IL-6 Revisited: From Rheumatoid Arthritis to CAR T Cell Therapy and COVID-19.

The diverse biological activity of interleukin-6 (IL-6) contributes to the maintenance of homeostasis. Emergent infection or tissue injury induces rapid production of IL-6 and activates host defense through augmentation of acute-phase proteins and immune responses. However, excessive IL-6 production and uncontrolled IL-6 receptor signaling are critical to pathogenesis. Over the years, therapeutic agents targeting IL-6 signaling, such as tocilizumab, a humanized anti-IL-6 receptor antibody, have shown remarkable efficacy for rheumatoid arthritis, Castleman disease, and juvenile idiopathic arthritis, and their efficacy in other diseases is continually being reported. Emerging evidence has demonstrated the benefit of tocilizumab for several types of acute inflammatory diseases, including cytokine storms induced by chimeric antigen receptor T cell therapy and coronavirus disease 2019 (COVID-19). Here, we refocus attention on the biology of IL-6 and summarize the distinct pathological roles of IL-6 signaling in several acute and chronic inflammatory diseases.

Signaling and Function of Interleukin-2 in T Lymphocytes.

The discovery of interleukin-2 (IL-2) changed the molecular understanding of how the immune system is controlled. IL-2 is a pleiotropic cytokine, and dissecting the signaling pathways that allow IL-2 to control the differentiation and homeostasis of both pro- and anti-inflammatory T cells is fundamental to determining the molecular details of immune regulation. The IL-2 receptor couples to JAK tyrosine kinases and activates the STAT5 transcription factors. However, IL-2 does much more than control transcriptional programs; it is a key regulator of T cell metabolic programs. The development of global phosphoproteomic approaches has expanded the understanding of IL-2 signaling further, revealing the diversity of phosphoproteins that may be influenced by IL-2 in T cells. However, it is increasingly clear that within each T cell subset, IL-2 will signal within a framework of other signal transduction networks that together will shape the transcriptional and metabolic programs that determine T cell fate.

Maternal inflammation regulates fetal emergency myelopoiesis.

Neonates are highly susceptible to inflammation and infection. Here, we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPCs) respond to inflammation, testing the hypothesis that deficits in the engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical adult-like EM transcriptional program. Moreover, we find that fetal HSPCs can respond to EM-inducing inflammatory stimuli in vitro but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that the loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of fetal demise. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection.

Crystal Formation in Inflammation.

The formation and accumulation of crystalline material in tissues is a hallmark of many metabolic and inflammatory conditions. The discovery that the phase transition of physiologically soluble substances to their crystalline forms can be detected by the immune system and activate innate immune pathways has revolutionized our understanding of how crystals cause inflammation. It is now appreciated that crystals are part of the pathogenesis of numerous diseases, including gout, silicosis, asbestosis, and atherosclerosis. In this review we discuss current knowledge of the complex mechanisms of crystal formation in diseased tissues and their interplay with the nutrients, metabolites, and immune cells that account for crystal-induced inflammation.

Immunity to the microbiota promotes sensory neuron regeneration.

Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.

A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

Interleukin-22: immunobiology and pathology.

Interleukin-22 (IL-22) is a recently described IL-10 family cytokine that is produced by T helper (Th) 17 cells, γδ T cells, NKT cells, and newly described innate lymphoid cells (ILCs). Knowledge of IL-22 biology has evolved rapidly since its discovery in 2000, and a role for IL-22 has been identified in numerous tissues, including the intestines, lung, liver, kidney, thymus, pancreas, and skin. IL-22 primarily targets nonhematopoietic epithelial and stromal cells, where it can promote proliferation and play a role in tissue regeneration. In addition, IL-22 regulates host defense at barrier surfaces. However, IL-22 has also been linked to several conditions involving inflammatory tissue pathology. In this review, we assess the current understanding of this cytokine, including its physiologic and pathologic effects on epithelial cell function.

Insights into cytokine-receptor interactions from cytokine engineering.

Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine-receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4.

Facile discovery of surrogate cytokine agonists.

Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This "cytokine med-chem" approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems.

Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis.

Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Microglial Remodeling of the Extracellular Matrix Promotes Synapse Plasticity.

