draining and mesenteric LNs as well as hypocellularity in other immune populations at these sites, but not in the spleen or thymus. These results suggested that DTR transgene expression in the absence of DT treatment had marked effects on immune cell populations. Reduced DC frequencies and hypocellularity were also found in some, but not all, DC-specific DTR models, suggesting that this phenomenon was not an artifact generated in the Clec9a+/CreROSAiDTR model. Specific expression of DTR on mouse B cells did not adversely impact the frequencies of different immune cell populations or total cell numbers in LNs, suggesting this phenotype may be limited to mouse models with DC-specific expression of DTR. Together, these results suggest that DTR expression in DCs has previously unforeseen biological consequences on the immune system that must be taken into account when interpreting data generated using this specific ablation strategy.
Turn Down Those TLRs
Evidence in the chronic lymphocytic leukemia (CLL) field suggests that some CLL clones are transformed autoreactive B cells. Tan et al. (p. 101) hypothesized that CLL clones expressing autoreactive BCRs bind Ag from apoptotic cells and internalize released nucleic acids that stimulate TLR7 and TLR9, thereby triggering CLL proliferation. The authors investigated whether rolipram, a type 4 cAMP phosphodiesterase inhibitor that has been shown to induce apoptosis in CLL cells, was able to disrupt these signals from TLR7 and TLR9 and halt CLL activation. Rolipram successfully inhibited TLR7- and TLR9-induced CD40 and CD54 upregulation on CLL cells, as well as proliferation in response to culture with irradiated autologous cells or multiple TLR agonists. Interestingly, rolipram was effective even on CLL clones carrying unmutated IgH V regions, which is an indicator of poor prognosis. Clones with unmutated IgH V regions were resistant to apoptosis following TLR9 stimulation, but treatment with rolipram abrogated this effect. The investigators showed that rolipram interfered with TLR7 and TLR9 signaling by preventing nuclear translocation of NF-κB and IRF5. Furthermore, rolipram was able to reduce or inhibit cytokine secretion following TLR stimulation in both CLL and healthy cells. These findings suggest that phosphodiesterase inhibitors such as rolipram could be used to dampen TLR signaling as therapy for autoimmunity as well as CLL.
β-catenin Alters DC Decisions
Different dendritic cell (DC) subsets serve specific functions in the initiation and regulation of adaptive immune responses. In the context of autoimmunity, recent work has implicated β-catenin signaling in promoting a tolerogenic DC phenotype. In this issue, Cohen et al. (p. 210) show that β-catenin can also promote proinflammatory DC maturation in response to pathogens. The authors employed a mouse model expressing phosphorylation-resistant (constitutively active) β-catenin specifically in CD11c+ cells. In these “mutant mice,” stabilized β-catenin in CD11c+ cells skewed differentiation toward CD8α+, plasmacytoid, and CD103+CD11b– DCs through the action of IRF8. The authors then investigated the impact of this altered DC subset distribution on T.gondii or vaccinia virus infection. In both cases, CD4+ T cells and NK cells from mutant mice produced more IFN-γ than those from littermate controls. Ag-specific CD8+ T cell numbers and IFN-γ secretion were also elevated following infection with vaccinia virus, possibly due to cross-priming by CD8α+ DCs. Consistent with an aberrantly strong proinflammatory response, mutant mice succumbed to T. gondii infection, despite a parasite burden comparable to that of littermate controls. These results highlight an unexpected role for β-catenin in influencing the response to infection through promoting the maturation of proinflammatory DCs.
- Copyright © 2014 by The American Association of Immunologists, Inc.
