Enteric helminth coinfection enhances host susceptibility to neurotropic flaviviruses via a tuft cell-IL-4 receptor signaling axis.

Although enteric helminth infections modulate immunity to mucosal pathogens, their effects on systemic microbes remain less established. Here, we observe increased mortality in mice coinfected with the enteric helminth Heligmosomoides polygyrus bakeri (Hpb) and West Nile virus (WNV). This enhanced susceptibility is associated with altered gut morphology and transit, translocation of commensal bacteria, impaired WNV-specific T cell responses, and increased virus infection in the gastrointestinal tract and central nervous system. These outcomes were due to type 2 immune skewing, because coinfection in Stat6-/- mice rescues mortality, treatment of helminth-free WNV-infected mice with interleukin (IL)-4 mirrors coinfection, and IL-4 receptor signaling in intestinal epithelial cells mediates the susceptibility phenotypes. Moreover, tuft cell-deficient mice show improved outcomes with coinfection, whereas treatment of helminth-free mice with tuft cell-derived cytokine IL-25 or ligand succinate worsens WNV disease. Thus, helminth activation of tuft cell-IL-4-receptor circuits in the gut exacerbates infection and disease of a neurotropic flavivirus.

A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression.

Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.

Interleukin-4 downregulates transcription factor BCL6 to promote memory B cell selection in germinal centers.

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

Type 2 cytokine signaling in macrophages protects from cellular senescence and organismal aging.

Accumulation of senescent cells in organs and tissues is a hallmark of aging and known to contribute to age-related diseases. Although aging-associated immune dysfunction, or immunosenescence, is known to contribute to this process, the underlying mechanism remains elusive. Here, we report that type 2 cytokine signaling deficiency accelerated aging and, conversely, that the interleukin-4 (IL-4)-STAT6 pathway protected macrophages from senescence. Mechanistically, activated STAT6 promoted the expression of genes involved in DNA repair both via homologous recombination and Fanconi anemia pathways. Conversely, STAT6 deficiency induced release of nuclear DNA into the cytoplasm to promote tissue inflammation and organismal aging. Importantly, we demonstrate that IL-4 treatment prevented macrophage senescence and improved the health span of aged mice to an extent comparable to senolytic treatment, with further additive effects when combined. Together, our findings support that type 2 cytokine signaling protects macrophages from immunosenescence and thus hold therapeutic potential for improving healthy aging.

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch.

Mammals have evolved neurophysiologic reflexes, such as coughing and scratching, to expel invading pathogens and noxious environmental stimuli. It is well established that these responses are also associated with chronic inflammatory diseases, including asthma and atopic dermatitis. However, the mechanisms by which inflammatory pathways promote sensations such as itch remain poorly understood. Here, we show that type 2 cytokines directly activate sensory neurons in both mice and humans. Further, we demonstrate that chronic itch is dependent on neuronal IL-4Rα and JAK1 signaling. We also observe that patients with recalcitrant chronic itch that failed other immunosuppressive therapies markedly improve when treated with JAK inhibitors. Thus, signaling mechanisms previously ascribed to the immune system may represent novel therapeutic targets within the nervous system. Collectively, this study reveals an evolutionarily conserved paradigm in which the sensory nervous system employs classical immune signaling pathways to influence mammalian behavior.

The epigenetic state of IL-4-polarized macrophages enables inflammatory cistromic expansion and extended synergistic response to TLR ligands.

Prior exposure to microenvironmental signals could fundamentally change the response of macrophages to subsequent stimuli. It is believed that T helper-2 (Th2)-cell-type cytokine interleukin-4 (IL-4) and Toll-like receptor (TLR) ligand-activated transcriptional programs mutually antagonize each other, and no remarkable convergence has been identified between them. In contrast, here, we show that IL-4-polarized macrophages established a hyperinflammatory gene expression program upon lipopolysaccharide (LPS) exposure. This phenomenon, which we termed extended synergy, was supported by IL-4-directed epigenomic remodeling, LPS-activated NF-κB-p65 cistrome expansion, and increased enhancer activity. The EGR2 transcription factor contributed to the extended synergy in a macrophage-subtype-specific manner. Consequently, the previously alternatively polarized macrophages produced increased amounts of immune-modulatory factors both in vitro and in vivo in a murine Th2 cell-type airway inflammation model upon LPS exposure. Our findings establish that IL-4-induced epigenetic reprogramming is responsible for the development of inflammatory hyperresponsiveness to TLR activation and contributes to lung pathologies.

Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation.

