Immunosensation: Neuroimmune Cross Talk in the Skin.

Classically, skin was considered a mere structural barrier protecting organisms from a diversity of environmental insults. In recent decades, the cutaneous immune system has become recognized as a complex immunologic barrier involved in both antimicrobial immunity and homeostatic processes like wound healing. To sense a variety of chemical, mechanical, and thermal stimuli, the skin harbors one of the most sophisticated sensory networks in the body. However, recent studies suggest that the cutaneous nervous system is highly integrated with the immune system to encode specific sensations into evolutionarily conserved protective behaviors. In addition to directly sensing pathogens, neurons employ novel neuroimmune mechanisms to provide host immunity. Therefore, given that sensation underlies various physiologies through increasingly complex reflex arcs, a much more dynamic picture is emerging of the skin as a truly systemic organ with highly coordinated physical, immunologic, and neural functions in barrier immunology.

Sensory neurons promote immune homeostasis in the lung.

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPß was dependent on JAK1 in the vagus nerve, and CGRPß suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

A basophil-neuronal axis promotes itch.

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.

IL-33 potentiates histaminergic itch.

BACKGROUND: Itch is a common symptom that can greatly diminish quality of life. Histamine is a potent endogenous pruritogen, and while antihistamines are often the first-line treatment for itch, in conditions like chronic spontaneous urticaria (CSU), many patients remain symptomatic while receiving maximal doses. Mechanisms that drive resistance to antihistamines are poorly defined. OBJECTIVES: Signaling of the alarmin cytokine IL-33 in sensory neurons is postulated to drive chronic itch by inducing neuronal sensitization to pruritogens. Thus, we sought to determine if IL-33 can augment histamine-induced (histaminergic) itch. METHODS: Itch behavior was assessed in response to histamine after IL-33 or saline administration. Various stimuli and conditional and global knockout mice were utilized to dissect cellular mechanisms. Multiple existing transcriptomic data sets were evaluated, including single-cell RNA sequencing of human and mouse skin, microarrays of isolated mouse mast cells at steady state and after stimulation with IL-33, and microarrays of skin biopsy samples from subjects with CSU and healthy controls. RESULTS: IL-33 amplifies histaminergic itch independent of IL-33 signaling in sensory neurons. Mast cells are the top expressors of the IL-33 receptor in both human and mouse skin. When stimulated by IL-33, mouse mast cells significantly increase IL-13 levels. Enhancement of histaminergic itch by IL-33 relies on a mast cell- and IL-13-dependent mechanism. IL-33 receptor expression is increased in lesional skin of subjects with CSU compared to healthy controls. CONCLUSIONS: Our findings suggest that IL-33 signaling may be a key driver of histaminergic itch in mast cell-associated pruritic conditions such as CSU.

Difelikefalin suppresses itch and reduces scratching independent of inflammation in a murine model of atopic dermatitis.

BACKGROUND: Therapies specifically targeting nonhistaminergic pruritus are largely lacking. Difelikefalin (DFK) has been found to reduce itch in various chronic pruritic conditions, including atopic dermatitis (AD). OBJECTIVE: We sought to investigate the ability of DFK to impact scratching behavior, inflammatory mediators, and neuronal signaling in a murine model of AD. METHODS: The ears of C57BL/6 mice were topically treated with MC903 for 12 consecutive days to induce AD-like inflammation and itch. Before MC903 treatment, mice were treated with either DFK (0.5 mg/kg, intraperitoneal injection twice daily) or vehicle (saline). Skin ear thickness, histological analysis, flow cytometry, RNA-sequencing, and differential gene expression analyses of mouse ear skin were used to examine the effect of DFK on skin inflammation. Scratching behavior was quantified to measure itch behavior in mice that were topically treated with MC903 for 6 consecutive days; then, mice received a single injection of either DFK (1.0 mg/kg, intraperitoneal injection) or saline. Calcium imaging and single-cell RNA-sequencing were used in mouse dorsal root ganglia neurons to determine the size of the neurons activated with DFK treatment. Statistical significance was determined by Mann-Whitney test, unless otherwise noted. RESULTS: DFK rapidly suppressed itch without altering AD-like skin inflammation in MC903 (calcipotriol)-treated mice. In vitro Ca2+ influx trace of dorsal root ganglia suggested that a major target for DFK is the larger-diameter mechanoreceptors (eg, Aꞵ-fibers), rather than small-diameter pruriceptive C-fibers. CONCLUSIONS: These studies support a potential neuromodulatory role of DFK for reducing itch associated with AD in mice.

IL-33 signaling in sensory neurons promotes dry skin itch.

