Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

NLRP1B and NLRP3 Control the Host Response following Colonization with the Commensal Protist Tritrichomonas musculis.

Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on the host's intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis, a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. In this study, we report that colonization with T. musculis results in an increase in luminal extracellular ATP that is followed by increased caspase activity, higher cell death, elevated levels of IL-1ß, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. musculis These findings demonstrate that T. musculis-mediated host protection requires sensors of extracellular and intracellular ATP to confer resistance to enteric Salmonella infections.

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.

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.

ILC2 Activation by Protozoan Commensal Microbes.

Group 2 innate lymphoid cells (ILC2s) are a member of the ILC family and are involved in protective and pathogenic type 2 responses. Recent research has highlighted their involvement in modulating tissue and immune homeostasis during health and disease and has uncovered critical signaling circuits. While interactions of ILC2s with the bacterial microbiome are rather sparse, other microbial members of our microbiome, including helminths and protozoans, reveal new and exciting mechanisms of tissue regulation by ILC2s. Here we summarize the current field on ILC2 activation by the tissue and immune environment and highlight particularly new intriguing pathways of ILC2 regulation by protozoan commensals in the intestinal tract.

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.

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.

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.

Commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota.

Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus and intestinal bacteria in healthy and B-cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with Tritrichomonas musculus functional changes, which were accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single-cell transcriptomics identified distinct Tritrichomonas musculus life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable data sets to drive future mechanistic studies.

The commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota.

Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus ( T. mu ) and intestinal bacteria in healthy and B cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with T. mu functional changes, accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single cell transcriptomics identified distinct T. mu life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable datasets to drive future mechanistic studies.

Microbial energy metabolism fuels an intestinal macrophage niche in solitary isolated lymphoid tissues through purinergic signaling.

Maintaining macrophage (MΦ) heterogeneity is critical to ensure intestinal tissue homeostasis and host defense. The gut microbiota and host factors are thought to synergistically guide intestinal MΦ development, although the exact nature, regulation, and location of such collaboration remain unclear. Here, we report that microbial biochemical energy metabolism promotes colony-stimulating factor 2 (CSF2) production by group 3 innate lymphoid cells (ILC3s) within solitary isolated lymphoid tissues (SILTs) in a cell-extrinsic, NLRP3/P2X7R-dependent fashion in the steady state. Tissue-infiltrating monocytes accumulating around SILTs followed a spatially constrained, distinct developmental trajectory into SILT-associated MΦs (SAMs). CSF2 regulated the mitochondrial membrane potential and reactive oxygen species production of SAMs and contributed to the antimicrobial defense against enteric bacterial infections. Collectively, these findings identify SILTs and CSF2-producing ILC3s as a microanatomic niche for intestinal MΦ development and functional programming fueled by the integration of commensal microbial energy metabolism.

Rapid isolation of mouse ILCs from murine intestinal tissues.

Tissue-resident immune cells like innate lymphoid cells (ILCs) are regulators of local immunity and tissue homeostasis. Similar to Natural Killer (NK) cells, ILCs express germline-encoded natural cytotoxicity receptors (NCRs) that facilitate the rapid execution of effector functions. Recent advances using transgenic animal models have further uncovered the developmental, transcriptional, epigenetic, and functional differences between members of the ILC family. Isolation of ILCs, which are particularly enriched in non-lymphoid tissues, can often be challenging and time consuming. Here, we provide a simple and rapid protocol for the isolation of NK cells and ILCs from murine intestinal tissues. This protocol is suitable for Fluorescence Activated Cell Sorting (FACS) and intracellular analysis of cytokine and transcription factor expression using flow and mass cytometry.

Isolation of mononuclear phagocytes from the mouse gut.

The intestinal tract is home to trillions of microbes that make up the gut microbiota and is a major source of environmental antigens that can be derived from food, commensal microorganisms, and potential pathogens. Amidst this complex environment, myeloid cells, including macrophages (MPs) and dendritic cells (DCs), are key immunological sentinels that locally maintain both tissue and immune homeostasis. Recent research has revealed substantial functional and developmental heterogeneity within the intestinal DC and MP compartments, with evidence pointing to their regulation by the microbiota. DCs are classically divided into three subsets based on their CD103 and CD11b expression: CD103+CD11b-(XCR1+) cDC1s, CD103+CD11b+ cDC2s, and CD103-CD11b+ cDC2s. Meanwhile, mature gut MPs have recently been classified by their expression of Tim-4 and CD4 into a long-lived, self-maintaining Tim-4+CD4+ population and short-lived, monocyte-derived Tim-4-CD4+ and Tim-4-CD4- populations. In this chapter, we provide experimental procedures to classify and isolate these myeloid subsets from the murine intestinal lamina propria for functional characterization.

Going green with solar-powered ILC3 homeostasis.

Circadian clock proteins BMAL1 and REV-ERBα harmonize the development and function of ILC3 (see related articles by Teng et al. and Wang et al.).