B cells within germinal centers (GCs) enter cycles of antibody affinity maturation or exit the GC as memory cells or plasma cells. Here, we examined the contribution of interleukin (IL)-4 on B cell fate decisions in the GC. Single-cell RNA-sequencing identified a subset of light zone GC B cells expressing high IL-4 receptor-a (IL4Ra) and CD23 and lacking a Myc-associated signature. These cells could differentiate into pre-memory cells. B cell-specific deletion of IL4Ra or STAT6 favored the pre-memory cell trajectory, and provision of exogenous IL-4 in a wild-type context reduced pre-memory cell frequencies. IL-4 acted during antigen-specific interactions but also influenced bystander cells. Deletion of IL4Ra from follicular dendritic cells (FDCs) increased the availability of IL-4 in the GC, impaired the selection of affinity-matured B cells, and reduced memory cell generation. We propose that GC FDCs establish a niche that limits bystander IL-4 activity, focusing IL-4 action on B cells undergoing selection and enhancing memory cell differentiation.

Brief homogeneous TCR signals instruct common iNKT progenitors whose effector diversification is characterized by subsequent cytokine signaling.

Innate-like T cell populations expressing conserved TCRs play critical roles in immunity through diverse developmentally acquired effector functions. Focusing on the prototypical lineage of invariant natural killer T (iNKT) cells, we sought to dissect the mechanisms and timing of fate decisions and functional effector differentiation. Utilizing induced expression of the semi-invariant NKT cell TCR on double positive thymocytes, an initially highly synchronous wave of iNKT cell development was triggered by brief homogeneous TCR signaling. After reaching a uniform progenitor state characterized by IL-4 production potential and proliferation, effector subsets emerged simultaneously, but then diverged toward different fates. While NKT17 specification was quickly completed, NKT1 cells slowly differentiated and expanded. NKT2 cells resembled maturing progenitors, which gradually diminished in numbers. Thus, iNKT subset diversification occurs in dividing progenitor cells without acute TCR input but utilizes multiple active cytokine signaling pathways. These data imply a two-step model of iNKT effector differentiation.

Cross-tissue single-cell landscape of human monocytes and macrophages in health and disease.

Mononuclear phagocytes (MNPs) encompass dendritic cells, monocytes, and macrophages (MoMac), which exhibit antimicrobial, homeostatic, and immunoregulatory functions. We integrated 178,651 MNPs from 13 tissues across 41 datasets to generate a MNP single-cell RNA compendium (MNP-VERSE), a publicly available tool to map MNPs and define conserved gene signatures of MNP populations. Next, we generated a MoMac-focused compendium that revealed an array of specialized cell subsets widely distributed across multiple tissues. Specific pathological forms were expanded in cancer and inflammation. All neoplastic tissues contained conserved tumor-associated macrophage populations. In particular, we focused on IL4I1+CD274(PD-L1)+IDO1+ macrophages, which accumulated in the tumor periphery in a T cell-dependent manner via interferon-γ (IFN-γ) and CD40/CD40L-induced maturation from IFN-primed monocytes. IL4I1_Macs exhibited immunosuppressive characteristics through tryptophan degradation and promoted the entry of regulatory T cell into tumors. This integrated analysis provides a robust online-available platform for uniform annotation and dissection of specific macrophage functions in healthy and pathological states.

Age-Related Dopaminergic Innervation Augments T Helper 2-Type Allergic Inflammation in the Postnatal Lung.

Young children are more susceptible to developing allergic asthma than adults. As neural innervation of the peripheral tissue continues to develop after birth, neurons may modulate tissue inflammation in an age-related manner. Here we showed that sympathetic nerves underwent a dopaminergic-to-adrenergic transition during post-natal development of the lung in mice and humans. Dopamine signaled through a specific dopamine receptor (DRD4) to promote T helper 2 (Th2) cell differentiation. The dopamine-DRD4 pathway acted synergistically with the cytokine IL-4 by upregulating IL-2-STAT5 signaling and reducing inhibitory histone trimethylation at Th2 gene loci. In murine models of allergen exposure, the dopamine-DRD4 pathway augmented Th2 inflammation in the lungs of young mice. However, this pathway operated marginally after sympathetic nerves became adrenergic in the adult lung. Taken together, the communication between dopaminergic nerves and CD4+ T cells provides an age-related mechanism underlying the susceptibility to allergic inflammation in the early lung.

The alternative macrophage relay: STAT6 passes the baton to EGR2.

Alternative polarization of macrophages induced by IL-4 is important for homeostasis and tissue repair. Downstream from IL-4 receptor signaling, STAT6 activation is transient, but induces stable transcriptional changes. These data suggest that STAT6 induces second messengers to carry out the alternative transcriptional program. In this issue of Genes & Development, Daniel and colleagues (pp. 1474-1492) identify EGR2 as a downstream regulator of STAT6 with broad functionality that further induces many transcription factors associated with alternative polarization. Identification of high EGR2 expression in a subset of mouse and human alveolar macrophages further highlights EGR2 as a conserved marker of alternatively activated macrophages.