BACKGROUND: Chronic pruritus, or itch, is common and debilitating, but the neuroimmune mechanisms that drive chronic itch are only starting to be elucidated. Recent studies demonstrate that the IL-33 receptor (IL-33R) is expressed by sensory neurons. However, whether sensory neuron-restricted activity of IL-33 is necessary for chronic itch remains poorly understood. OBJECTIVES: We sought to determine if IL-33 signaling in sensory neurons is critical for the development of chronic itch in 2 divergent pruritic disease models. METHODS: Plasma levels of IL-33 were assessed in patients with atopic dermatitis (AD) and chronic pruritus of unknown origin (CPUO). Mice were generated to conditionally delete IL-33R from sensory neurons. The contribution of neuronal IL-33R signaling to chronic itch development was tested in mouse models that recapitulate key pathologic features of AD and CPUO, respectively. RESULTS: IL-33 was elevated in both AD and CPUO as well as their respective mouse models. While neuron-restricted IL-33R signaling was dispensable for itch in AD-like disease, it was required for the development of dry skin itch in a mouse model that mirrors key aspects of CPUO pathology. CONCLUSIONS: These data highlight how IL-33 may be a predominant mediator of itch in certain contexts, depending on the tissue microenvironment. Further, this study provides insight into future therapeutic strategies targeting the IL-33 pathway for chronic itch.

Ubiquitin regulates caspase recruitment domain-mediated signaling by nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2.

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.

RAG suppresses group 2 innate lymphoid cells.

Antigen specificity is the central trait distinguishing adaptive from innate immune function. Assembly of antigen-specific T cell and B cell receptors occurs through V(D)J recombination mediated by the Recombinase Activating Gene endonucleases RAG1 and RAG2 (collectively called RAG). In the absence of RAG, mature T and B cells do not develop and thus RAG is critically associated with adaptive immune function. In addition to adaptive T helper 2 (Th2) cells, group 2 innate lymphoid cells (ILC2s) contribute to type 2 immune responses by producing cytokines like Interleukin-5 (IL-5) and IL-13. Although it has been reported that RAG expression modulates the function of innate natural killer (NK) cells, whether other innate immune cells such as ILC2s are affected by RAG remains unclear. We find that in RAG-deficient mice, ILC2 populations expand and produce increased IL-5 and IL-13 at steady state and contribute to increased inflammation in atopic dermatitis (AD)-like disease. Further, we show that RAG modulates ILC2 function in a cell-intrinsic manner independent of the absence or presence of adaptive T and B lymphocytes. Lastly, employing multiomic single cell analyses of RAG1 lineage-traced cells, we identify key transcriptional and epigenomic ILC2 functional programs that are suppressed by a history of RAG expression. Collectively, our data reveal a novel role for RAG in modulating innate type 2 immunity through suppression of ILC2s.

A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain.

Microglia, resident macrophages of the CNS, are essential to brain development, homeostasis, and disease. Microglial activation and proliferation are hallmarks of many CNS diseases, including neuropathic pain. However, molecular mechanisms that govern the spinal neuroimmune axis in the setting of neuropathic pain remain incompletely understood. Here, we show that genetic ablation or pharmacological blockade of transient receptor potential vanilloid type 4 (TRPV4) markedly attenuated neuropathic pain-like behaviors in a mouse model of spared nerve injury. Mechanistically, microglia-expressed TRPV4 mediated microglial activation and proliferation and promoted functional and structural plasticity of excitatory spinal neurons through release of lipocalin-2. Our results suggest that microglial TRPV4 channels reside at the center of the neuroimmune axis in the spinal cord, which transforms peripheral nerve injury into central sensitization and neuropathic pain, thereby identifying TRPV4 as a potential new target for the treatment of chronic pain.

Debaryomyces is enriched in Crohn's disease intestinal tissue and impairs healing in mice.

Alterations of the mycobiota composition associated with Crohn's disease (CD) are challenging to link to defining elements of pathophysiology, such as poor injury repair. Using culture-dependent and -independent methods, we discovered that Debaryomyces hansenii preferentially localized to and was abundant within incompletely healed intestinal wounds of mice and inflamed mucosal tissues of CD human subjects. D. hansenii cultures from injured mice and inflamed CD tissues impaired colonic healing when introduced into injured conventionally raised or gnotobiotic mice. We reisolated D. hansenii from injured areas of these mice, fulfilling Koch's postulates. Mechanistically, D. hansenii impaired mucosal healing through the myeloid cell-specific type 1 interferon-CCL5 axis. Taken together, we have identified a fungus that inhabits inflamed CD tissue and can lead to dysregulated mucosal healing.

Crystal structure of a complex of NOD1 CARD and ubiquitin.

The Caspase Recruitment Domain (CARD) from the innate immune receptor NOD1 was crystallized with Ubiquitin (Ub). NOD1 CARD was present as a helix-swapped homodimer similar to other structures of NOD1 CARD, and Ub monomers formed a homodimer similar in conformation to Lys48-linked di-Ub. The interaction between NOD1 CARD and Ub in the crystal was mediated by novel binding sites on each molecule. Comparisons of these sites to previously identified interaction surfaces on both molecules were made along with discussion of their potential functional significance.