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Eosinophilic esophagitis (EoE) is a chronic and progressive immune-mediated disease of the esophagus associated with antigen-driven type 2 inflammation and symptoms of esophageal dysfunction. Our understanding of EoE pathophysiology has evolved since its initial recognition more than 20 years ago and has translated into diagnostic and novel therapeutic approaches that are affecting patient care. The mechanisms underlying disease development and progression are influenced by diverse factors, such as genetics, age, allergic comorbidities, and allergen exposures. Central to EoE pathophysiology is a dysregulated feed-forward cycle that develops between the esophageal epithelium and the immune system. Allergen-induced, type 2-biased immune activation by the esophageal epithelium propagates a cycle of impaired mucosal barrier integrity and allergic inflammation, eventually leading to tissue remodeling and progressive organ dysfunction. Herein, we review the current understanding of fundamental pathophysiological mechanisms contributing to EoE pathogenesis.

Background and aims In clinical and experimental NASH, the origin of the scar-forming myofibroblast is the HSC. We used foz/foz mice on a Western diet to characterize in detail the phenotypic changes of HSCs in a NASH model. Approach and results We examined the single-cell expression profiles (scRNA sequencing) of HSCs purified from the normal livers of foz/foz mice on a chow diet, in NASH with fibrosis of foz/foz mice on a Western diet, and in livers during regression of NASH after switching back to a chow diet. Selected genes were analyzed using immunohistochemistry, quantitative real-time PCR, and short hairpin RNA knockdown in primary mouse HSCs. Our analysis of the normal liver identified two distinct clusters of quiescent HSCs that correspond to their acinar position of either pericentral vein or periportal vein. The NASH livers had four distinct HSC clusters, including one representing the classic fibrogenic myofibroblast. The three other HSC clusters consisted of a proliferating cluster, an intermediate activated cluster, and an immune and inflammatory cluster. The livers with NASH regression had one cluster of inactivated HSCs, which was similar to, but distinct from, the quiescent HSCs. Conclusions Analysis of single-cell RNA sequencing in combination with an interrogation of previous studies revealed an unanticipated heterogeneity of HSC phenotypes under normal and injured states.

Noncoding genetic variation is a major driver of phenotypic diversity but determining the underlying mechanisms and the cell types in which it acts remain challenging problems. Here, we investigate the impact of natural genetic variation provided by phenotypically diverse inbred strains of mice on gene expression and epigenetic landscapes of Kupffer cells. Analysis of gene expression in Kupffer cells and other liver cell types derived from C57BL/6J, BALB/cJ and A/J mice provided evidence for strain-specific differences in environmental factors influencing Kupffer cell phenotypes, including preferential Leptin signaling in BALB/cJ Kupffer cells. Systematic analysis of transcriptomic and epigenetic data from F1 hybrids of these mice, and transcriptomic data from strain-specific Kupffer cells engrafted into a common host enabled quantitative assessment of cis versus trans effects of genetic variation on gene expression and an estimate of cell autonomous versus non cell autonomous effects. Under homeostatic conditions, trans effects of genetic variation were dominant, with the majority of trans regulation being non cell autonomous. In contrast, strain specific responses to acutely administered LPS were primarily associated with genetic variation acting in cis to modify response elements for lineage determining and signal dependent transcription factors. Collectively, these findings reveal cell intrinsic and environmental effects of natural genetic variation on gene expression, demonstrate the use of enhancers as detectors of trans effects of genetic variation, and provide a new resource for understanding the impact of genetic variation on gene expression in Kupffer cells.

Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1(Enh/Enh)). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia—even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1(Enh/−) mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

The development and functional potential of metazoan cells is dependent on combinatorial roles of transcriptional enhancers and promoters. Macrophages provide exceptionally powerful model systems for investigation of mechanisms underlying the activation of cell-specific enhancers that drive transitions in cell fate and cell state. Here, we review recent advances that have expanded appreciation of the diversity of macrophage phenotypes in health and disease, emphasizing studies of liver, adipose tissue, and brain macrophages as paradigms for other tissue macrophages and cell types. Studies of normal tissue-resident macrophages and macrophages associated with cirrhosis, obese adipose tissue, and neurodegenerative disease illustrate the major roles of tissue environment in remodeling enhancer landscapes to specify the development and functions of distinct macrophage phenotypes. We discuss the utility of quantitative analysis of environment-dependent changes in enhancer activity states as an approach to discovery of regulatory transcription factors and upstream signaling pathways.

Summary Integrative analysis of next-generation sequencing data can help understand disease mechanisms. Specifically, ChIP-seq can illuminate where transcription regulators bind to regulate transcription. A major obstacle to performing this assay on primary cells is the low numbers obtained from tissues. The extensively validated ChIP-seq protocol presented here uses small volumes and single-pot on-bead library preparation to generate diverse high-quality ChIP-seq data. This protocol allows for medium-to-high-throughput ChIP-seq of low-abundance cells and can also be applied to other mammalian cells. For complete details on the use and execution of this protocol, please refer to Brigidi et al. (2019), Carlin et al. (2018), Heinz et al. (2018), Nott et al. (2019), Sakai et al. (2019), and Seidman et al. (2020)., Graphical abstract, Highlights • Cost-effective rapid, high-quality ChIP-seq prep (∼$40/library) • 8-well tube strip format for medium- to high-throughput ChIP-seq • Wide input range (0.1–5 million cells per IP) • Recommendations for antibody choice, assay optimization, and troubleshooting, Integrative analysis of next-generation sequencing data can help understand disease mechanisms. Specifically, ChIP-seq can illuminate where transcription regulators bind to regulate transcription. A major obstacle to performing this assay on primary cells is the low numbers obtained from tissues. The extensively validated ChIP-seq protocol presented here uses small volumes and single-pot on-bead library preparation to generate diverse high-quality ChIP-seq data. This protocol allows for medium-to-high-throughput ChIP-seq of low-abundance cells and can also be applied to other mammalian cells.

Macrophages respond to microbial ligands and various noxious cues by initiating an inflammatory response aimed at eliminating the original pathogenic insult. Transition of macrophages from a proinflammatory state to a reparative state, however, is vital for resolution of inflammation and return to homeostasis. The molecular players governing this transition remain poorly defined. Here, we find that the reparative macrophage transition is dictated by B-cell adapter for PI3K (BCAP). Mice harboring a macrophage-specific deletion of BCAP fail to recover from and succumb to dextran sulfate sodium-induced colitis due to prolonged intestinal inflammation and impaired tissue repair. Following microbial stimulation, gene expression in WT macrophages switches from an early inflammatory signature to a late reparative signature, a process that is hampered in BCAP-deficient macrophages. We find that absence of BCAP hinders inactivation of FOXO1 and GSK3β, which contributes to their enhanced inflammatory state. BCAP deficiency also results in defective aerobic glycolysis and reduced lactate production. This translates into reduced histone lactylation and decreased expression of reparative macrophage genes. Thus, our results reveal BCAP to be a critical cell-intrinsic switch that regulates transition of inflammatory macrophages to reparative macrophages by imprinting epigenetic changes.

Tissue resident macrophages and recruited monocyte-derived macrophages contribute to host defense but also play pathological roles in a diverse range of human diseases. Multiple macrophage phenotypes are often represented in a diseased tissue, but we lack a deep understanding of the mechanisms that control diversification. Here we use a combination of genetic, genomic, and imaging approaches to investigate the origins and epigenetic trajectories of hepatic myeloid cells during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induces striking changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss is compensated by gain of adjacent monocyte derived macrophages that exhibit convergent epigenomes, transcriptomes and functions. NASH-induced changes in Kupffer cell enhancers are driven by AP-1 and Egr factors that reprogram the Kupffer cell specific functions of Liver X receptors. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct programs of gene expression and corresponding phenotypes.