The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization.

Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages.

The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1ß production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.

The Transcription Factor Ets1 Suppresses T Follicular Helper Type 2 Cell Differentiation to Halt the Onset of Systemic Lupus Erythematosus.

Single-nucleotide polymorphisms in ETS1 are associated with systemic lupus erythematosus (SLE). Ets1-/- mice develop SLE-like symptoms, suggesting that dysregulation of this transcription factor is important to the onset or progression of SLE. We used conditional deletion approaches to examine the impact of Ets1 expression in different immune cell types. Ets1 deletion on CD4+ T cells, but not B cells or dendritic cells, resulted in the SLE autoimmunity, and this was associated with the spontaneous expansion of T follicular helper type 2 (Tfh2) cells. Ets1-/- Tfh2 cells exhibited increased expression of GATA-3 and interleukin-4 (IL-4), which induced IgE isotype switching in B cells. Neutralization of IL-4 reduced Tfh2 cell frequencies and ameliorated disease parameters. Mechanistically, Ets1 suppressed signature Tfh and Th2 cell genes, including Cxcr5, Bcl6, and Il4ra, thus curbing the terminal Tfh2 cell differentiation process. Tfh2 cell frequencies in SLE patients correlated with disease parameters, providing evidence for the relevance of these findings to human disease.

Mitochondrial Membrane Potential Regulates Nuclear Gene Expression in Macrophages Exposed to Prostaglandin E2.

Metabolic engagement is intrinsic to immune cell function. Prostaglandin E2 (PGE2) has been shown to modulate macrophage activation, yet how PGE2 might affect metabolism is unclear. Here, we show that PGE2 caused mitochondrial membrane potential (Δψm) to dissipate in interleukin-4-activated (M(IL-4)) macrophages. Effects on Δψm were a consequence of PGE2-initiated transcriptional regulation of genes, particularly Got1, in the malate-aspartate shuttle (MAS). Reduced Δψm caused alterations in the expression of 126 voltage-regulated genes (VRGs), including those encoding resistin-like molecule α (RELMα), a key marker of M(IL-4) cells, and genes that regulate the cell cycle. The transcription factor ETS variant 1 (ETV1) played a role in the regulation of 38% of the VRGs. These results reveal ETV1 as a Δψm-sensitive transcription factor and Δψm as a mediator of mitochondrial-directed nuclear gene expression.

The Nuclear Receptor PPARγ Controls Progressive Macrophage Polarization as a Ligand-Insensitive Epigenomic Ratchet of Transcriptional Memory.

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

RAS P21 Protein Activator 3 (RASA3) Specifically Promotes Pathogenic T Helper 17 Cell Generation by Repressing T-Helper-2-Cell-Biased Programs.

Pathogenic Th17 (pTh17) cells drive inflammation and immune-pathology, but whether pTh17 cells are a Th17 cell subset whose generation is under specific molecular control remains unaddressed. We found that Ras p21 protein activator 3 (RASA3) was highly expressed by pTh17 cells relative to non-pTh17 cells and was required specifically for pTh17 generation in vitro and in vivo. Mice conditionally deficient for Rasa3 in T cells showed less pathology during experimental autoimmune encephalomyelitis. Rasa3-deficient T cells acquired a Th2 cell-biased program that dominantly trans-suppressed pTh17 cell generation via interleukin 4 production. The Th2 cell bias of Rasa3-deficient T cells was due to aberrantly elevated transcription factor IRF4 expression. RASA3 promoted proteasome-mediated IRF4 protein degradation by facilitating interaction of IRF4 with E3-ubiquitin ligase Cbl-b. Therefore, a RASA3-IRF4-Cbl-b pathway specifically directs pTh17 cell generation by balancing reciprocal Th17-Th2 cell programs. These findings indicate that a distinct molecular program directs pTh17 cell generation and reveals targets for treating pTh17 cell-related pathology and diseases.

Epigenomic regulation of macrophage polarization: Where do the nuclear receptors belong?

Our laboratory has a long-standing research interest in understanding how lipid-activated transcription factors, nuclear hormone receptors, contribute to dendritic cell and macrophage gene expression regulation, subtype specification, and responses to a changing extra and intracellular milieu. This journey in the last more than two decades took us from identifying target genes for various RXR heterodimers to systematically mapping nuclear receptor-mediated pathways in dendritic cells to identifying hierarchies of transcription factors in alternative polarization in macrophages to broaden the role of nuclear receptors beyond strictly ligand-regulated gene expression. We detail here the milestones of the road traveled and draw conclusions regarding the unexpectedly broad role of nuclear hormone receptors as epigenomic components of dendritic cell and macrophage gene regulation as we are getting ready for the next challenges.