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) alpha/FIZZ1; however, the influence of these molecules on the development, progression, or resolution of inflammatory diseases is unknown. We describe the generation of RELM-alpha-deficient (Retnla(-/-)) mice and use a model of T helper type 2 (Th2) cytokine-dependent lung inflammation to identify an immunoregulatory role for RELM-alpha. 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-alpha could bind to macrophages and effector CD4(+) Th2 cells and inhibited Th2 cytokine production in a Bruton's 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-alpha in limiting the pathogenesis of Th2 cytokine-mediated pulmonary inflammation, in part through the regulation of CD4(+) T cell responses.

Intestinal epithelial cells (IECs) produce thymic stromal lymphopoietin (TSLP); however, the in vivo influence of TSLP-TSLP receptor (TSLPR) interactions on immunity and inflammation in the intestine remains unclear. We show that TSLP-TSLPR interactions are critical for immunity to the intestinal pathogen Trichuris. Monoclonal antibody-mediated neutralization of TSLP or deletion of the TSLPR in normally resistant mice resulted in defective expression of Th2 cytokines and persistent infection. Susceptibility was accompanied by elevated expression of interleukin (IL) 12/23p40, interferon (IFN) gamma, and IL-17A, and development of severe intestinal inflammation. Critically, neutralization of IFN-gamma in Trichuris-infected TSLPR(-/-) mice restored Th2 cytokine responses and resulted in worm expulsion, providing the first demonstration of TSLPR-independent pathways for Th2 cytokine production. Additionally, TSLPR(-/-) mice displayed elevated production of IL-12/23p40 and IFN-gamma, and developed heightened intestinal inflammation upon exposure to dextran sodium sulfate, demonstrating a previously unrecognized immunoregulatory role for TSLP in a mouse model of inflammatory bowel disease.

Dendritic cells can prime naive CD4+ T cells; however, here we demonstrate that dendritic cell-mediated priming was insufficient for the development of T helper type 2 cell-dependent immunity. We identify basophils as a dominant cell population that coexpressed major histocompatibility complex class II and interleukin 4 message after helminth infection. Basophilia was promoted by thymic stromal lymphopoietin, and depletion of basophils impaired immunity to helminth infection. Basophils promoted antigen-specific CD4+ T cell proliferation and interleukin 4 production in vitro, and transfer of basophils augmented the population expansion of helminth-responsive CD4+ T cells in vivo. Collectively, our studies suggest that major histocompatibility complex class II-dependent interactions between basophils and CD4+ T cells promote T helper type 2 cytokine responses and immunity to helminth infection.

The migration of lymphocytes to the small intestine is controlled by expression of the integrin α4β7 and the chemokine receptor CCR9. However, the molecules that specifically regulate migration to the large intestine remain unclear. Immunity to infection with the large intestinal helminth parasite Trichuris muris is dependent upon CD4(+) T cells that migrate to the large intestine. We examine the role of specific chemokine receptors, adhesion molecules and glycosyltransferases in the development of protective immunity to Trichuris. Mice deficient in expression of the chemokine receptors CCR2 or CCR6 were resistant to infection with Trichuris. Similarly, loss of CD34, CD43, CD44 or PSGL-1 had no effect on resistance to infection. In contrast, simultaneous deletion of the Core2 β1,6-N-acetylglucosaminyltransferase (C2GnT) enzymes C2GnT1 and C2Gnt2 resulted in delayed expulsion of worms. These results suggest that C2GnT-dependent modifications may play a role in migration of protective immune cells to the large intestine.

The cytokine interleukin (IL) 25 has been implicated in the initiation of type 2 immunity by driving the expression of type 2 cytokines such as IL-5 and IL-13, although its role in the regulation of immunity and infection-induced inflammation is unknown. Here, we identify a dual function for IL-25: first, in promoting type 2 cytokine-dependent immunity to gastrointestinal helminth infection and, second, in limiting proinflammatory cytokine production and chronic intestinal inflammation. Treatment of genetically susceptible mice with exogenous IL-25 promoted type 2 cytokine responses and immunity to Trichuris. IL-25 was constitutively expressed by CD4+ and CD8+ T cells in the gut of mouse strains that are resistant to Trichuris, and IL-25-deficient mice on a genetically resistant background failed to develop a type 2 immune response or eradicate infection. Furthermore, chronically infected IL-25(-/-) mice developed severe infection-induced intestinal inflammation associated with heightened expression of interferon-gamma and IL-17, identifying a role for IL-25 in limiting pathologic inflammation at mucosal sites. Therefore, IL-25 is not only a critical mediator of type 2 immunity, but is also required for the regulation of inflammation in the gastrointestinal tract.