Synapse remodeling is essential to encode experiences into neuronal circuits. Here, we define a molecular interaction between neurons and microglia that drives experience-dependent synapse remodeling in the hippocampus. We find that the cytokine interleukin-33 (IL-33) is expressed by adult hippocampal neurons in an experience-dependent manner and defines a neuronal subset primed for synaptic plasticity. Loss of neuronal IL-33 or the microglial IL-33 receptor leads to impaired spine plasticity, reduced newborn neuron integration, and diminished precision of remote fear memories. Memory precision and neuronal IL-33 are decreased in aged mice, and IL-33 gain of function mitigates age-related decreases in spine plasticity. We find that neuronal IL-33 instructs microglial engulfment of the extracellular matrix (ECM) and that its loss leads to impaired ECM engulfment and a concomitant accumulation of ECM proteins in contact with synapses. These data define a cellular mechanism through which microglia regulate experience-dependent synapse remodeling and promote memory consolidation.

Trans-ethnic and Ancestry-Specific Blood-Cell Genetics in 746,667 Individuals from 5 Global Populations.

Most loci identified by GWASs have been found in populations of European ancestry (EUR). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EUR individuals, we identified 5,552 trait-variant associations at p < 5 × 10-9, including 71 novel associations not found in EUR populations. We also identified 28 additional novel variants in ancestry-specific, non-EUR meta-analyses, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL-7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EUR-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations and compared genetic architecture and the effect of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies.

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.

A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion.

Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.

Diet Diurnally Regulates Small Intestinal Microbiome-Epithelial-Immune Homeostasis and Enteritis.

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10+ lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.

Migratory Neural Crest Cells Phagocytose Dead Cells in the Developing Nervous System.

During neural tube closure and spinal cord development, many cells die in both the central and peripheral nervous systems (CNS and PNS, respectively). However, myeloid-derived professional phagocytes have not yet colonized the trunk region during early neurogenesis. How apoptotic cells are removed from this region during these stages remains largely unknown. Using live imaging in zebrafish, we demonstrate that neural crest cells (NCCs) respond rapidly to dying cells and phagocytose cellular debris around the neural tube. Additionally, NCCs have the ability to enter the CNS through motor exit point transition zones and clear debris in the spinal cord. Surprisingly, NCCs phagocytosis mechanistically resembles macrophage phagocytosis and their recruitment toward cellular debris is mediated by interleukin-1ß. Taken together, our results reveal a role for NCCs in phagocytosis of debris in the developing nervous system before the presence of professional phagocytes.

Neutrophils Driving Unconventional T Cells Mediate Resistance against Murine Sarcomas and Selected Human Tumors.

Neutrophils are a component of the tumor microenvironment and have been predominantly associated with cancer progression. Using a genetic approach complemented by adoptive transfer, we found that neutrophils are essential for resistance against primary 3-methylcholantrene-induced carcinogenesis. Neutrophils were essential for the activation of an interferon-γ-dependent pathway of immune resistance, associated with polarization of a subset of CD4- CD8- unconventional αß T cells (UTCαß). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTCαß associated with neutrophil-dependent anti-sarcoma immunity. In selected human tumors, including undifferentiated pleomorphic sarcoma, CSF3R expression, a neutrophil signature and neutrophil infiltration were associated with a type 1 immune response and better clinical outcome. Thus, neutrophils driving UTCαß polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors.

Interleukin-2 signaling in the regulation of T cell biology in autoimmunity and cancer.

Interleukin-2 (IL-2) is a critical cytokine for T cell peripheral tolerance and immunity. Here, we review how IL-2 interaction with the high-affinity IL-2 receptor (IL-2R) supports the development and homeostasis of regulatory T cells and contributes to the differentiation of helper, cytotoxic, and memory T cells. A critical element for each T cell population is the expression of CD25 (Il2rα), which heightens the receptor affinity for IL-2. Signaling through the high-affinity IL-2R also reinvigorates CD8+ exhausted T (Tex) cells in response to checkpoint blockade. We consider the molecular underpinnings reflecting how IL-2R signaling impacts these various T cell subsets and the implications for enhancing IL-2-dependent immunotherapy of autoimmunity, other inflammatory disorders, and cancer.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity.

Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of ß-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1ß and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery.