Sean Stevens

Innate and Adaptive Interleukin-22 Protects Mice from Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease thought to be mediated by dysfunctional innate and/or adaptive immunity. This aberrant immune response leads to the secretion of harmful cytokines that destroy the epithelium of the gastrointestinal tract and thus cause further inflammation. Interleukin-22 (IL-22) is a T helper 17 (Th17) T cell-associated cytokine that is bifunctional in that it has both proinflammatory and protective effects on tissues depending on the inflammatory context. We show herein that IL-22 protected mice from IBD. Interestingly, not only was this protection mediated by CD4+ T cells, but IL-22-expressing natural killer (NK) cells also conferred protection. In addition, IL-22 expression was differentially regulated between NK cell subsets. Thus, both the innate and adaptive immune responses have developed protective mechanisms to counteract the damaging effects of inflammation on tissues.

Humanized mice for modeling human infectious disease: challenges, progress, and outlook.

Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.

F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses

A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laser-assisted injection of either inbred or hybrid ES cells into eight cell–stage embryos efficiently yields F0 generation mice that are fully ES cell–derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.

Alternatively activated macrophage-derived RELM-α is a negative regulator of type 2 inflammation in the lung

Differentiation and recruitment of alternatively activated macrophages (AAMacs) are hallmarks of several inflammatory conditions associated with infection, allergy, diabetes, and cancer. AAMacs are defined by the expression of Arginase 1, chitinase-like molecules, and resistin-like molecule (RELM) α/FIZZ1; however, the influence of these molecules on the development, progression, or resolution of inflammatory diseases is unknown. We describe the generation of RELM-α–deficient (Retnla−/−) mice and use a model of T helper type 2 (Th2) cytokine-dependent lung inflammation to identify an immunoregulatory role for RELM-α. After challenge with Schistosoma mansoni (Sm) eggs, Retnla−/− mice developed exacerbated lung inflammation compared with their wild-type counterparts, characterized by excessive pulmonary vascularization, increased size of egg-induced granulomas, and elevated fibrosis. Associated with increased disease severity, Sm egg–challenged Retnla−/− mice exhibited elevated expression of pathogen-specific CD4+ T cell–derived Th2 cytokines. Consistent with immunoregulatory properties, recombinant RELM-α could bind to macrophages and effector CD4+ Th2 cells and inhibited Th2 cytokine production in a Brutons tyrosine kinase–dependent manner. Additionally, Retnla−/− AAMacs promoted exaggerated antigen-specific Th2 cell differentiation. Collectively, these data identify a previously unrecognized role for AAMac-derived RELM-α in limiting the pathogenesis of Th2 cytokine-mediated pulmonary inflammation, in part through the regulation of CD4+ T cell responses.

Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. We generated RAG2−/−γc−/− mice in which we replaced the gene encoding mouse TPO by its human homolog. Homozygous humanization of TPO led to increased levels of human engraftment in the bone marrow of the hosts, and multilineage differentiation of hematopoietic cells was improved, with an increased ratio of myelomonocytic verus lymphoid lineages. Moreover, maintenance of human stem and progenitor cells was improved, as demonstrated by serial transplantation. Therefore, RAG2−/−γc−/− TPO-humanized mice represent a useful model to study human hematopoiesis in vivo.

Unique functions of the type II interleukin 4 receptor identified in mice lacking the interleukin 13 receptor α1 chain

The interleukin 4 receptor (IL-4R) is a central mediator of T helper type 2 (TH2)–mediated disease and associates with either the common γ-chain to form the type I IL-4R or with the IL-13R α1 chain (IL-13Rα1) to form the type II IL-4R. Here we used Il13ra1−/− mice to characterize the distinct functions of type I and type II IL-4 receptors in vivo. In contrast to Il4ra−/− mice, which have weak TH2 responses, Il13ra1−/− mice had exacerbated TH2 responses. Il13ra1−/− mice showed much less mortality after infection with Schistosoma mansoni and much more susceptibility to Nippostrongylus brasiliensis. IL-13Rα1 was essential for allergen-induced airway hyperreactivity and mucus hypersecretion but not for fibroblast or alternative macrophage activation. Thus, type I and II IL-4 receptors exert distinct effects on immune responses.

Hyperalgesia, synovitis and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis

Background: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 β (IL1β) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis. Objective: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1α and IL1β) on pain, synovitis and systemic inflammatory biomarkers. Methods: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation. Results: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling. Conclusions: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice

Significance The accompanying paper describes the precise, in situ replacement of six megabases of mouse immune genes with the corresponding human immune genes. This manuscript shows that this genomic engineering feat resulted in a unique kind of “HumAb” mouse. Dubbed VelocImmune, these mice efficiently generate antibodies that can be rapidly reformatted into therapeutics. VelocImmune mice have proven to be extraordinarily efficient and productive, generating over a dozen therapeutic candidates that have already progressed into human clinical trials for a variety of important diseases. Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

Goblet Cell-Derived Resistin-Like Molecule β Augments CD4+ T Cell Production of IFN-γ and Infection-Induced Intestinal Inflammation

The secreted goblet cell-derived protein resistin-like molecule β (RELMβ) has been implicated in divergent functions, including a direct effector function against parasitic helminths and a pathogenic function in promoting inflammation in models of colitis and ileitis. However, whether RELMβ influences CD4+ T cell responses in the intestine is unknown. Using a natural model of intestinal inflammation induced by chronic infection with gastrointestinal helminth Trichuris muris, we identify dual functions for RELMβ in augmenting CD4+ Th1 cell responses and promoting infection-induced intestinal inflammation. Following exposure to low-dose Trichuris, wild-type C57BL/6 mice exhibit persistent infection associated with robust IFN-γ production and intestinal inflammation. In contrast, infected RELMβ−/− mice exhibited a significantly reduced expression of parasite-specific CD4+ T cell-derived IFN-γ and TNF-α and failed to develop Trichuris-induced intestinal inflammation. In in vitro T cell differentiation assays, recombinant RELMβ activated macrophages to express MHC class II and secrete IL-12/23p40 and enhanced their ability to mediate Ag-specific IFN-γ expression in CD4+ T cells. Taken together, these data suggest that goblet cell-macrophage cross-talk, mediated in part by RELMβ, can promote adaptive CD4+ T cell responses and chronic inflammation following intestinal helminth infection.

The transcription factor NFATc2 controls IL-6–dependent T cell activation in experimental colitis

The nuclear factor of activated T cells (NFAT) family of transcription factors controls calcium signaling in T lymphocytes. In this study, we have identified a crucial regulatory role of the transcription factor NFATc2 in T cell–dependent experimental colitis. Similar to ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-induced chronic intestinal inflammation. Furthermore, NFATc2 deficiency suppressed colitis induced by oxazolone administration. This finding was associated with enhanced T cell apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by mucosal T lymphocytes. Further studies using knockout mice showed that IL-6, rather than IL-23 and -17, are essential for oxazolone colitis induction. Administration of hyper-IL-6 blocked the protective effects of NFATc2 deficiency in experimental colitis, suggesting that IL-6 signal transduction plays a major pathogenic role in vivo. Finally, adoptive transfer of IL-6 and wild-type T cells demonstrated that oxazolone colitis is critically dependent on IL-6 production by T cells. Collectively, these results define a unique regulatory role for NFATc2 in colitis by controlling mucosal T cell activation in an IL-6–dependent manner. NFATc2 in T cells thus emerges as a potentially new therapeutic target for inflammatory bowel diseases.