Retinoic acid and TGF-ß orchestrate organ-specific programs of tissue residency.

Tissue-resident memory T (TRM) cells are integral to tissue immunity, persisting in diverse anatomical sites where they adhere to a common transcriptional framework. How these cells integrate distinct local cues to adopt the common TRM cell fate remains poorly understood. Here, we show that whereas skin TRM cells strictly require transforming growth factor ß (TGF-ß) for tissue residency, those in other locations utilize the metabolite retinoic acid (RA) to drive an alternative differentiation pathway, directing a TGF-ß-independent tissue residency program in the liver and synergizing with TGF-ß to drive TRM cells in the small intestine. We found that RA was required for the long-term maintenance of intestinal TRM populations, in part by impeding their retrograde migration. Moreover, enhanced RA signaling modulated TRM cell phenotype and function, a phenomenon mirrored in mice with increased microbial diversity. Together, our findings reveal RA as a fundamental component of the host-environment interaction that directs immunosurveillance in tissues.

Helminth Infection-Induced Increase in Virtual Memory CD8 T Cells Is Transient, Driven by IL-15, and Absent in Aged Mice.

CD8 virtual memory T (TVM) cells are Ag-naive CD8 T cells that have undergone partial differentiation in response to common γ-chain cytokines, particularly IL-15 and IL-4. TVM cells from young individuals are highly proliferative in response to TCR and cytokine stimulation but, with age, they lose TCR-mediated proliferative capacity and exhibit hallmarks of senescence. Helminth infection can drive an increase in TVM cells, which is associated with improved pathogen clearance during subsequent infectious challenge in young mice. Given the cytokine-dependent profile of TVM cells and their age-associated dysfunction, we traced proliferative and functional changes in TVM cells, compared with true naive CD8 T cells, after helminth infection of young and aged C57BL/6 mice. We show that IL-15 is essential for the helminth-induced increase in TVM cells, which is driven only by proliferation of existing TVM cells, with negligible contribution from true naive cell differentiation. Additionally, TVM cells showed the greatest proliferation in response to helminth infection and IL-15 compared with other CD8 T cells. Furthermore, TVM cells from aged mice did not undergo expansion after helminth infection due to both TVM cell-intrinsic and -extrinsic changes associated with aging.

Modulating inflammation with interleukin 37 treatment ameliorates murine Autosomal Dominant Polycystic Kidney Disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of kidney failure and is associated with substantial morbidity and mortality. Interstitial inflammation is attributed to the action of infiltrating macrophages and is a feature thought to aggravate disease progression. Here, we investigated the therapeutic potential of the anti-inflammatory IL37b cytokine as a treatment for ADPKD using genetic mouse models, demonstrating that transgenic expression of human IL37b reduced collecting duct cyst burden in both early and adult-onset ADPKD rodent models. Moreover, injection of recombinant human IL37b could also reduce cyst burden in early onset ADPKD mice, an observation not associated with increased macrophage number at early stages of cyst formation. Interestingly, transgenic IL37b expression also did not alter macrophage numbers in advanced disease. Whole kidney RNA-seq highlighted an IL37b-mediated upregulation of the interferon signaling pathway and single-cell RNA-seq established that these changes originate at least partly from kidney resident macrophages. We further found that blocking type I interferon signaling in mice expressing IL37b resulted in increased cyst number, confirming this as an important pathway by which IL37b exerts its beneficial effects. Thus, our studies show that IL37b promotes interferon signaling in kidney resident macrophages which suppresses cyst initiation, identifying this protein as a potential therapy for ADPKD.

Intestinal-epithelial LSD1 controls goblet cell maturation and effector responses required for gut immunity to bacterial and helminth infection.

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.

c-Rel employs multiple mechanisms to promote the thymic development and peripheral function of regulatory T cells in mice.

The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel-/- ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel-/- mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel-/- tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.

MHC class II-dependent basophil-CD4+ T cell interactions promote T(H)2 cytokine-dependent immunity.

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.

HIC1 links retinoic acid signalling to group 3 innate lymphoid cell-dependent regulation of intestinal immunity and homeostasis.

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.

Mastering gut permeability: New roles for old friends.

Mast cells are innate immune cells that respond rapidly to infection in barrier tissues such as the skin and intestinal mucosa. Expulsion of parasitic worms in the gut involves a robust type 2 host response, and an acute mastocytosis is often generated at the site of infection. However, the role of mast cells in resistance to worm infections appears to be parasite specific. Mast cells are also involved in tissue repair, but the long-term contribution of mast cell activation after worm expulsion has not been definitively studied. In this issue of European Journal of Immunology, Sorobetea et al. [Eur. J. Immunol. 2017. 47: 257-268] demonstrate that activated mast cells persist in the large intestinal lamina propria and intraepithelial compartment long after worm expulsion, resulting in continued local and systemic presence of the mast cell protease mast cell protease 1 (MCPt-1) and enhanced intestinal permeability. In this commentary, we discuss these findings in the wider context of mast cell function in health and disease.

Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection.

The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7-/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/ß-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection.

Loss of Vascular CD34 Results in Increased Sensitivity to Lung Injury.

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular, and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild-type (WT) and Cd34-/- mice by bleomycin administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared with WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared with WT mice reconstituted with Cd34-/- cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together, our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced tissue damage.

Dendritic-cell expression of Ship1 regulates Th2 immunity to helminth infection in mice.

In mouse models of infection with the gastrointestinal parasite Trichuris muris, appropriate dendritic-cell (DC) Ag sampling, migration, and presentation to T cells are necessary to mount a protective Th2-polarized adaptive immune response, which is needed to clear infection. SH2-containing inositol 5'-phosphatase 1 (SHIP-1) has been shown to be an important regulator of DC function in vitro through the negative regulation of the phosphoinositide 3-kinase (PI3K) pathway, but its role in vivo is relatively unexplored. In the current work, mice with a specific deletion of SHIP-1 in DCs (Ship1(ΔDC) ) were infected with the parasite T. muris. Ship1(ΔDC) mice were susceptible to infection due to ineffective priming of Th2-polarized responses. This is likely due to an increased production of interleukin (IL) 12p40 by SHIP-1-deficient DCs, as in vivo antibody blockade of IL-12p40 was able to facilitate the clearing of infection in Ship1(ΔDC) mice. Our results describe a critical role for SHIP-1 in regulating the ability of DCs to efficiently prime Th2-type responses.

Chronic Trichuris muris infection alters hematopoiesis and causes IFN-γ-expressing T-cell accumulation in the mouse bone marrow.

Proinflammatory cytokines produced during immune responses to infectious stimuli are well-characterized to have secondary effects on the function of hematopoietic progenitor cells in the BM. However, these effects on the BM are poorly characterized during chronic infection with intestinal helminth parasites. In this study, we use the Trichuris muris model of infection and show that Th1 cell-associated, but not acute Th2 cell-associated, responses to chronic T. muris infection cause a major, transient expansion of CD48- CD150- multipotent progenitor cells in the BM that is dependent on the presence of adaptive immune cells and IFN-γ signaling. Chronic T. muris infection also broadly stimulated proliferation of BM progenitor cells including CD48- CD150+ hematopoietic stem cells. This shift in progenitor activity during chronic T. muris infection correlated with a functional increase in myeloid colony formation in vitro as well as neutrophilia in the BM and peripheral blood. In parallel, we observed an accumulation of CD4+ , CD8+ , and CD4- CD8- (double negative) T cells that expressed IFN-γ, displaying activated and central memory-type phenotypes in the bone marrow during chronic infection. Thus, these results demonstrate that Th1 cell-driven responses in the intestine during chronic helminth infection potently influence upstream hematopoietic processes in the BM via IFN-γ.

Goblet Cell Derived RELM-ß Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation.

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)-ß 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-ß's role in host defense is unclear. Thus, wildtype and RELM-ß 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-ß did not directly increase IEC proliferation, rather RELM-ß 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-ß 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-ß 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.

(R)-PFI-2 is a potent and selective inhibitor of SETD7 methyltransferase activity in cells.

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.

Low-Dose Intestinal Trichuris muris Infection Alters the Lung Immune Microenvironment and Can Suppress Allergic Airway Inflammation.

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.

Lineage-specific regulation of allergic airway inflammation by the lipid phosphatase Src homology 2 domain-containing inositol 5-phosphatase (SHIP-1).

BACKGROUND: Inpp5d (Src homology 2 domain-containing inositol-5-phosphatase [Ship1])-deficient mice experience spontaneous airway inflammation and have enhanced sensitivity to allergen-induced airway inflammation. OBJECTIVE: We hypothesized that lineage-specific deletion of Ship1 expression in cells known to be crucial for adaptive TH2 responses would uncover distinct roles that could either positively or negatively regulate susceptibility to allergic airway inflammation (AAI). METHODS: Ship1 expression was deleted in B cells, T cells, or dendritic cells (DCs), and the resulting Ship1(ΔB cell), Ship1(ΔT cell), Ship1(ΔDC), or Ship1(F/F) (wild-type) control mice were evaluated in a model of house dust mite (HDM)-induced AAI. RESULTS: Unlike germline panhematopoietic Ship1 deletion, deletion of Ship1 selectively in either the B-cell, T-cell, or DC lineages did not result in spontaneous airway inflammation. Strikingly, although loss of Ship1 in the B-cell lineage did not affect HDM-induced AAI, loss of Ship1 in either of the T-cell or DC lineages protected mice from AAI by skewing the typical TH2 immune response toward a TH1 response. CONCLUSIONS: Although panhematopoietic deletion of Ship1 leads to spontaneous lung inflammation, selective deletion of Ship1 in T cells or DCs impairs the formation of an adaptive TH2 response and protects animals from HDM-induced AAI.

