ILC2s chew the fat.

In this issue of JEM, Rana et al. (https://doi.org/10.1084/jem.20190689) report that adipose tissue multipotent stromal cells (MSCs) provide multifaceted support for adipose tissue-resident ILC2s through contact-mediated proliferation and IL-33-mediated stress-induced activation

Leishmania major LACK Antigen Is Required for Efficient Vertebrate Parasitization

The Leishmania major LACK antigen is a key target of the immune response in susceptible BALB/c mice and remains a viable vaccine candidate for human leishmaniasis. We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting. Parasites containing a single lack gene in either the upstream or downstream locus grew comparably to wild-type promastigotes in vitro, but failed to parasitize BALB/c mice efficiently, even in a T cell–deficient environment. The replication of single copy lack mutants as amastigotes was attenuated in macrophages in vitro, and parasites failed to increase in numbers in immunodeficient mice, despite their persistence over months. Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes. Conversely, attempts to generate lack-null parasites failed, suggesting that LACK is required for parasite viability. These data suggest that LACK is critical for effective mammalian parasitization and thus represents a potential drug target for leishmaniasis

Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s.

Group 2 innate lymphoid cells (ILC2s) and type 2 helper T cells (Th2 cells) are the primary source of interleukin 5 (IL-5) and IL-13 during type 2 (allergic) inflammation in the lung. In Th2 cells, T cell receptor (TCR) signaling activates the transcription factors nuclear factor of activated T cells (NFAT), nuclear factor κB (NF-κB), and activator protein 1 (AP-1) to induce type 2 cytokines. ILC2s lack a TCR and respond instead to locally produced cytokines such as IL-33. Although IL-33 induces AP-1 and NF-κB, NFAT signaling has not been described in ILC2s. In this study, we report a nonredundant NFAT-dependent role for lipid-derived leukotrienes (LTs) in the activation of lung ILC2s. Using cytokine reporter and LT-deficient mice, we find that complete disruption of LT signaling markedly diminishes ILC2 activation and downstream responses during type 2 inflammation. Type 2 responses are equivalently attenuated in IL-33- and LT-deficient mice, and optimal ILC2 activation reflects potent synergy between these pathways. These findings expand our understanding of ILC2 regulation and may have important implications for the treatment of airways disease

Efficient generation of long-distance conditional alleles using recombineering and a dual selection strategy in replicate plates

<p>Abstract</p> <p>Background</p> <p>Conditional knockout mice are a useful tool to study the function of gene products in a tissue-specific or inducible manner. Classical approaches to generate targeting vectors for conditional alleles are often limited by the availability of suitable restriction sites. Furthermore, plasmid-based targeting vectors can only cover a few kB of DNA which precludes the generation of targeting vectors where the two <it>lox</it>P sites are placed far apart. These limitations have been overcome in the recent past by using homologous recombination of bacterial artificial chromosomes (BACs) in <it>Escherichia coli </it>to produce large targeting vector containing two different <it>lox</it>P-flanked selection cassettes so that a single targeting event is sufficient to introduce <it>lox</it>P-sites a great distances into the mouse genome. However, the final targeted allele should be free of selection cassettes and screening for correct removal of selection cassettes can be a laborious task. Therefore, we developed a new strategy to rapidly identify ES cells containing the desired allele.</p> <p>Results</p> <p>Using BAC recombineering we generated a single targeting vector which contained two different selection cassettes that were flanked by <it>lox</it>P-<it>lox</it>P sites or by FRT-FRT/<it>lox</it>P sites so that they could be deleted sequentially by Cre- and FLPe-recombinases, respectively. Transfected ES cells were first selected in the presence of both antibiotics <it>in vitro </it>before correctly targeted clones were identified by Southern blot. After transfection of a Cre recombinase expression plasmid ES cell clones were selected on replicate plates to identify those clones which maintained the FRT-FRT/<it>lox</it>P flanked cassette and lost the <it>lox</it>P-<it>lox</it>P flanked cassette. Using this strategy facilitated the identification of ES cell clones containing the desired allele before blastocyst injection.</p> <p>Conclusion</p> <p>The strategy of ES cell cultures in replicate plates proved to be very efficient in identifying ES cells that had undergone the correct recombination event. This approach facilitates the generation of conditional knock-out mice when large parts of the genome are intended to be flanked by <it>lox</it>P sites.</p

Type 2 immunity is controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system