Oxidized phospholipids (OxPLs), which arise due to oxidative stress, are pro-inflammatory and pro-atherogenic, but their roles in non-alcoholic steatohepatitis (NASH) are unknown. Here, we show that OxPLs accumulate in human and mouse NASH. Using a transgenic mouse that expresses a functional single chain variable fragment of E06, a natural antibody that neutralizes OxPLs, we demonstrate the casual role of OxPLs in NASH. Targeting OxPLs in hyperlipidemic Ldlr(−/−) mice improved multiple aspects of NASH, including steatosis, inflammation, fibrosis, hepatocyte death and progression to hepatocellular carcinoma. Mechanistically, we found that OxPLs promote ROS accumulation to induce mitochondrial dysfunction in hepatocytes. Neutralizing OxPLs in AMLN diet-fed Ldlr(−/−) mice reduced oxidative stress, improved hepatic and adipose tissue mitochondrial function and fatty acid oxidation. These results suggest targeting OxPLs may be an effective therapeutic strategy for NASH.

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macrophage phenotypes are represented in diseased tissues, but we lack deep understanding of mechanisms controlling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derived macrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-induced changes in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions required for Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct gene expression programs and corresponding functions.

SETD5, a gene linked to intellectual disability (ID) and autism spectrum disorder (ASD), is a member of the SET-domain family and encodes a putative histone methyltransferase (HMT). To date, the mechanism by which SETD5 haploinsufficiency causes ASD/ID remains an unanswered question. Setd5 is the highly conserved mouse homolog, and although the Setd5 null mouse is embryonic lethal, the heterozygote is viable. Morphological tracing and multielectrode array was used on cultured cortical neurons. MRI was conducted of adult mouse brains and immunohistochemistry of juvenile mouse brains. RNA-Seq was used to investigate gene expression in the developing cortex. Behavioral assays were conducted on adult mice. Setd5+/− cortical neurons displayed significantly reduced synaptic density and neuritic outgrowth in vitro, with corresponding decreases in network activity and synchrony by electrophysiology. A specific subpopulation of fetal Setd5+/− cortical neurons showed altered gene expression of neurodevelopment-related genes. Setd5+/− animals manifested several autism-like behaviors, including hyperactivity, cognitive deficit, and altered social interactions. Anatomical differences were observed in Setd5+/− adult brains, accompanied by a deficit of deep-layer cortical neurons in the developing brain. Our data converge on a picture of abnormal neurodevelopment driven by Setd5 haploinsufficiency, consistent with a highly penetrant risk factor.

Significance The beneficial effects of LXR-pathway activation have long been appreciated, but clinical application of synthetic LXR ligands has been limited by coactivation of SREBP1c and consequent hypertriglyceridemia. Natural LXR ligands such as desmosterol do not promote hypertriglyceridemia because of coordinate down-regulation of the SREBP pathway. Here we demonstrate that synthetic desmosterol mimetics activate LXR in macrophages both in vitro and in vivo while suppressing SREBP target genes. Unexpectedly, desmosterol and synthetic desmosterol mimetics have almost no effect on LXR activity in hepatocytes in comparison with conventional synthetic LXR ligands. These findings reveal cell-specific differences in LXR responses to natural and synthetic ligands in macrophages and liver cells that provide a conceptually new basis for future drug development., Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause marked hypertriglyceridemia by inducing the expression of sterol regulatory element-binding protein (SREBP)1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c-induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytes in vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophages in vitro and in vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in the liver that lead to hypertriglyceridemia.