Regulation of CD4 T-cell differentiation and inflammation by repressive histone methylation.

Repressive epigenetic modifications such as dimethylation and trimethylation histone H3 at lysine 9 (H3K9me2 and H3K9me3) and H3K27me3 have been shown to be critical for embryonic stem (ES) cell differentiation by silencing cell lineage-promiscuous genes. CD4(+) T helper (T(H)) cell differentiation is a powerful model to study the molecular mechanisms associated with cellular lineage choice in adult cells. Naïve T(H) cells have the capacity to differentiate into one of the several phenotypically and functionally distinct and stable lineages. Although some repressive epigenetic mechanisms have a critical role in T(H) cell differentiation in a similar manner to that in ES cells, it is clear that there are disparate functions for certain modifications between ES cells and T(H) cells. Here we review the role of repressive histone modifications in the differentiation and function of T(H) cells in health and disease.

Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2-inducing allergen exposures.

BACKGROUND: Allergic inflammation involves the sensitization of naive CD4(+) T cells to allergens, resulting in a TH2-skewed inflammatory response. Although antigen presentation by dendritic cells to T cells in the lymph node is crucial for TH2 cell development, the innate signals that initiate adaptive type 2 inflammation and the role of group 2 innate lymphoid cells (ILC2s) are poorly understood. OBJECTIVE: We sought to investigate the influence of ILC2s and the route of priming on the development of an adaptive type 2 immune response to lung allergens. METHODS: Wild-type and ILC2-deficient mice were exposed intranasally or systemically to the TH2-inducing antigens house dust mite or ovalbumin in a model of allergic airway inflammation or the TH17-inducing bacterial antigen Saccharopolyspora rectivirgula in a model of hypersensitivity pneumonitis. The formation of an adaptive immune response was evaluated based on serum antibody titers and production of T cell-derived cytokines (IL-4, IL-5, IL-13 and IL-17A). RESULTS: We find that lung ILC2s play a critical role in priming the adaptive type 2 immune response to inhaled allergens, including the recruitment of eosinophils, TH2 cytokine production and serum IgE levels. Surprisingly, systemic priming with ovalbumin, with or without adjuvants, circumvents the requirement for ILC2s in inducing TH2-driven lung inflammation. ILC2s were also found to be dispensable for the sensitization to TH1- or TH17-inducing antigens. CONCLUSION: These data highlight a critical role for ILC2s in the development of adaptive type 2 responses to local, but not systemic, antigen exposure.

Lymph node hypertrophy following Leishmania major infection is dependent on TLR9.

Control of the protozoan parasite Leishmania major is dependent on establishing a robust T cell response. An early event in the development of an effective T cell response is the expansion (or hypertrophy) of the lymph node draining the site of infection, although the mechanisms involved in this response are not completely understood. In this study, we show that lymph node hypertrophy following L. major infection in mice is associated with increased recruitment of lymphocytes to the lymph node from the blood, and that CD62L-deficient mice, which are unable to recruit cells to the lymph node, develop a chronic infection with L. major. Injection of L. major-activated dendritic cells promoted lymph node hypertrophy, and this correlated with an increase in the expression of CCR7 on dendritic cells, although the upregulation of CCR7 occurred on the bystander (uninfected) dendritic cells rather than those containing parasites. We found that increased CCR7 expression was TLR9-dependent, that TLR9(-/-) dendritic cells migrated less efficiently to the draining lymph node, and that TLR9(-/-) mice exhibited a deficit in lymph node expansion following L. major infection, as well as increased susceptibility. Taken together, to our knowledge, these results are the first to demonstrate that activation of dendritic cells via TLR9 is essential for the induction of lymph node hypertrophy in leishmaniasis.

DOT1L maintains NK cell phenotype and function for optimal tumor control.

Histone methyltransferases (HMTs) are crucial in gene regulation and function, yet their role in natural killer (NK) cell biology within the tumor microenvironment (TME) remains largely unknown. We demonstrate that the HMT DOT1L limits NK cell conversion to CD49a+ CD49b+ intILC1, a subset that can be observed in the TME in response to stimulation with transforming growth factor (TGF)-ß and is correlated with impaired tumor control. Deleting Dot1l in NKp46-expressing cells reveals its pivotal role in maintaining NK cell phenotype and function. Loss of DOT1L skews NK cells toward intILC1s even in the absence of TGF-ß. Transcriptionally, DOT1L-null NK cells closely resemble intILC1s and ILC1s, correlating with altered NK cell responses and impaired solid tumor control. These findings deepen our understanding of NK cell biology and could inform approaches to prevent NK cell conversion to intILC1s in adoptive NK cell therapies for cancer.