ARPC1B binds WASP to control actin polymerization and curtail tonic signaling in B cells.

Immune cells exhibit low-level, constitutive signaling at rest (tonic signaling). Such tonic signals are required for fundamental processes, including the survival of B lymphocytes, but when they are elevated by genetic or environmental causes, they can lead to autoimmunity. Events that control ongoing signal transduction are, therefore, tightly regulated by submembrane cytoskeletal polymers like F-actin. The actin-binding proteins that underpin the process, however, are poorly described. By investigating patients with ARPC1B deficiency, we report that ARPC1B-containing ARP2/3 complexes are stimulated by Wiskott Aldrich Syndrome protein (WASP) to nucleate the branched actin networks that control tonic signaling from the B cell receptor (BCR). Despite an upregulation of ARPC1A, ARPC1B-deficient cells were not capable of WASP-mediated nucleation by ARP2/3, and this caused the loss of WASP-dependent structures, including podosomes in macrophages and lamellipodia in B cells. In the B cell compartment, ARPC1B deficiency also led to weakening of the cortical F-actin cytoskeleton that normally curtails the diffusion of BCRs and ultimately resulted in increased tonic lipid signaling, oscillatory calcium release from the endoplasmic reticulum (ER), and phosphorylated Akt. These events contributed to skewing the threshold for B cell activation in response to microbial-associated molecular patterns (MAMPs). Thus, ARPC1B is critical for ARP2/3 complexes to control steady-state signaling of immune cells.

The E3 ubiquitin ligase UBR5 interacts with TTC7A and may be associated with very early onset inflammatory bowel disease.

Very early onset inflammatory bowel disease (VEOIBD) denotes children with onset of IBD before six years of age. A number of monogenic disorders are associated with VEOIBD including tetratricopeptide repeat domain 7A (TTC7A) deficiency. TTC7A-deficiency is characterized by apoptotic colitis in milder cases with severe intestinal atresia and immunodeficiency in cases with complete loss of protein. We used whole exome sequencing in a VEOIBD patient presenting with colitis characterized by colonic apoptosis and no identified known VEOIBD variants, to identify compound heterozygous deleterious variants in the Ubiquitin protein ligase E3 component N-recognin 5 (UBR5) gene. Functional studies demonstrated that UBR5 co-immunoprecipitates with the TTC7A and the UBR5 variants had reduced interaction between UBR5 and TTC7A. Together this implicates UBR5 in regulating TTC7A signaling in VEOIBD patients with apoptotic colitis.

Human interleukin-4-treated regulatory macrophages promote epithelial wound healing and reduce colitis in a mouse model.

Murine alternatively activated macrophages can exert anti-inflammatory effects. We sought to determine if IL-4-treated human macrophages [i.e., hM(IL4)] would promote epithelial wound repair and can serve as a cell transfer treatment for inflammatory bowel disease (IBD). Blood monocytes from healthy volunteers and patients with active and inactive IBD were converted to hM(IL4)s. IL-4 treatment of blood-derived macrophages from healthy volunteers and patients with inactive IBD resulted in a characteristic CD206+CCL18+CD14low/- phenotype (RNA-seq revealed IL-4 affected expression of 996 genes). Conditioned media from freshly generated or cryopreserved hM(IL4)s promoted epithelial wound healing in part by TGF, and reduced cytokine-driven loss of epithelial barrier function in vitro. Systemic delivery of hM(IL4) to dinitrobenzene sulphonic acid (DNBS)-treated Rag1-/- mice significantly reduced disease. These findings from in vitro and in vivo analyses provide proof-of-concept support for the development of autologous M(IL4) transfer as a cellular immunotherapy for IBD.

Drug Screen Identifies Leflunomide for Treatment of Inflammatory Bowel Disease Caused by TTC7A Deficiency.

