FOXP3⁺ Tregs require WASP to restrain Th2-mediated food allergy

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Abstract

In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen–free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3⁺ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3⁺ Tregs, but not in naive-like FOXP3⁺ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens.

Authors

Willem S. Lexmond, Jeremy A. Goettel, Jonathan J. Lyons, Justin Jacobse, Marion M. Deken, Monica G. Lawrence, Thomas H. DiMaggio, Daniel Kotlarz, Elizabeth Garabedian, Paul Sackstein, Celeste C. Nelson, Nina Jones, Kelly D. Stone, Fabio Candotti, Edmond H.H.M. Rings, Adrian J. Thrasher, Joshua D. Milner, Scott B. Snapper, Edda Fiebiger

EGFR regulates macrophage activation and function in bacterial infection

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Abstract

EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

Authors

Dana M. Hardbower, Kshipra Singh, Mohammad Asim, Thomas G. Verriere, Danyvid Olivares-Villagómez, Daniel P. Barry, Margaret M. Allaman, M. Kay Washington, Richard M. Peek Jr., M. Blanca Piazuelo, Keith T. Wilson

Methanobactin reverses acute liver failure in a rat model of Wilson disease

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Abstract

In Wilson disease (WD), functional loss of ATPase copper-transporting β (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration– and European Medicines Agency–approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.

Authors

Josef Lichtmannegger, Christin Leitzinger, Ralf Wimmer, Sabine Schmitt, Sabine Schulz, Yaschar Kabiri, Carola Eberhagen, Tamara Rieder, Dirk Janik, Frauke Neff, Beate K. Straub, Peter Schirmacher, Alan A. DiSpirito, Nathan Bandow, Bipin S. Baral, Andrew Flatley, Elisabeth Kremmer, Gerald Denk, Florian P. Reiter, Simon Hohenester, Friedericke Eckardt-Schupp, Norbert A. Dencher, Jerzy Adamski, Vanessa Sauer, Christoph Niemietz, Hartmut H.J. Schmidt, Uta Merle, Daniel Nils Gotthardt, Guido Kroemer, Karl Heinz Weiss, Hans Zischka

Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function

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Abstract

Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT₄ agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT₄–mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation.

Authors

Kara Gross Margolis, Zhishan Li, Korey Stevanovic, Virginia Saurman, Narek Israelyan, George M. Anderson, Isaac Snyder, Jeremy Veenstra-VanderWeele, Randy D. Blakely, Michael D. Gershon

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22

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Abstract

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1β, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1β secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1β release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1β. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1β levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

Authors

Marianne R. Spalinger, Stephanie Kasper, Claudia Gottier, Silvia Lang, Kirstin Atrott, Stephan R. Vavricka, Sylvie Scharl, Petrus M. Gutte, Markus G. Grütter, Hans-Dietmar Beer, Emmanuel Contassot, Andrew C. Chan, Xuezhi Dai, David J. Rawlings, Florian Mair, Burkhard Becher, Werner Falk, Michael Fried, Gerhard Rogler, Michael Scharl

Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

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Abstract

Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing the transcriptome of IECs from IBD patients using a genome-wide approach. We observed disease-specific alterations in IECs with markedly impaired Rho-A signaling in active IBD patients. Localization of epithelial Rho-A was shifted to the cytosol in IBDs, and inflammation was associated with suppressed Rho-A activation due to reduced expression of the Rho-A prenylation enzyme geranylgeranyltransferase-I (GGTase-I). Functionally, we found that mice with conditional loss of Rhoa or the gene encoding GGTase-I, Pggt1b, in IECs exhibit spontaneous chronic intestinal inflammation with accumulation of granulocytes and CD4⁺ T cells. This phenotype was associated with cytoskeleton rearrangement and aberrant cell shedding, ultimately leading to loss of epithelial integrity and subsequent inflammation. These findings uncover deficient prenylation of Rho-A as a key player in the pathogenesis of IBDs. As therapeutic triggering of Rho-A signaling suppressed intestinal inflammation in mice with GGTase-I–deficient IECs, our findings suggest new avenues for treatment of epithelial injury and mucosal inflammation in IBD patients.

Authors

Rocío López-Posadas, Christoph Becker, Claudia Günther, Stefan Tenzer, Kerstin Amann, Ulrike Billmeier, Raja Atreya, Gionata Fiorino, Stefania Vetrano, Silvio Danese, Arif B. Ekici, Stefan Wirtz, Veronika Thonn, Alastair J.M. Watson, Cord Brakebusch, Martin Bergö, Markus F. Neurath, Imke Atreya

Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis

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Abstract

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1^–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3⁺CD4⁺IL-17⁺ and reduced tolerogenic Foxp3⁺ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.

Authors

Charlotte E. Egan, Chhinder P. Sodhi, Misty Good, Joyce Lin, Hongpeng Jia, Yukihiro Yamaguchi, Peng Lu, Congrong Ma, Maria F. Branca, Samantha Weyandt, William B. Fulton, Diego F. Niño, Thomas Prindle Jr., John A. Ozolek, David J. Hackam

A collagen VI–dependent pathogenic mechanism for Hirschsprung’s disease

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Abstract

Hirschsprung’s disease (HSCR) is a severe congenital anomaly of the enteric nervous system (ENS) characterized by functional intestinal obstruction due to a lack of intrinsic innervation in the distal bowel. Distal innervation deficiency results from incomplete colonization of the bowel by enteric neural crest cells (eNCCs), the ENS precursors. Here, we report the generation of a mouse model for HSCR — named Holstein — that contains an untargeted transgenic insertion upstream of the collagen-6α4 (Col6a4) gene. This insertion induces eNCC-specific upregulation of Col6a4 expression that increases total collagen VI protein levels in the extracellular matrix (ECM) surrounding both the developing and the postnatal ENS. Increased collagen VI levels during development mainly result in slower migration of eNCCs. This appears to be due to the fact that collagen VI is a poor substratum for supporting eNCC migration and can even interfere with the migration-promoting effects of fibronectin. Importantly, for a majority of patients in a HSCR cohort, the myenteric ganglia from the ganglionated region are also specifically surrounded by abundant collagen VI microfibrils, an outcome accentuated by Down syndrome. Collectively, our data thus unveil a clinically relevant pathogenic mechanism for HSCR that involves cell-autonomous changes in ECM composition surrounding eNCCs. Moreover, as COL6A1 and COL6A2 are on human Chr.21q, this mechanism is highly relevant to the predisposition of patients with Down syndrome to HSCR.

Authors

Rodolphe Soret, Mathilde Mennetrey, Karl F. Bergeron, Anne Dariel, Michel Neunlist, Franziska Grunder, Christophe Faure, David W. Silversides, Nicolas Pilon, for the Ente-Hirsch study group

Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis

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Abstract

Mesenchymal stem cell (MSC) therapy is an emerging field of regenerative medicine; however, it is often unclear how these cells mediate repair. Here, we investigated the use of MSCs in the treatment of intestinal disease and modeled abnormal repair by creating focal wounds in the colonic mucosa of prostaglandin-deficient mice. These wounds developed into ulcers that infiltrated the outer intestinal wall. We determined that penetrating ulcer formation in this model resulted from increased hypoxia and smooth muscle wall necrosis. Prostaglandin I₂ (PGI₂) stimulated VEGF-dependent angiogenesis to prevent penetrating ulcers. Treatment of mucosally injured WT mice with a VEGFR inhibitor resulted in the development of penetrating ulcers, further demonstrating that VEGF is critical for mucosal repair. We next used this model to address the role of transplanted colonic MSCs (cMSCs) in intestinal repair. Compared with intravenously injected cMSCs, mucosally injected cMSCs more effectively prevented the development of penetrating ulcers, as they were more efficiently recruited to colonic wounds. Importantly, mucosally injected cMSCs stimulated angiogenesis in a VEGF-dependent manner. Together, our results reveal that penetrating ulcer formation results from a reduction of local angiogenesis and targeted injection of MSCs can optimize transplantation therapy. Moreover, local MSC injection has potential for treating diseases with features of abnormal angiogenesis and repair.

Authors

Nicholas A. Manieri, Madison R. Mack, Molly D. Himmelrich, Daniel L. Worthley, Elaine M. Hanson, Lars Eckmann, Timothy C. Wang, Thaddeus S. Stappenbeck

Engineering the gut microbiota to treat hyperammonemia

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Abstract

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.

Authors

Ting-Chin David Shen, Lindsey Albenberg, Kyle Bittinger, Christel Chehoud, Ying-Yu Chen, Colleen A. Judge, Lillian Chau, Josephine Ni, Michael Sheng, Andrew Lin, Benjamin J. Wilkins, Elizabeth L. Buza, James D. Lewis, Yevgeny Daikhin, Ilana Nissim, Marc Yudkoff, Frederic D. Bushman, Gary D. Wu