Volumetric muscle loss (VML) is associated with loss of skeletal muscle function, and current treatments show limited efficacy. Here we show that bioconstructs suffused with genetically-labelled muscle stem cells (MuSCs) and other muscle resident cells (MRCs) are effective to treat VML injuries in mice. Imaging of bioconstructs implanted in damaged muscles indicates MuSCs survival and growth, and ex vivo analyses show force restoration of treated muscles. Histological analysis highlights myofibre formation, neovascularisation, but insufficient innervation. Both innervation and in vivo force production are enhanced when implantation of bioconstructs is followed by an exercise regimen. Significant improvements are also observed when bioconstructs are used to treat chronic VML injury models. Finally, we demonstrate that bioconstructs made with human MuSCs and MRCs can generate functional muscle tissue in our VML model. These data suggest that stem cell-based therapies aimed to engineer tissue in vivo may be effective to treat acute and chronic VML.

The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity.

Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

Successful T-dependent humoral responses require the production of antibody-secreting plasmablasts, as well as the formation of germinal centers which eventually form high-affinity B cell memory. The ability of B cells to differentiate into germinal center and plasma cells, as well as the ability to tailor responses to different pathogens, is driven by transcription factors. In T cells, the T-box transcription factors T-bet and Eomesodermin (Eomes) regulate effector and memory T cell differentiation, respectively. While T-bet has a critical role in regulating anti-viral B cell responses, a role for Eomes in B cells has yet to be described. We therefore investigated whether Eomes was required for B cell differentiation during either Th1 or Th2 cell-biased immune responses. Here, we demonstrate that deletion of Eomes specifically in B cells did not affect B cell differentiation in response to vaccination, as well as following viral or helminth infection. In contrast to its established role in CD8+ T cells, Eomes did not influence memory B cell differentiation. Finally, the use of an Eomes reporter mouse confirmed the lack of Eomes expression during immune responses. Thus, germinal center and plasma cell differentiation and the formation of isotype-switched memory B cells in response to infection are independent of Eomes expression.

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis.

Immunological cross talk between mucosal tissues such as the intestine and the lung is poorly defined during homeostasis and disease. Here, we show that a low-dose infection with the intestinally restricted helminth parasite Trichuris muris results in the production of Th1 cell-dependent gamma interferon (IFN-γ) and myeloid cell-derived interleukin-10 (IL-10) in the lung without causing overt airway pathology. This cross-mucosal immune response in the lung inhibits the development of papain-induced allergic airway inflammation, an innate cell-mediated type 2 airway inflammatory disease. Thus, we identify convergent and nonredundant roles of adaptive and innate immunity in mediating cross-mucosal suppression of type 2 airway inflammation during low-dose helminth-induced intestinal inflammation. These results provide further insight in identifying novel intersecting immune pathways elicited by gut-to-lung mucosal cross talk.

Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-β is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-β's role in host defense is unclear. Thus, wildtype and RELM-β gene deficient mice (Retnlb-/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb-/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb-/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-β did not directly increase IEC proliferation, rather RELM-β acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb-/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-β to Retnlb-/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-β and goblet cells play an unexpected, yet critical role in recruiting CD4+ T cells to the colon to protect against an enteric pathogen, in part via the induction of increased IEC proliferation.

The current approaches to reduce the burden of chronic helminth infections in endemic areas are adequate sanitation and periodic administration of deworming drugs. Yet, resistance against some deworming drugs and reinfection can still rapidly occur even after treatment. A vaccine against helminths would be an effective solution at preventing reinfection. However, vaccines against helminth parasites have yet to be successfully developed. While T helper cells and innate lymphoid cells have been established as important components of the protective type 2 response, the roles of B cells and antibodies remain the most controversial. Here, we review the roles of B cells during intestinal helminth infection. We discuss the potential factors that contribute to the context-specific roles for B cells in protection against diverse intestinal helminth parasite species, using evidence from well-defined murine model systems. Understanding the precise roles of B cells during resistance and susceptibility to helminth infection may offer a new perspective of type 2 protective immunity.

Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.

The development of cell therapy for repairing damaged or diseased skeletal muscle has been hindered by the inability to significantly expand immature, transplantable myogenic stem cells (MuSCs) in culture. To overcome this limitation, a deeper understanding of the mechanisms regulating the transition between activated, proliferating MuSCs and differentiation-primed, poorly engrafting progenitors is needed. Here, we show that methyltransferase Setd7 facilitates such transition by regulating the nuclear accumulation of β-catenin in proliferating MuSCs. Genetic or pharmacological inhibition of Setd7 promotes in vitro expansion of MuSCs and increases the yield of primary myogenic cell cultures. Upon transplantation, both mouse and human MuSCs expanded with a Setd7 small-molecule inhibitor are better able to repopulate the satellite cell niche, and treated mouse MuSCs show enhanced therapeutic potential in preclinical models of muscular dystrophy. Thus, Setd7 inhibition may help bypass a key obstacle in the translation of cell therapy for muscle disease.