Nippostrongylus brasiliensis infection and ovalbumin-induced allergic lung pathology are highly interleukin (IL)-4/IL-13 dependent, but the contributions of IL-4/IL-13 from adaptive (T helper [Th]2 cells) and innate (eosinophil, basophils, and mast cells) immune cells remain unknown. Although required for immunoglobulin (Ig)E induction, IL-4/IL-13 from Th2 cells was not required for worm expulsion, tissue inflammation, or airway hyperreactivity. In contrast, innate hematopoietic cell–derived IL-4/IL-13 was dispensable for Th2 cell differentiation in lymph nodes but required for effector cell recruitment and tissue responses. Eosinophils were not required for primary immune responses. Thus, components of type 2 immunity mediated by IL-4/IL-13 are partitioned between T cell–dependent IgE and an innate non-eosinophil tissue component, suggesting new strategies for interventions in allergic immunity

A Two-Step Process for Cytokine Production Revealed by IL-4 Dual-Reporter Mice

SummaryTo monitor IL-4 expression at the single-cell level, we generated mice with insertions of different reporter genes into both copies of the Il4 gene that permitted the simultaneous analysis of IL-4 transcripts via GFP and IL-4 protein secretion by use of huCD2. Innate and adaptive cells competent for IL-4 production were marked by GFP, while cells that presently or recently secreted IL-4 additionally displayed huCD2. After challenge with the strictly enteric helminth, Heligmosomoides polygyrus, GFP-positive innate and adaptive cells disseminated widely, but IL-4 secretion was predominantly mediated by CD4+ T cells in the intestines and draining lymphoid organs. IL-4-competent cells persisted in cured animals, and memory responses reflected rapid cytokine production at the site of rechallenge. These data reveal a two-step process for cytokine production: the first generating poised cells that disseminate systemically and the second inducing the rapid production of the cytokine in response to local stimulation

MicroRNA regulation of type 2 innate lymphoid cell homeostasis and function in allergic inflammation.

MicroRNAs (miRNAs) exert powerful effects on immunity through coordinate regulation of multiple target genes in a wide variety of cells. Type 2 innate lymphoid cells (ILC2s) are tissue sentinel mediators of allergic inflammation. We established the physiological requirements for miRNAs in ILC2 homeostasis and immune function and compared the global miRNA repertoire of resting and activated ILC2s and T helper type 2 (TH2) cells. After exposure to the natural allergen papain, mice selectively lacking the miR-17∼92 cluster in ILC2s displayed reduced lung inflammation. Moreover, miR-17∼92-deficient ILC2s exhibited defective growth and cytokine expression in response to IL-33 and thymic stromal lymphopoietin in vitro. The miR-17∼92 cluster member miR-19a promoted IL-13 and IL-5 production and inhibited expression of several targets, including SOCS1 and A20, signaling inhibitors that limit IL-13 and IL-5 production. These findings establish miRNAs as important regulators of ILC2 biology, reveal overlapping but nonidentical miRNA-regulated gene expression networks in ILC2s and TH2 cells, and reinforce the therapeutic potential of targeting miR-19 to alleviate pathogenic allergic responses

Parasitic helminths induce fetal-like reversion in the intestinal stem cell niche.

Epithelial surfaces form critical barriers to the outside world and are continuously renewed by adult stem cells1. Whereas dynamics of epithelial stem cells during homeostasis are increasingly well understood, how stem cells are redirected from a tissue-maintenance program to initiate repair after injury remains unclear. Here we examined infection by Heligmosomoides polygyrus, a co-evolved pathosymbiont of mice, to assess the epithelial response to disruption of the mucosal barrier. H. polygyrus disrupts tissue integrity by penetrating the duodenal mucosa, where it develops while surrounded by a multicellular granulomatous infiltrate2. Crypts overlying larvae-associated granulomas did not express intestinal stem cell markers, including Lgr53, in spite of continued epithelial proliferation. Granuloma-associated Lgr5- crypt epithelium activated an interferon-gamma (IFN-γ)-dependent transcriptional program, highlighted by Sca-1 expression, and IFN-γ-producing immune cells were found in granulomas. A similar epithelial response accompanied systemic activation of immune cells, intestinal irradiation, or ablation of Lgr5+ intestinal stem cells. When cultured in vitro, granuloma-associated crypt cells formed spheroids similar to those formed by fetal epithelium, and a sub-population of H. polygyrus-induced cells activated a fetal-like transcriptional program, demonstrating that adult intestinal tissues can repurpose aspects of fetal development. Therefore, re-initiation of the developmental program represents a fundamental mechanism by which the intestinal crypt can remodel itself to sustain function after injury