Cytokine Networks between Innate Lymphoid Cells and Myeloid Cells.

Innate lymphoid cells (ILCs) are an essential component of the innate immune system in vertebrates. They are developmentally rooted in the lymphoid lineage and can diverge into at least three transcriptionally distinct lineages. ILCs seed both lymphoid and non-lymphoid tissues and are locally self-maintained in tissue-resident pools. Tissue-resident ILCs execute important effector functions making them key regulator in tissue homeostasis, repair, remodeling, microbial defense, and anti-tumor immunity. Similar to T lymphocytes, ILCs possess only few sensory elements for the recognition of non-self and thus depend on extrinsic cellular sensory elements residing within the tissue. Myeloid cells, including mononuclear phagocytes (MNPs), are key sentinels of the tissue and are able to translate environmental cues into an effector profile that instructs lymphocyte responses. The adaptation of myeloid cells to the tissue state thus influences the effector program of ILCs and serves as an example of how environmental signals are integrated into the function of ILCs via a tissue-resident immune cell cross talks. This review summarizes our current knowledge on the role of myeloid cells in regulating ILC functions and discusses how feedback communication between ILCs and myeloid cells contribute to stabilize immune homeostasis in order to maintain the healthy state of an organ.

The ion channel TRPM7 is required for B cell lymphopoiesis.

The transient receptor potential (TRP) family is a large family of widely expressed ion channels that regulate the intracellular concentration of ions and metals and respond to various chemical and physical stimuli. TRP subfamily M member 7 (TRPM7) is unusual in that it contains both an ion channel and a kinase domain. TRPM7 is a divalent cation channel with preference for Ca2+ and Mg2+ It is required for the survival of DT40 cells, a B cell line; however, deletion of TRPM7 in T cells does not impair their development. We found that expression of TRPM7 was required for B cell development in mice. Mice that lacked TRPM7 in B cells failed to generate peripheral B cells because of a developmental block at the pro-B cell stage. The loss of TRPM7 kinase activity alone did not affect the proportion of peripheral mature B cells or the development of B cells in the bone marrow. However, supplementation with a high concentration of extracellular Mg2+ partially rescued the development of TRPM7-deficient B cells in vitro. Thus, our findings identify a critical role for TRPM7 ion channel activity in B cell development.

Measurement of Cohesion and Adhesion of Semiconducting Polymers by Scratch Testing: Effect of Side-Chain Length and Degree of Polymerization.

Most advantages of organic electronic materials are enabled by mechanical deformability, as flexible (and stretchable) devices made from these materials must be able to withstand roll-to-roll printing and survive mechanical insults from the external environment. Cohesion and adhesion are two properties that dictate the mechanical reliability of a flexible organic electronic device. In this paper, progressive-load scratch tests are used for the first time to correlate the cohesive and adhesive behavior of poly(3-alkylthiophenes) (P3ATs) with respect to two molecular parameters: length of the alkyl side chain and molecular weight. In contrast to metrological techniques based on buckling or pull testing of pseudofreestanding films, scratch tests reveal information about both the cohesive and adhesive properties of thin polymeric films from a single procedure. Our data show a decrease in cohesion and adhesion, that is, a decrease in overall mechanical robustness, with increasing length of the side chain. This behavior is likely due to increases in free volume and concomitant decreases in the glass transition temperature. In contrast, we observe increases in both the cohesion and adhesion with increasing molecular weight. This behavior is attributed to an increased density of entanglements with high molecular weight, which manifests as increased extensibility. These observations are consistent with the results of molecular dynamics simulations. Interestingly, the normal (applied) forces associated with cohesive and adhesive failure are directly proportional to the average degree of polymerization, as opposed to simply the molecular weight, as the length of the alkyl side chain increases the molecular weight without increasing the degree of polymerization.

Neutrophils instruct homeostatic and pathological states in naive tissues.

Immune protection relies on the capacity of neutrophils to infiltrate challenged tissues. Naive tissues, in contrast, are believed to remain free of these cells and protected from their toxic cargo. Here, we show that neutrophils are endowed with the capacity to infiltrate multiple tissues in the steady-state, a process that follows tissue-specific dynamics. By focusing in two particular tissues, the intestine and the lungs, we find that neutrophils infiltrating the intestine are engulfed by resident macrophages, resulting in repression of Il23 transcription, reduced G-CSF in plasma, and reinforced activity of distant bone marrow niches. In contrast, diurnal accumulation of neutrophils within the pulmonary vasculature influenced circadian transcription in the lungs. Neutrophil-influenced transcripts in this organ were associated with carcinogenesis and migration. Consistently, we found that neutrophils dictated the diurnal patterns of lung invasion by melanoma cells. Homeostatic infiltration of tissues unveils a facet of neutrophil biology that supports organ function, but can also instigate pathological states.

G9a regulates group 2 innate lymphoid cell development by repressing the group 3 innate lymphoid cell program.

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.