BACKGROUND & AIMS: Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient and develop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe and untreatable, and it recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease. METHODS: We developed a high-throughput screen to identify compounds approved by the US Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout (TTC7A-KO) HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsy samples of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport. RESULTS: TTC7A-KO HAP1 cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-KO cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and X-linked inhibitor of apoptosis (XIAP); incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a-/- zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function. CONCLUSIONS: In a drug screen, we identified leflunomide as an agent that reduces apoptosis and activates AKT signaling in TTC7A-KO cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.

Association of Indoor Tanning Exposure With Age at Melanoma Diagnosis and BRAF V600E Mutations.

There is limited information on how indoor tanning promotes melanoma development. We investigated indoor tanning use in patients with melanomas in sun-exposed skin and studied the clinicopathological and molecular characteristics in relation to indoor tanning exposure. Patients from a multidisciplinary clinic for cutaneous cancers completed standardized questionnaires on risk factors for melanoma as a component of medical history at their initial consultations. For this study, we included patients from December 2013 to May 2015. The 114 patients who reported indoor tanning exposure were younger at diagnosis than the 222 patients who did not (51.5 vs 64.0 years, two-sided P < .001). BRAF V600E genotype was more prevalent in ever-users than in nonusers (42.9% vs 28.3%, two-sided P = .04) and higher in ever-users who initiated indoor tanning prior to age 25 years compared with age 25 years or older (62.2% vs 31.1%, two-sided P = .003). There were more melanomas in intermittently sun-exposed skin in ever-users than nonusers (65.7% vs 51.9%, respectively, two-sided P = .02). Our data suggest indoor tanning may promote melanomas that arise in skin with low-chronic sun-induced damage through BRAF V600E-mediated melanomagenesis.

Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Macrophages play central roles in immunity as early effectors and modulating adaptive immune reponses; we implicated macrophages in the anticolitic effect of infection with the tapeworm Hymenolepis diminuta. Here, gene arrays revealed that H. diminuta antigen (HdAg) evoked a program in murine macrophages distinct from that elicited by IL-4. Further, HdAg suppressed LPS-evoked release of TNF-α and IL-1ß from macrophages via autocrine IL-10 signaling. In assessing the ability of macrophages treated in vitro with an extract of H. diminuta [M(HdAg)] to affect disease, intravenous, but not peritoneal, injection of M(HdAg) protected wild-type but not RAG1-/- mice from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Administration of splenic CD4+ T cells from in vitro cocultures with M(HdAg), but not those cocultured with M(IL-4) cells, inhibited DNBS-induced colitis; fractionation of the T-cell population indicated that the CD4+CD25+ T cells from cocultures with M(HdAg) drove the suppression of DNBS-induced colitis. Use of IL-4-/- or IL-10-/- CD4+ T cells revealed that neither cytokine alone from the donor cells was essential for the anticolitic effect. These data illustrate that HdAg evokes a unique regulatory program in macrophages, identifies HdAg-evoked IL-10 suppression of macrophage activation, and reveals the ability of HdAg-treated macrophages to educate ( i.e., condition) and mobilize CD4+CD25+ T cells, which could be deployed to treat colonic inflammation.-Reyes, J. L., Lopes, F., Leung, G., Jayme, T. S., Matisz, C. E., Shute, A., Burkhard, R., Carneiro, M., Workentine, M. L., Wang, A., Petri, B., Beck, P. L., Geuking, M. B., McKay, D. M., Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Monogenic Intestinal Epithelium Defects and the Development of Inflammatory Bowel Disease.

The incidence of inflammatory bowel disease (IBD) is increasing worldwide, most notably in young children. The development of disease is a combination of several factors, including genetics, environment, the microbiota, and immune system. Recently, next-generation sequencing has allowed for the identification of novel genetic causes for intestinal disease, including pediatric inflammatory bowel disease (IBD). These IBD genes can generally be grouped into genes causing either primary immunodeficiency or intestinal epithelial defects (the focus of this review). Most of these genes have been functionally validated with in vitro and/or animal models, and have been demonstrated to cause intestinal disease. Intestinal epithelial IBD genes are of particular interest since they are the least amenable to current therapies; therefore, further research is warranted to develop potential therapies. A number of cellular pathways are impacted with intestinal epithelial IBD genes, including intestinal epithelial cell adhesion and generation of reactive oxygen species. Here, we describe the currently known IBD risk alleles and monogenic causal intestinal epithelial genes, their putative roles in preserving intestinal epithelial cell homeostasis, and their implications for IBD pathophysiology.

Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease.

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.

Treatment with Cestode Parasite Antigens Results in Recruitment of CCR2+ Myeloid Cells, the Adoptive Transfer of Which Ameliorates Colitis.

Awareness of the immunological underpinnings of host-parasite interactions may reveal immune signaling pathways that could be used to treat inflammatory disease in humans. Previously we showed that infection with the rat tapeworm, Hymenolepis diminuta, used as a model helminth, or systemic delivery of worm antigen (HdAg) significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Extending these analyses, intraperitoneal injection of HdAg dose-dependently suppressed dextran sodium sulfate (DSS)-induced colitis, and this was paralleled by reduced gamma interferon (IFN-γ), interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-α) production and increased IL-10 production from mitogen-activated splenocytes. Treatment with HdAg resulted in a CCR2-dependent recruitment of CDllb+ F4/80+ Ly6Chi Gr-1lo monocyte-like cells into the peritoneum 24 h later that were predominantly programmed death ligand 1 (PD-L1) positive and CXCR2 negative. In vitro assays indicated that these cells were unable to suppress T cell proliferation but enhanced IL-10 and IL-4 production from activated T cells. Adoptive transfer of the HdAg-recruited monocytic cells into naive mice blocked DSS-induced colitis. These findings add to the variety of means by which treatment with parasitic helminth-derived antigens can ameliorate concomitant disease. A precise understanding of the mechanism(s) of action of HdAg and other helminth-derived antigens (and a parallel consideration of putative side effects) may lead to the development of novel therapies for human idiopathic disorders such as inflammatory bowel disease.

IL-22 Restrains Tapeworm-Mediated Protection against Experimental Colitis via Regulation of IL-25 Expression.

Interleukin (IL)-22, an immune cell-derived cytokine whose receptor expression is restricted to non-immune cells (e.g. epithelial cells), can be anti-inflammatory and pro-inflammatory. Mice infected with the tapeworm Hymenolepis diminuta are protected from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Here we assessed expulsion of H. diminuta, the concomitant immune response and the outcome of DNBS-induced colitis in wild-type (WT) and IL-22 deficient mice (IL-22-/-) ± infection. Interleukin-22-/- mice had a mildly impaired ability to expel the worm and this correlated with reduced or delayed induction of TH2 immunity as measured by splenic and mesenteric lymph node production of IL-4, IL-5 and IL-13 and intestinal Muc-2 mRNA and goblet cell hyperplasia; in contrast, IL-25 increased in the small intestine of IL-22-/- mice 8 and 12 days post-infection compared to WT mice. In vitro experiments revealed that H. diminuta directly evoked epithelial production of IL-25 that was inhibited by recombinant IL-22. Also, IL-10 and markers of regulatory T cells were increased in IL-22-/- mice that displayed less DNBS (3 mg, ir. 72h)-induced colitis. Wild-type mice infected with H. diminuta were protected from colitis, as were infected IL-22-/- mice and the latter to a degree that they were almost indistinguishable from control, non-DNBS treated mice. Finally, treatment with anti-IL-25 antibodies exaggerated DNBS-induced colitis in IL-22-/- mice and blocked the anti-colitic effect of infection with H. diminuta. Thus, IL-22 is identified as an endogenous brake on helminth-elicited TH2 immunity, reducing the efficacy of expulsion of H. diminuta and limiting the effectiveness of the anti-colitic events mobilized following infection with H. diminuta in a non-permissive host.

Cryopreserved Interleukin-4-Treated Macrophages Attenuate Murine Colitis in an Integrin ß7 - Dependent Manner.

