Single-Cell Transcriptomic Analysis Links Nonmyelinating Schwann Cells to Proinflammatory Response in the Lung.

The lung is a barrier tissue with constant exposure to the inhaled environment. Therefore, innate immunity against particulates and pathogens is of critical importance to maintain tissue homeostasis. Although the lung harbors both myelinating and nonmyelinating Schwann cells (NMSCs), NMSCs represent the most abundant Schwann cell (SC) population in the lung. However, their contribution to lung physiology remains largely unknown. In this study, we used the human glial fibrillary acidic protein promoter driving tdTomato expression in mice to identify SCs in the peripheral nervous system and determine their location within the lung. Single-cell transcriptomic analysis revealed the existence of two NMSC populations (NMSC1 and NMSC2) that may participate in pathogen recognition. We demonstrated that these pulmonary SCs produce chemokines and cytokines upon LPS stimulation using in vitro conditions. Furthermore, we challenged mouse lungs with LPS and found that NMSC1 exhibits an enriched proinflammatory response among all SC subtypes. Collectively, these findings define the molecular profiles of lung SCs and suggest a potential role for NMSCs in lung inflammation.

Cross-species transcriptomic atlas of dorsal root ganglia reveals species-specific programs for sensory function.

Sensory neurons of the dorsal root ganglion (DRG) are critical for maintaining tissue homeostasis by sensing and initiating responses to stimuli. While most preclinical studies of DRGs are conducted in rodents, much less is known about the mechanisms of sensory perception in primates. We generated a transcriptome atlas of mouse, guinea pig, cynomolgus monkey, and human DRGs by implementing a common laboratory workflow and multiple data-integration approaches to generate high-resolution cross-species mappings of sensory neuron subtypes. Using our atlas, we identified conserved core modules highlighting subtype-specific biological processes related to inflammatory response. We also identified divergent expression of key genes involved in DRG function, suggesting species-specific adaptations specifically in nociceptors that likely point to divergent function of nociceptors. Among these, we validated that TAFA4, a member of the druggable genome, was expressed in distinct populations of DRG neurons across species, highlighting species-specific programs that are critical for therapeutic development.

Tetrahydrofuran-Based Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists: Ligand-Based Discovery, Activity in a Rodent Asthma Model, and Mechanism-of-Action via Cryogenic Electron Microscopy.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

Interleukin-17: A Social Cytokine.

Some children with autism spectrum disorder (ASD) show behavioral improvements when experiencing inflammation accompanied by fever; however, little is known about the mechanisms that underlie these beneficial effects. In a recent issue of Nature, Reed and colleagues demonstrate that the production of interleukin-17 (IL-17) during inflammation promotes social behavior in mouse models of neurodevelopmental disorders.

Behavioral characterization of a CRISPR-generated TRPA1 knockout rat in models of pain, itch, and asthma.

The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1 KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.

Inhibition of the kinase ITK in a mouse model of asthma reduces cell death and fails to inhibit the inflammatory response.

Interleukin-2 (IL-2)-inducible T cell kinase (ITK) mediates T cell receptor (TCR) signaling primarily to stimulate the production of cytokines, such as IL-4, IL-5, and IL-13, from T helper 2 (TH2) cells. Compared to wild-type mice, ITK knockout mice are resistant to asthma and exhibit reduced lung inflammation and decreased amounts of TH2-type cytokines in the bronchoalveolar lavage fluid. We found that a small-molecule selective inhibitor of ITK blocked TCR-mediated signaling in cultured TH2 cells, including the tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) and the secretion of IL-2 and TH2-type cytokines. Unexpectedly, inhibition of the kinase activity of ITK during or after antigen rechallenge in an ovalbumin-induced mouse model of asthma failed to reduce airway hyperresponsiveness and inflammation. Rather, in mice, pharmacological inhibition of ITK resulted in T cell hyperplasia and the increased production of TH2-type cytokines. Thus, our studies predict that inhibition of the kinase activity of ITK may not be therapeutic in patients with asthma.

The Regulation of Immunological Processes by Peripheral Neurons in Homeostasis and Disease.

The nervous system and the immune system are the principal sensory interfaces between the internal and external environment. They are responsible for recognizing, integrating, and responding to varied stimuli, and have the capacity to form memories of these encounters leading to learned or 'adaptive' future responses. We review current understanding of the cross-regulation between these systems. The autonomic and somatosensory nervous systems regulate both the development and deployment of immune cells, with broad functions that impact on hematopoiesis as well as on priming, migration, and cytokine production. In turn, specific immune cell subsets contribute to homeostatic neural circuits such as those controlling metabolism, hypertension, and the inflammatory reflex. We examine the contribution of the somatosensory system to autoimmune, autoinflammatory, allergic, and infectious processes in barrier tissues and, in this context, discuss opportunities for therapeutic manipulation of neuro-immune interactions.

