Joanne L. Viney

Mucosal CD8α+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties

Repetitive stimulation of naïve T cells by immature splenic dendritic cells (DC) can result in the differentiation of T‐cell lines with regulatory properties. In the present study we identified a population of DC in the mucosae that exhibit the plasmacytoid phenotype, secrete interferon‐α (IFN‐α) following stimulation with oligodeoxynucleotides containing certain cytosine‐phosphate‐guanosine (CpG) motifs and can differentiate naïve T cells into cells that exhibit regulatory properties. Although these DC appear to be present in both spleen and mesenteric lymph nodes (MLN), only CpG‐matured DC from the MLN (but not the spleen) were able to differentiate naïve T cells into T regulatory 1‐like cells with regulatory properties. The activity of these DC failed to sustain robust T‐cell proliferation and thereby enhanced the suppressive efficacy of CD4+ CD25+ T regulatory cells. These DC are the major CD8α+ DC population in the Peyers patches (PP). Given their significant presence in mucosal tissue, we propose that these DC may provide a mechanistic basis for the homeostatic regulation in the gut by eliciting regulatory cell suppressor function and poorly supporting T helper cell proliferation at a site of high antigenic stimulation like the intestine.

Regulation of mucosal dendritic cell function by receptor activator of NF-kappa B (RANK)/RANK ligand interactions: impact on tolerance induction.

The mucosal immune system is uniquely equipped to discriminate between potentially invasive pathogens and innocuous food proteins. While the mechanisms responsible for induction of mucosal immunity vs tolerance are not yet fully delineated, recent studies have highlighted mucosal dendritic cells (DC) as being important in determining the fate of orally administered Ag. To further investigate the DC:T cell signals involved in regulating the homeostatic balance between mucosal immunity and tolerance, we have examined the expression and function of the TNFR family member receptor activator of NF-κB (RANK) and its cognate ligand, RANKL, in vitro and in vivo. Our data show that although DC isolated from mucosal lymphoid tissues expressed similar levels of surface RANK compared with DC isolated from peripheral lymphoid tissues, DC from the distinct anatomical sites displayed differential responsiveness to RANK engagement with soluble RANKL. Whereas splenic DC responded to RANKL stimulation with elevated IL-12 p40 mRNA expression, Peyer’s patch DC instead preferentially displayed increased IL-10 mRNA expression. Our data also show that the in vivo functional capacity of mucosal DC can be modulated by RANKL. Treatment with RANKL in vivo at the time of oral administration of soluble OVA enhanced the induction of tolerance in two different mouse models. These studies underscore the functional differences between mucosal and peripheral DC and highlight a novel role for RANK/RANKL interactions during the induction of mucosal immune responses.

BTNL2, a Butyrophilin/B7-Like Molecule, Is a Negative Costimulatory Molecule Modulated in Intestinal Inflammation

Butyrophilin-like 2 (BTNL2) is a butyrophilin family member with homology to the B7 costimulatory molecules, polymorphisms of which have been recently associated through genetic analyses to sporadic inclusion body myositis and sarcoidosis. We have characterized the full structure, expression, and function of BTNL2. Structural analysis of BTNL2 shows a molecule with an extracellular region containing two sets of two Ig domains, a transmembrane region, and a previously unreported cytoplasmic tail. Unlike most other butyrophilin members, BTNL2 lacks the prototypical B30.2 ring domain. TaqMan and Northern blot analysis indicate BTNL2 is predominantly expressed in digestive tract tissues, in particular small intestine and Peyer’s patches. Immunohistochemistry with BTNL2-specific Abs further localizes BTNL2 to epithelial and dendritic cells within these tissues. Despite its homology to the B7 family, BTNL2 does not bind any of the known B7 family receptors such as CD28, CTLA-4, PD-1, ICOS, or B and T lymphocyte attenuator. Because of its localization in the gut and potential role in the immune system, BTNL2 expression was analyzed in a mouse model of inflammatory bowel disease. BTNL2 is overexpressed during both the asymptomatic and symptomatic phase of the Mdr1a knockout model of spontaneous colitis. In functional assays, soluble BTNL2-Fc protein inhibits the proliferation of murine CD4+ T cells from the spleen, mesenteric lymph node, and Peyer’s patch. In addition, BTNL2-Fc reduces proliferation and cytokine production from T cells activated by anti-CD3 and B7-related protein 1. These data suggest a role for BTNL2 as a negative costimulatory molecule with implications for inflammatory disease.

High antigen dose and activated dendritic cells enable Th cells to escape regulatory T cell-mediated suppression in vitro.

CD4+CD25+ regulatory T cells (Tregs) are critical for peripheral tolerance and prevention of autoimmunity. In vitro coculture studies have revealed that increased costimulation breaks Treg‐mediated suppression in response to anti‐CD3 or antigen. However, it was unclear whether loss of suppression arose from inactivation of Tregs or whether increased stimulationcaused Th cells to escape suppression. We have investigated conditions that allow or override Treg‐mediated suppression using DO11.10 TCR‐transgenic T cells and chicken ovalbumin peptide 323–339‐pulsed antigen‐presenting cells. Treg suppression of Th proliferation is broken with potent stimulation, using activated spleen cells and high antigen dose, but is intact at low antigen dose. Costimulation with CD80 and CD86 expressed on activated dendritic cells was essential for Th cell escape from suppression at a high antigen dose. Potently stimulated Tregs were functional since they reducedlevels of IL‐2, IFN‐γ, IL‐4 and Th CD25 expression in cocultures. Furthermore, Tregs responding to high antigen dose and activated splenocytes retained the ability to suppress proliferation, but only of Th cells responding to a sub‐optimal dose of independent antigen. Together, our results demonstrate that under conditions of strong antigen‐specific stimulation, Tregs remain functional, but Th cells escape Treg‐mediated suppression.