Innate lymphoid cells (ILCs) are emerging as important regulators of homeostatic and disease-associated immune processes. Despite recent advances in defining the molecular pathways that control development and function of ILCs, the epigenetic mechanisms that regulate ILC biology are unknown. Here, we identify a role for the lysine methyltransferase G9a in regulating ILC2 development and function. Mice with a hematopoietic cell-specific deletion of G9a (Vav.G9a(-/-) mice) have a severe reduction in ILC2s in peripheral sites, associated with impaired development of immature ILC2s in the bone marrow. Accordingly, Vav.G9a(-/-) mice are resistant to the development of allergic lung inflammation. G9a-dependent dimethylation of histone 3 lysine 9 (H3K9me2) is a repressive histone mark that is associated with gene silencing. Genome-wide expression analysis demonstrated that the absence of G9a led to increased expression of ILC3-associated genes in developing ILC2 populations. Further, we found high levels of G9a-dependent H3K9me2 at ILC3-specific genetic loci, demonstrating that G9a-mediated repression of ILC3-associated genes is critical for the optimal development of ILC2s. Together, these results provide the first identification of an epigenetic regulatory mechanism in ILC development and function.

Despite the regenerative capacity of muscle, tissue volume is not restored after volumetric muscle loss (VML), perhaps due to a loss-of-structural extracellular matrix. We recently demonstrated the structural and functional restoration of muscle tissue in a mouse model of VML using an engineered "bioconstruct," comprising an extracellular matrix scaffold (decellularized muscle), muscle stem cells (MuSCs), and muscle-resident cells (MRCs). To test the ability of the cell-based bioconstruct to restore whole-muscle biomechanics, we measured biomechanical parameters in uninjured muscles, muscles injured to produce VML lesions, and in muscles that were injured and then treated by implanting either the scaffolds alone or with bioconstructs containing the scaffolds, MuSCs, and MRCs. We measured the active and passive forces over a range of lengths, viscoelastic force relaxation, optimal length, and twitch dynamics. Injured muscles showed a narrowed length-tension curve or lower force over a narrower range of muscle lengths, and increased passive force. When treated with bioconstructs, but not with scaffolds alone, injured muscles showed active and passive length-tension relationships that were not different from uninjured muscles. Moreover, injured muscles treated with bioconstructs exhibited reduced fibrosis compared to injured muscles either untreated or treated with scaffolds alone. The cell-based bioconstruct is a promising treatment approach for future translational efforts to restore whole-muscle biomechanics in muscles with VML lesions.

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4+ T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond.

In the intestine, the integrin CD103 is expressed on a subset of T regulatory (T(reg)) cells and a population of dendritic cells (DCs) that produce retinoic acid and promote immune homeostasis. However, the role of CD103 during intestinal helminth infection has not been tested.

Interleukin (IL) 31Ralpha (glycoprotein 130-like monocyte receptor and glycoprotein 130-like receptor) heterodimerizes with oncostatin M receptor beta to bind IL-31, a cytokine expressed preferentially by CD4(+) T helper type 2 (Th2) cells. However, the functions of IL-31-IL-31R signaling in immune regulation remain unknown. Here, we identify a novel role for IL-31R in limiting type 2 inflammation in the lung. After intravenous injection of Schistosoma mansoni eggs, IL-31Ralpha(-/-) mice developed severe pulmonary inflammation, characterized by an increase in the area of granulomatous inflammation, increased numbers of resistin-like molecule alpha(+) cells, and enhanced collagen deposition compared to WT counterparts. In vitro, macrophages generated from IL-31Ralpha(-/-) mice promoted enhanced ovalbumin-specific CD4(+) T cell proliferation and purified naive CD4(+) T cells from IL-31Ralpha(-/-) mice exhibited enhanced proliferation and expression of Th2 cytokines, identifying a T cell- and macrophage-intrinsic regulatory function for IL-31R signaling. In contrast, the generation of CD4(+) T cell-mediated Th1 responses were normal in IL-31Ralpha(-/-) mice, suggesting that the regulatory role of IL-31R signaling is limited to type 2 responses. Together, these data implicate IL-31R signaling as a novel negative regulatory pathway that specifically limits type 2 inflammation.