The adoptive transfer of alternatively activated macrophages (AAMs) has proven to attenuate inflammation in multiple mouse models of colitis; however, the effect of cryopreservation on AAMs, the ability of previously frozen AAMs to block dinitrobenzene sulfonic acid (DNBS) (Th1) and oxazolone (Th2) colitis and their migration postinjection remains unknown. Here we have found that while cryopreservation reduced mRNA expression of canonical markers of interleukin (IL)-4-treated macrophages [M(IL-4)], this step did not translate to reduced protein or activity, and the cells retained their capacity to drive the suppression of colitis. The anticolitic effect of M(IL-4) adoptive transfer required neither T or B cell nor peritoneal macrophages in the recipient. After injection into the peritoneal cavity, M(IL-4)s migrated to the spleen, mesenteric lymph nodes and colon of DNBS-treated mice. The chemokines CCL2, CCL4 and CX3CL1 were expressed in the colon during the course of DNBS-induced colitis. The expression of integrin ß7 on transferred M(IL-4)s was required for their anticolitic effect, whereas the presence of the chemokine receptors CCR2 and CX3CR1 were dispensable in this model. Collectively, the data show that M(IL-4)s can be cryopreserved M(IL-4)s and subsequently used to suppress colitis in an integrin ß7-dependent manner, and we suggest that these proof-of-concept studies may lead to new cellular therapies for human inflammatory bowel disease.

Adoptive transfer of helminth antigen-pulsed dendritic cells protects against the development of experimental colitis in mice.

Infection with helminth parasites and treatment with worm extracts can suppress inflammatory disease, including colitis. Postulating that dendritic cells (DCs) participated in the suppression of inflammation and seeking to move beyond the use of helminths per se, we tested the ability of Hymenolepis diminuta antigen-pulsed DCs to suppress colitis as a novel cell-based immunotherapy. Bone marrow derived DCs pulsed with H. diminuta antigen (HD-DCs), or PBS-, BSA-, or LPS-DCs as controls, were transferred into wild-type (WT), interleukin-10 (IL-10) knock-out (KO), and RAG-1 KO mice, and the impact on dinitrobenzene sulphonic acid (DNBS)-induced colitis and splenic cytokine production assessed 72 h later. Mice receiving HD-DCs were significantly protected from DNBS-induced colitis and of the experimental groups only these mice displayed increased Th2 cytokines and IL-10 production. Adoptive transfer of HD-DCs protected neither RAG-1 nor IL-10 KO mice from DNBS-colitis. Furthermore, the transfer of CD4(+) splenocytes from recipients of HD-DCs protected naïve mice against DNBS-colitis, in an IL-10 dependent manner. Thus, HD-DCs are a novel anti-colitic immunotherapy that can educate anti-colitic CD4(+) T cells: mechanistically, the anti-colitic effect of HD-DCs requires that the host has an adaptive immune response and the ability to mobilize IL-10.

Enteric epithelial cells support growth of Hymenolepis diminuta in vitro and trigger TH2-promoting events in a species-specific manner.

Knowledge of the gut epithelium in modulating immune responses to cestode parasites is scant. Hymenolepis diminuta causes no damage to its rodent host and is expelled from mice. Hymenolepis diminuta (scolex+2cm strobila) was cultured with rat (IEC6), human (T84) or mouse (IEC4) epithelial cell lines: all promoted worm survival, but those cultured on IEC6 (rat is a permissive host) were the healthiest. In contrast, production of Th2 polarising cytokines, IL-17e, IL-33 and TSLP, was greatest in IEC4 cells, less in T84 epithelia and almost negligible in IEC6 cells. Thus, the enteric epithelium is a key determinant of the response to infection with H. diminuta and is predictive of host permissiveness.

Splenic B cells from Hymenolepis diminuta-infected mice ameliorate colitis independent of T cells and via cooperation with macrophages.