Dual Infection with Helicobacter bilis and Helicobacter hepaticus in P-Glycoprotein-Deficient mdr1a−/− Mice Results in Colitis that Progresses to Dysplasia

Patients with inflammatory bowel disease (IBD) are at increased risk for developing high-grade dysplasia and colorectal cancer. Animal IBD models that develop dysplasia and neoplasia may help elucidate the link between inflammation and colorectal cancer. Mdr1a-/- mice lack the membrane efflux pump p-glycoprotein and spontaneously develop IBD that can be modulated by infection with Helicobacter sp: H. bilis accelerates development of colitis while H. hepaticus delays disease. In this study, we determined if H. hepaticus infection could prevent H. bilis-induced colitis. Unexpectedly, a proportion of dual-infected mdr1a-/- mice showed IBD with foci of low- to high-grade dysplasia. A group of dual-infected mdr1a-/- animals were maintained long term (39 weeks) by intermittent feeding of medicated wafers to model chronic and relapsing disease. These mice showed a higher frequency of high-grade crypt dysplasia, including invasive adenocarcinoma, possibly because H. hepaticus, in delaying the development of colitis, allows time for transformation of epithelial cells. Colonic epithelial preparations from co-infected mice showed increased expression of c-myc (5- to 12-fold) and interleukin-1alpha/beta (600-fold) by real-time polymerase chain reaction relative to uninfected wild-type and mdr1a-/- animals. This animal model may have particular relevance to human IBD and colorectal cancer because certain human MDR1 polymorphisms have been linked to ulcerative colitis and increased risk for colorectal cancer.

The mdr1a-/- mouse model of spontaneous colitis : A relevant and appropriate animal model to study inflammatory bowel disease

There are many types of colitis models in animals that researchers use to elucidate the mechanism of action of human inflammatory bowel disease (IBD). These models are also used to test novel therapeutics and therapeutic treatment regimens. Here, we will review the characteristics of the mdr1a−/− model of spontaneous colitis that we believe make this model an important part of the IBD researchers toolbox. We will also share new data that will reinforce the fact that this model is relevant in the study of IBD. Mdrla−/− mice lack the murine multiple drug resistance gene for P-glyco-protein 170 that is normally expressed in multiple tissues including intestinal epithelial cells. These mice spontaneously develop a form of colitis at around 12 wk of age. The fact that the complexity of this model mirrors the complexity of disease in humans, as well as recent literature that links MDR1 polymorphisms in humans to Crohns Disease and Ulcerative Colitis, makes this an appropriate animal model to study.

Regulation of costimulation in the era of butyrophilins

The butyrophilin and butyrophilin-like superfamily of molecules has garnered attention in the immunology world in the past few years as a result of the observation that the butyrophilin-like 2 molecule, BTNL2, can alter T cell responsiveness. Additional interest in this superfamily solidified following the discovery that genetic polymorphisms in BTNL2 are associated with predisposition to many human diseases. In this review, we will provide an overview of the members comprising the butyrophilin superfamily of molecules. We will then discuss BTNL2 immunomodulatory function, and BTNL2 structural associations with other costimulatory molecules. We will then draw your attention to some of the lesser-known butyrophilin superfamily members by describing the expression patterns of these molecules in human tissues and cells. And we will finish by hypothesizing on the potential influence on general immune homeostasis that might be mediated by this, thus-far little-studied, family of molecules.

Immune modulation by butyrophilins

The B7 family of co-stimulatory molecules has an important role in driving the activation and inhibition of immune cells. Evolving data have shown that a related family of molecules — the butyrophilins — have similar immunomodulatory functions to B7 family members and may represent a novel subset of co-stimulatory molecules. These studies have taken the field by surprise, as the butyrophilins were previously thought to only be important in lactation and milk production. In this Review, we describe the expression patterns of the various members of the butyrophilin family and explore their immunomodulatory functions. In particular, we emphasize the contribution of butyrophilins to immune homeostasis and discuss the potential of targeting these molecules for therapeutic purposes.

Methods of Inducing Inflammatory Bowel Disease in Mice

Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of disease, identifying new targets for therapeutic intervention, and testing novel therapeutic agents. This unit provides detailed protocols for four of the most commonly used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten‐induced colitis via 2,4,6‐trinitrobenzene sulfonic acid (TNBS), Helicobacter–induced colitis in mdr1a−/− mice, and the CD4+ CD45RBhi SCID transfer colitis model. Curr. Protoc. Pharmacol. 47:5.58.1‐5.58.37.

Getting to the guts of immune regulation.

The mechanisms controlling the balance between tolerance and active immunity in mucosal tissues are critical, but not well understood. Specifically, the normal healthy gut exhibits tolerance both to beneficial antigens derived from food and to innocuous antigens derived from commensal flora, but will mount active and protective immune responses against detrimental and damaging antigens from invading gut pathogens. When such immune control mechanisms go awry, the consequences can be devastating. Loss of tolerance to food antigens can manifest as food allergies, such as coeliac disease, that may afflict about 1 in every 300 persons in several countries in the Western World. Likewise, loss of tolerance to commensal flora may manifest as inflammatory bowel disease.