Activation of liver X receptors (LXRs) with synthetic agonists promotes reverse cholesterol transport and protects against atherosclerosis in mouse models. Most synthetic LXR agonists also cause marked hypertriglyceridemia by inducing the expression of SREBP1c and downstream genes that drive fatty acid biosynthesis. Recent studies demonstrated that desmosterol, an intermediate in the cholesterol biosynthetic pathway that suppresses SREBP processing by binding to SCAP, also binds and activates LXRs and is the most abundant LXR ligand in macrophage foam cells. Here, we explore the potential of increasing endogenous desmosterol production or mimicking its activity as a means of inducing LXR activity while simultaneously suppressing SREBP1c induced hypertriglyceridemia. Unexpectedly, while desmosterol strongly activated LXR target genes and suppressed SREBP pathways in mouse and human macrophages, it had almost no activity in mouse or human hepatocytesin vitro. We further demonstrate that sterol-based selective modulators of LXRs have biochemical and transcriptional properties predicted of desmosterol mimetics and selectively regulate LXR function in macrophagesin vitroandin vivo. These studies thereby reveal cell-specific discrimination of endogenous and synthetic regulators of LXRs and SREBPs, providing a molecular basis for dissociation of LXR functions in macrophages from those in liver that lead to hypertriglyceridemia.SIGNIFICANCE STATEMENTThe beneficial effects of LXR pathway activation in the prevention of atherosclerotic heart disease have long been appreciated. However, efforts to translate those effects in humans with synthetic LXR ligands has been met with the unintended consequence of hypertriglyceridemia, a product of co-activation of SREBP1c. Natural LXR ligands such as desmosterol do not promote hypertriglyceridemia because of coordinate down-regulation of the SREBP pathway. Here, we demonstrate that synthetic desmosterol mimetics activate LXR pathways macrophages both in vitro and in vivo without co-stimulation of SREBP1c. Unexpectedly, desmosterol and synthetic desmosterol mimetics almost no effect on LXR activity in hepatocytes in comparison to conventional synthetic LXR ligands. These findings reveal cell-specific differences in LXR responses to natural and synthetic ligands in macrophages and liver cells that provide a conceptually new basis for future drug development.

Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes. Analyses of genetic variation in macrophages from inbred mouse strains reveal how a complex network of transcription factors influence cis-regulatory elements to impact differentiation and responses to environmental change.

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.

Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of mouse CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis plays an important role in translating TCR signal strength into proportional activation of enhancers and genes essential for T cell function. DOI: http://dx.doi.org/10.7554/eLife.10134.001, eLife digest T helper cells recognize and respond to bacteria, viruses and other invading microbes and thus play a central role in the adaptive immune system. These cells have a receptor on their surface that binds to fragments of proteins – known as oligopeptides – from the microbes that have been digested and presented on the surfaces of other immune cells. Once active, T helper cells multiply, grow and release signals that regulate genes in other cells to promote immune responses. Previous studies suggest that a T helper cell’s response is binary – that is, either on or off. However, this does not explain how the strength of the T cell response to infection can vary. Allison et al. used a technique called high-throughput sequencing to examine the activity of genes in T helper cells from mice that had been genetically engineered to only produce one type of T cell receptor. For the experiments, the T cells were exposed to various concentrations of different peptides known to bind either well or poorly to the receptor. Allison et al. found that, once activated, the response of an individual T cell was not binary, but instead was related to the strength of the signal it received through its receptor. Further experiments showed that although a subset of the genes activated in T helper cells do respond in a binary fashion, the activities of many other genes involved in immune responses and cell metabolism were related to the strength of the signal from the receptor. This “analog” gene activation depends on the level of activity of the MAP kinase signaling pathway. Together, Allison et al.’s findings help us to understand how T cells are able to fine-tune immune responses to invading microbes. The next challenge will be to investigate the mechanisms underlying binary and analog gene activity in T cells. DOI: http://dx.doi.org/10.7554/eLife.10134.002

Although macrophages can be polarized to distinct phenotypes in vitro with individual ligands, in vivo they encounter multiple signals that control their varied functions in homeostasis, immunity, and disease. Here, we identify roles of Rev-erb nuclear receptors in regulating responses of mouse macrophages to complex tissue damage signals and wound repair. Rather than reinforcing a specific program of macrophage polarization, Rev-erbs repress subsets of genes that are activated by TLR ligands, IL4, TGFβ, and damage-associated molecular patterns (DAMPS). Unexpectedly, a complex damage signal promotes co-localization of NF-κB, Smad3, and Nrf2 at Rev-erb-sensitive enhancers and drives expression of genes characteristic of multiple polarization states in the same cells. Rev-erb-sensitive enhancers thereby integrate multiple damage-activated signaling pathways to promote a wound repair phenotype. DOI: http://dx.doi.org/10.7554/eLife.13024.001