A solvent-free thermosponge nanoparticle platform for efficient delivery of labile proteins.

Protein therapeutics have gained attention recently for treatment of a myriad of human diseases due to their high potency and unique mechanisms of action. We present the development of a novel polymeric thermosponge nanoparticle for efficient delivery of labile proteins using a solvent-free polymer thermo-expansion mechanism with clinical potential, capable of effectively delivering a range of therapeutic proteins in a sustained manner with no loss of bioactivity, with improved biological half-lives and efficacy in vivo.

Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.

The skin has a dual function as a barrier and a sensory interface between the body and the environment. To protect against invading pathogens, the skin harbours specialized immune cells, including dermal dendritic cells (DDCs) and interleukin (IL)-17-producing γδ T (γδT17) cells, the aberrant activation of which by IL-23 can provoke psoriasis-like inflammation. The skin is also innervated by a meshwork of peripheral nerves consisting of relatively sparse autonomic and abundant sensory fibres. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear. We exposed the skin of mice to imiquimod, which induces IL-23-dependent psoriasis-like inflammation. Here we show that a subset of sensory neurons expressing the ion channels TRPV1 and Nav1.8 is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of DDCs, the principal source of IL-23, is in close contact with these nociceptors. Upon selective pharmacological or genetic ablation of nociceptors, DDCs failed to produce IL-23 in imiquimod-exposed skin. Consequently, the local production of IL-23-dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were markedly reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with DDCs and restored the inflammatory response. These findings indicate that TRPV1(+)Nav1.8(+) nociceptors, by interacting with DDCs, regulate the IL-23/IL-17 pathway and control cutaneous immune responses.

Detecting apoptosis of leukocytes in mouse lymph nodes.

Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6-6.5 h for preparation and analysis plus an additional 34-40 h to allow apoptosis of the injected DCs in the PLN.

Chemokine CXCL12 uses CXCR4 and a signaling core formed by bifunctional Akt, extracellular signal-regulated kinase (ERK)1/2, and mammalian target of rapamycin complex 1 (mTORC1) proteins to control chemotaxis and survival simultaneously in mature dendritic cells.

Chemokines control several cell functions in addition to chemotaxis. Although much information is available on the involvement of specific signaling molecules in the control of single functions controlled by chemokines, especially chemotaxis, the mechanisms used by these ligands to regulate several cell functions simultaneously are completely unknown. Mature dendritic cells (maDCs) migrate through the afferent lymphatic vessels to the lymph nodes, where they regulate the initiation of the immune response. As maDCs are exposed to chemokine CXCL12 (receptors CXCR4 and CXCR7) during their migration, its functions are amenable to be regulated by this ligand. We have used maDCs as a model system to analyze the mechanisms whereby CXCL12 simultaneously controls chemotaxis and survival in maDCs. We show that CXCL12 uses CXCR4, but not CXCR7, and the components of a signaling core that includes G(i)/Gßγ, PI3K-α/-δ/-γ, Akt, ERK1/2 and mammalian target of rapamycin complex 1 (mTORC1), which organize hierarchically to control both functions. Downstream of Akt, Forkhead box class O (FOXO) regulates CXCL12-dependent survival, but not chemotaxis, suggesting that downstream of the aforementioned signaling core, additional signaling molecules may control more selectively CXCL12-dependent chemotaxis or survival. Finally, the data obtained also show that CXCR4 uses a signaling signature that is different from that used by CCR7 to control similar functions.

The neuronal protein Kidins220/ARMS associates with ICAM-3 and other uropod components and regulates T-cell motility.

Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a protein that is mainly expressed in brain and neural cells where its function is only starting to be characterized. Here, we show that Kidins220/ARMS is also expressed in T lymphocytes where it is highly concentrated at the uropod of polarized T cells. In this cellular model, Kidins220/ARMS colocalizes with typical uropod T-cell molecules and coimmunoprecipitates with ICAM-3. Furthermore, Kidins220/ARMS associates with raft domains at the uropod and coimmunoprecipitates with caveolin-1, a molecule we show here to be also expressed in T cells. Importantly, induction of morphological polarization in primary T lymphocytes and Jurkat cells enhances Kidins220/ARMS colocalization with ICAM-3. Conversely, disruption of cell polarity provokes Kidins220/ARMS redistribution from the uropod to other cellular regions and drastically impairs its association with ICAM-3 in a protein kinase C-dependent manner. Finally, Kidins220/ARMS knockdown in human polarized T-cell lines promotes both basal and stromal cell-derived factor-1α-induced directed migration, identifying a novel function for this molecule. Altogether, our findings show that Kidins220/ARMS is a novel component of the uropod involved in the regulation of T-cell motility, an essential process for the immune response.

What is an immunological synapse?

Immunological synapses (IS) are emerging as highly organized 3D structures -formed by surface and cytoplasmic signalling and cytoskeletal molecules - that assemble at the zone of contact between a T cell and an antigen presenting cell (APC). The IS control functions that allow APC and T cells modulate the immune response.