Helminth parasites provoke multicellular immune responses in their hosts that can suppress concomitant disease. The gut lumen-dwelling tapeworm Hymenolepis diminuta, unlike other parasites assessed as helminth therapy, causes no host tissue damage while potently suppressing murine colitis. With the goal of harnessing the immunomodulatory capacity of infection with H. diminuta, we assessed the putative generation of anti-colitic regulatory B cells following H. diminuta infection. Splenic CD19(+) B cells isolated from mice infected 7 [HdBc(7(d))] and 14 d (but not 3 d) previously with H. diminuta and transferred to naive mice significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-, oxazolone-, and dextran-sodium sulfate-induced colitis. Mechanistic studies with the DNBS model, revealed the anti-colitic HdBc(7(d)) was within the follicular B cell population and its phenotype was not dependent on IL-4 or IL-10. The HdBc(7(d)) were not characterized by increased expression of CD1d, CD5, CD23, or IL-10 production, but did spontaneously, and upon LPS plus anti-CD40 stimulation, produce more TGF-ß than CD19(+) B cells from controls. DNBS-induced colitis in RAG1(-/-) mice was inhibited by administration of HdBc(7(d)), indicating a lack of a requirement for T and B cells in the recipient; however, depletion of macrophages in recipient mice abrogated the anti-colitic effect of HdBc(7(d)). Thus, in response to H. diminuta, a putatively unique splenic CD19(+) B cell with a functional immunoregulatory program is generated that promotes the suppression of colitis dominated by TH1, TH2, or TH1-plus-TH2 events, and may do so via the synthesis of TGF-ß and the generation of, or cooperation with, a regulatory macrophage.

The pro-inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated macrophages.

Macrophages are important innate immune cells that are associated with two distinct phenotypes: a pro-inflammatory (or classically activated) subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM)) subset linked with wound healing and tissue repair processes. In this study, we examined the effect of interlukein-6 on human and murine macrophage polarization. The results indicate that despite being commonly associated with pro-inflammatory functions and being implicated in the pathogenesis/pathophysiology of numerous inflammatory diseases, interleukin-6 can enhance the polarization of AAMs, based on increased expression of hallmark markers: arginase-1, Ym1 and CD206; this effect required the AAM differentiating cytokines, IL-4 and IL-13. Co-treatment of AAMs with IL-6 resulted in spontaneous release of IL-10, suppressed LPS-induced nitric oxide production and inhibited cytokine production by activated CD4+ T cells - immunoregulatory features not observed in the 'parent' IL-4+IL-13-induced AAM. The effect of IL-6 required signal transducer and activator of transcription (STAT)-3, was partially dependent on up-regulation of the IL4Rα chain, and was independent of autocrine IL-10. In the presence of IFNγ, IL-6 promoted the production of IL-1ß and TNFα suggesting that this cytokine can enhance the phenotype to which a macrophage has committed. This finding may explain the pleiotrophic nature of IL-6, where it is associated with the perpetuation and enhancement of disease in inflammatory situations, but is also necessary for resolution of inflammation and adequate wound healing to occur in others. Thus, the potential benefit of IL-6 in promoting an AAM, with its' anti-inflammatory and wound healing ability, may need to be considered in immunotherapies aimed at in vivo modulation or inhibition of IL-6.

Murine autoimmune arthritis is exaggerated by infection with the rat tapeworm, Hymenolepis diminuta.