TLRs are a family of pattern recognition receptors that recognize conserved molecular structures/products from a wide variety of microbes. Following recognition of ligands, TLRs recruit signaling adapters to initiate a pro-inflammatory signaling cascade culminating in the activation of several transcription factor families. Additionally, TLR signals lead to activation of PI3K, affecting many aspects of the cellular response, including cell survival, proliferation and regulation of the pro-inflammatory response. The recent discovery of BCAP as a TLR signaling adaptor, crucial for linking TLRs to PI3K activation, allows new questions of the importance of PI3K activation downstream of TLRs. Here, we summarize the current understanding of signaling pathways activated by TLRs and provide our perspective on TLR mediated activation of PI3K and its impact on regulating cellular processes.

The sensing and clearing of microbial infections by multicellular organisms is accomplished through specialized germ line-encoded receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are a group of PRRs that recognize conserved molecules or products derived from a plethora of pathogens such as bacteria, fungi, protozoa, and viruses. Following ligand recognition, TLRs function to induce acute inflammatory responses that contribute to the killing of invading pathogens. In addition, TLR activation also induces dendritic cell maturation and proinflammatory cytokine secretion, which act synergistically to activate and enhance pathogen-specific adaptive immune responses. The initiation of the functional TLR response depends upon the recruitment of TLR-signaling adaptor molecules containing Toll Interleukin-1 receptor domains. These TLR-signaling adaptors then initiate signaling cascades leading to the activation of AP-1, which are the transcription factor(s) activated by MAP kinases, nuclear translocation of NF-kB-light chain enhancer of activated B cells, and interferon-regulatory factor transcription factor families.

Toll like receptors (TLRs) use Toll–IL-1 receptor (TIR) domain-containing adapters, such as myeloid differentiation primary response gene 88 (MyD88) and TIR domain-containing adapter inducing IFN-β (TRIF), to induce activation of transcription factors, including NF-κB, MAP kinases, and IFN regulatory factors. TLR signaling also leads to activation of PI3K, but the molecular mechanism is not understood. Here we have discovered a unique role for B-cell adapter for PI3K (BCAP) in the TLR-signaling pathway. We find that BCAP has a functional N-terminal TIR homology domain and links TLR signaling to activation of PI3K. In addition, BCAP negatively regulates proinflammatory cytokine secretion upon TLR stimulation. In vivo, the absence of BCAP leads to exaggerated recruitment of inflammatory myeloid cells following infections and enhanced susceptibility to dextran sulfate sodium-induced colitis. Our results demonstrate that BCAP is a unique TIR domain-containing TLR signaling adapter crucial for linking TLRs to PI3K activation and regulating the inflammatory response.

SummaryActivation of pattern recognition receptors on dendritic cells (DCs) and macrophages leads to secretion of cytokines that control differentiation of CD4+ T cells. The current understanding is that interleukin-6 (IL-6) in combination with transforming growth factor-β (TGF-β) leads to generation of T helper 17 (Th17) lineage cells. Here, we have discovered that the cytokine requirements for Th17 cell polarization depend on the site of priming. Although IL-6 played a critical role in Th17 cell lineage priming in the skin and mucosal tissues, it was not required for Th17 cell priming in the spleen. In contrast, IL-1 played an irreplaceable role for priming of Th17 lineage cells in all tissues. Importantly, we have demonstrated that IL-6-independent and -dependent pathways of Th17 cell differentiation are guided by DCs residing in various tissues. These results reveal fundamental differences by which the systemic, mucosal, and cutaneous immune systems guide Th17 cell lineage commitment.