Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity.

Innate immunity provides the first line of defence against invading pathogens and provides important cues for the development of adaptive immunity. Type-2 immunity-responsible for protective immune responses to helminth parasites and the underlying cause of the pathogenesis of allergic asthma-consists of responses dominated by the cardinal type-2 cytokines interleukin (IL)4, IL5 and IL13 (ref. 5). T cells are an important source of these cytokines in adaptive immune responses, but the innate cell sources remain to be comprehensively determined. Here, through the use of novel Il13-eGFP reporter mice, we present the identification and functional characterization of a new innate type-2 immune effector leukocyte that we have named the nuocyte. Nuocytes expand in vivo in response to the type-2-inducing cytokines IL25 and IL33, and represent the predominant early source of IL13 during helminth infection with Nippostrongylus brasiliensis. In the combined absence of IL25 and IL33 signalling, nuocytes fail to expand, resulting in a severe defect in worm expulsion that is rescued by the adoptive transfer of in vitro cultured wild-type, but not IL13-deficient, nuocytes. Thus, nuocytes represent a critically important innate effector cell in type-2 immunity.

IL-33 is more potent than IL-25 in provoking IL-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction.

BACKGROUND: IL-25 and IL-33 belong to distinct cytokine families, but experimental mouse studies suggest their immunologic functions in type 2 immunity are almost entirely overlapping. However, only polymorphisms in the IL-33 pathway (IL1RL1 and IL33) have been significantly associated with asthma in large-cohort genome-wide association studies. OBJECTIVE: We sought to identify distinct pathways for IL-25 and IL-33 in the lung that might provide insight into their roles in asthma pathogenesis and potential for therapeutic intervention. METHODS: IL-25 receptor-deficient (Il17rb(-/-)), IL-33 receptor-deficient (ST2, Il1rl1(-/-)), and double-deficient (Il17rb(-/-)Il1rl1(-/-)) mice were analyzed in models of allergic asthma. Microarrays, an ex vivo lung slice airway contraction model, and Il13(+/eGFP) mice were then used to identify specific effects of IL-25 and IL-33 administration. RESULTS: Comparison of IL-25 and IL-33 pathway-deficient mice demonstrates that IL-33 signaling plays a more important in vivo role in airways hyperreactivity than IL-25. Furthermore, methacholine-induced airway contraction ex vivo increases after treatment with IL-33 but not IL-25. This is dependent on expression of the IL-33 receptor and type 2 cytokines. Confocal studies with Il13(+/eGFP) mice show that IL-33 more potently induces expansion of IL-13-producing type 2 innate lymphoid cells, correlating with airway contraction. This predominance of IL-33 activity is enforced in vivo because IL-33 is more rapidly expressed and released in comparison with IL-25. CONCLUSION: Our data demonstrate that IL-33 plays a critical role in the rapid induction of airway contraction by stimulating the prompt expansion of IL-13-producing type 2 innate lymphoid cells, whereas IL-25-induced responses are slower and less potent.

Chronic antigen stimulation alone is sufficient to drive CD8+ T cell exhaustion.

The failure of CD8(+) T cells to respond to chronic infection has been termed "exhaustion" and describes the condition in which CD8(+) T cells exhibit reduced differentiation, proliferation, and effector function. CD8(+) T cell exhaustion has been extensively studied in the murine model of chronic infection, lymphocytic choriomeningitis virus (LCMV). Although LCMV-based studies have yielded many interesting findings, they have not allowed for discrimination between the roles of cytokine- and Ag-driven exhaustion. We have created a system of chronic Ag stimulation using murine influenza A virus that leads to exhaustion and functional disability of virus-specific CD8(+) T cells, in the absence of high viral titers, sustained proinflammatory cytokine production and lymphocyte infection. Our findings show that Ag alone is sufficient to drive CD8(+) T cell impairment, that Ag-driven loss of virus-specific CD8(+) T cells is TRAIL mediated, and that removal of Ag reverses exhaustion. Although programmed death 1 was up-regulated on chronic Ag-stimulated CD8(+) T cells, it played no role in the exhaustion. These findings provide a novel insight into the mechanisms that control functional exhaustion of CD8(+) T cells in chronic infection.

Development of cell-based immunoassays to measure type I collagen in cultured fibroblasts.

Excessive deposition of type I collagen by activated fibroblasts is a hallmark of scarring and fibrotic pathologies. Quantitation of collagen I at the protein level is paramount to measure functionally relevant changes during pathological remodeling of the extracellular matrix. We describe two new cell-based assays to directly quantify the amount of collagen I incorporated into the extracellular matrix of primary human lung fibroblasts. Utilizing a monoclonal antibody specific to native human collagen I, we optimized conditions and parameters including incubation time, specificity and cell density to demonstrate dose-dependent induction of collagen I by transforming growth factor beta, as measured by in-cell enzyme linked immunosorbent assay. The results obtained by this assay were mimicked by an "In situ Quantitative Western Blot" on cultured cells using the same antibody. Results from these assays were comparable to those obtained with a commercial assay for collagen I N-propeptide, which is an index of collagen formation. These assays have been optimized for a 96-well format and provide a novel and useful approach for screening of anti-fibrotic agents in vitro. The assays described here also offer a significant improvement in throughput and specificity over conventional methods that primarily measure soluble collagen.