Infection with helminth parasites triggers strong and stereotypic immune responses in humans and mice, which can protect against specific experimentally-induced autoimmune diseases. We have shown that infection with the rat tapeworm, Hymenolepis diminuta, confers a protective effect on FCA-induced joint inflammation. Here, we investigated the effect of a prophylactic infection with H. diminuta on the K/BxN-serum model of polyarthritis in BALB/c mice. Mice were infected with 10 cysticercoids of H. diminuta by oral gavage and 8 days later arthritis was induced by i.p. injection of K/BxN arthritogenic serum. Joint swelling and pain measurements were recorded throughout a 13 day time course. At necropsy, joints and blood serum were collected. K/BxN-treated mice developed joint inflammation in the front paws, hind paws and knees as shown by increased swelling, mechanical allodynia and myeloperoxidase activity. Mice infected with H. diminuta had more severe disease, with increased eosinophil peroxidase activity in their paws and greater inflammatory infiltrate and synovitis in the knee joints. Hymenolepis diminuta-infected mice displayed significant increases in serum levels of C5a and mast cell protease-1 compared with K/BxN-serum only treatment, the latter being indicative of mast cell activation. In contrast to the protective effect of infection with H. diminuta in FCA-induced monoarthritis, infection with this helminth exacerbated K/BxN serum-induced polyarthritis in BALB/c mice. This correlated with increases in C5a and mast cell activation: factors critical in the development of K/BxN-induced arthritis. Thus, while data accumulate from animal models showing that infection with helminth parasites may be beneficial for a variety of auto-inflammatory diseases, our findings demonstrate the potential for helminths to exacerbate disease. Hence care is needed when helminth therapy is translated into a clinical setting.

Bone marrow-derived alternatively activated macrophages reduce colitis without promoting fibrosis: participation of IL-10.

Alternatively activated macrophages (AAMs) (or M2a) can inhibit colitis but may also be associated with fibrosis. Thus, by using the dinitrobenzene sulfonic (DNBS) murine model of colitis, this study aimed to determine whether 1) bone marrow (BM)-derived AAMs could reduce colitis, 2) any anticolitic effect of BM-AAMs was IL-10 dependent, and 3) repeated AAM treatments remained effective and were associated with fibrosis in the gut or other tissues. Balb/c mice received AAMs (10(6) intraperitoneally) from wild-type (WT) or IL-10(-/-) mice 48 h prior to DNBS (3 mg intrarectally) with disease assessed 72 h later, or they received three doses of DNBS at 2-wk intervals ± AAMs 6 h post-DNBS to mimic a treatment regimen. DNBS-treated mice developed colitis; this was significantly less severe in mice receiving WT AAMs and less so in animals given IL-10(-/-) AAMs, indicating a role for IL-10 in the inhibition of DNBS-driven colitis. Similarly, after the third AAM treatment lesser colonic histopathology was observed compared with time-matched DNBS-only-treated animals, and notably there was no evidence of increased fibroses in the colon, terminal ileum, lung, or liver of AAM-treated mice as assessed by quantitative PCR for prolyl-4-hydrolase, α-smooth muscle actin, and collagen (type IIIα) and histochemical and biochemical assessment of collagen deposition. This study provides mechanistic insight to the anticolitic capacity of AAMs and indicates that repeated adoptive transfer of ex vivo programmed BM-AAMs is safe and efficacious in the treatment of DNBS-induced murine colitis, providing additional support for their consideration as an immunotherapy.

Cestode regulation of inflammation and inflammatory diseases.

Helminth parasites are masters of immune regulation; a likely prerequisite for long-term survival by circumventing their hosts' attempt to eradicate them. From a translational perspective, knowledge of immune events as a response to infection with a helminth parasite could be used to reduce the intensity of unwanted inflammatory reactions. Substantial data have accumulated showing that inflammatory reactions that promote a variety of auto-inflammatory diseases are dampened as a consequence of infection with helminth parasites, via either the mobilization of an anti-worm spectrum of immune events or by the direct effect of secretory/excretory bioactive immunomodulatory molecules released from the parasite. However, many issues are outstanding in the definition of the mechanism(s) by which infection with helminth parasites can affect the outcome, positively or negatively, of concomitant disease. We focus on a subgroup of this complex group of metazoan parasites, the cestodes, summarizing studies from rodent models that illustrate if, and by what mechanisms, infection with tapeworms ameliorate or exaggerate disease in their host. The ability of infection with cestodes, or other classes of helminth, to worsen a disease course or confer susceptibility to intracellular pathogens should be carefully considered in the context of 'helminth therapy'. In addition, poorly characterised cestode extracts can regulate murine and human immunocyte function, yet the impact of these in the context of autoimmune or allergic diseases is poorly understood. Thus, studies with cestodes, as representative helminths, have helped cement the concept that infection with parasitic helminths can inhibit concomitant disease; however, issues relating to long-term effects, potential side-effects, mixed pathogen infections and purification of immunomodulatory molecules from the parasite remain as challenges that need to be addressed in order to achieve the use of helminths as anti-inflammatory agents for human diseases.

Reduced surface expression of epithelial E-cadherin evoked by interferon-gamma is Fyn kinase-dependent.

Interferon gamma (IFNγ) is an important regulatory cytokine that can exert a pro-inflammatory effect in the gut, where it has been shown to increase epithelial permeability via disruption of the tight junctions. Here we investigated the potential for IFNγ to regulate the adherens junction protein E-cadherin, an important mediator of normal epithelial tissue function, using the model T84 human colonic epithelial cell line. IFNγ (10 ng/ml) stimulated increased internalization of E-cadherin as assessed by immunofluorescence microscopy; internalization was reversed when cells were treated with PP1 (125 nM), a Src kinase-selective inhibitor. Immunoprecipitation studies demonstrated loss of E-cadherin from membrane fractions following IFNγ treatment and a corresponding increase in cytosolic E-cadherin and its binding partners, p120-catenin and beta-catenin: effects that were Src-kinase dependent. E-cadherin and p120-catenin phosphorylation was increased by IFNγ treatment and siRNA studies showed this was dependent upon the Src-kinase isoform Fyn. E-cadherin ubiquitinylation and subsequent proteasomal degradation stimulated by IFNγ was found to be dependent upon Fyn and the E-cadherin-selective ubiquitin ligase, Hakai. Use of Fyn and Hakai siRNA inhibited the internalization of E-cadherin as shown by immunoblotting and confocal fluorescence microscopy. Finally, IFNγ treatment resulted in a more fragile T84 cell monolayer with increased cell detachment in response to physical stress, which was prevented by PP1 and siRNA targeting Fyn or Hakai. Collectively, these results demonstrate a Fyn kinase-dependent mechanism through which IFNγ regulates E-cadherin stability and suggest a novel mechanism of disruption of epithelial cell contact, which could contribute to perturbed epithelial barrier function.

Exacerbation of oxazolone colitis by infection with the helminth Hymenolepis diminuta: involvement of IL-5 and eosinophils.

Substantial data show that infection with helminth parasites ameliorates colitis; however, oxazolone-induced colitis is exaggerated in mice infected with the tapeworm, Hymenolepis diminuta. We tested the hypothesis that the IL-5 response to helminth infection enhances the severity of oxazolone-induced colitis. Mice were infected with H. diminuta and 8 days later were treated with oxazolone ± anti-IL-5 antibodies. Colitis was assessed 72 hours postoxazolone treatment by disease activity scores, myeloperoxidase activity, and histopathology. Other mice received injections of a replication-deficient adenovirus that carried the IL-5 (Ad.IL-5) gene or a control adenovirus (Ad.delete) ± oxazolone. The effect of H. diminuta+oxazolone in CCL11/CCL22 (eotaxin-1 and 2) knockout (KO) mice was determined. Helminth infection and Ad.IL-5 treatment increased IL-5 and eosinophil numbers. In vivo neutralization of IL-5 significantly reduced the severity of colitis in H. diminuta+oxazolone-treated mice, and H. diminuta did not exaggerate oxazolone-induced colitis in CCL11/CCL22 KO mice. Mice receiving Ad.IL-5 only had no colitis, while oxazolone-induced colitis was more severe in animals cotreated with Ad.IL-5 (Ad.delete + oxazolone was not significantly different from oxazolone only). Thus, while there is much to be gleaned about antiinflammatory mechanisms from rodent-helminth model systems, these data illustrate the caveat that infection with helminth parasites as a therapy could be contraindicated in patients with eosinophilia or elevated IL-5 unless coupled to appropriate measures to block IL-5 and/or eosinophil activity.