What is the function of the dendritic cell side of the immunological synapse?

The adaptive immune response requires the formation of a specialized interface called the immunological synapse (IS), which is formed between a mature dendritic cell (DC) and a CD4(+) T cell in the lymph node. The IS involves organized motifs formed by cell-surface and cytoplasmic molecules at both the DC side (IS-DC) and the T cell side (IS-T) of the IS. Most studies of the functions of the IS have focused on the IS-T; however, to understand the function(s) of the entire IS, it is also necessary to gain insight into the role(s) of the IS-DC. Unlike T cells, which upon their activation leave the lymph node and return to the circulation, DCs largely become apoptotic and die in the node region. This latter observation and the known stability of the IS, which may last for hours, is consistent with the hypothesis that one of the functions of the IS-DC could be the temporal inhibition of the apoptosis of DCs, which would enable the activation of clonal T cells in the lymph nodes. Here, we discuss experimental data supporting the latter hypothesis, as well as the concept that the IS-DC is a signaling region that contributes to the functions of the IS.

IL-23 receptor regulates unconventional IL-17-producing T cells that control bacterial infections.

IL-23 plays an important role in autoimmune tissue inflammation and induces the generation of not fully characterized effector cells that mediate protection against pathogens. In this paper, we established the essential role of IL-23R in the host response against intracellular pathogens. IL-23 was critical for the expansion or maintenance of gammadelta and double negative (DN) alphabeta T cells. These cells were rapidly recruited to the site of infection and produced large amounts of IL-17, IFN-gamma, and TNF-alpha. Notably, DN T cells transferred into L. monocytogenes-infected RAG2(-/-) mice prevented bacterial growth, confirming their protective role against intracellular pathogens. Our results show that IL-23 regulates the function of IL-17-producing gammadelta and DN T cells, two essential components of the early protective immune response directed against intracellular pathogens.

The dendritic cell side of the immunological synapse: exploring terra incognita.

The activation and clonal expansion of naive T cells by antigen-loaded dendritic cells (DCs) in the lymph nodes is a key event during immune response. This activation involves the formation of a specialized cell-cell contact region, formed between a mature DC and a CD4 T cell, which is called immunological synapse (IS). The IS includes a DC and a T cell side that we call IS(DC) and IS(T cell), respectively. Most studies on the IS have focused on the IS(T cell) and the information gathered on the IS(DC) is sparse. However, lines of emerging evidence indicate that the IS(DC), likewise the IS(T cell), is a signaling and functional region that makes important contribution to T cell activation and immune response.

Immunological synapse formation inhibits, via NF-kappaB and FOXO1, the apoptosis of dendritic cells.

The immunological synapse (IS) is a cell-cell junction formed between CD4(+) T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-kappaB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.

Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells.

IL-23, an IL-12 family member, has been implicated in the development of Th17 cells and the progression of autoimmune diseases. However, due to the lack of availability of sensitive Ab reagents specific for the IL-23 receptor (IL-23R), it has been difficult to characterize the cell types that express the IL-23R and are responsive to IL-23 in vivo. To address the role of IL-23 in vivo, we have generated a novel "knock-in" mouse in which we have replaced the intracellular domain of the IL-23R with the GFP. We show that in addition to Th17 cells, a subset of myeloid cells express IL-23R and respond to IL-23 by producing IL-17 and IL-22. Our studies further demonstrate that IL-23R expression is crucial for generation of encephalitogenic Th17 cells, but its expression on the innate immune system is dispensible in the development of experimental autoimmune encephalomyelitis.

The multiple personalities of the chemokine receptor CCR7 in dendritic cells.

CCR7 was described initially as a potent leukocyte chemotactic receptor that was later shown to be responsible of directing the migration of dendritic cells (DCs) to the lymph nodes where these cells play an important role in the initiation of the immune response. Recently, a variety of reports have indicated that, apart from chemotaxis, CCR7 controls the cytoarchitecture, the rate of endocytosis, the survival, the migratory speed, and the maturation of the DCs. Some of these functions of CCR7 and additional ones also have been described in other cell types. Herein we discuss how this receptor may contribute to modulate the immune response by regulating different functions in DCs. Finally, we also suggest a possible mechanism whereby CCR7 may control its multiple tasks in these cells.

The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed.

CCR7 is necessary to direct dendritic cells (DCs) to secondary lymphoid nodes and to elicit an adaptative immune response. Despite its importance, little is known about the molecular mechanisms used by CCR7 to direct DCs to lymph nodes. In addition to chemotaxis, CCR7 regulates the migratory speed of DCs. We investigated the intracellular pathways that regulate CCR7-dependent chemotaxis and migratory speed. We found that CCR7 induced a G(i)-dependent activation of MAPK members ERK1/2, JNK, and p38, with ERK1/2 and p38 controlling JNK. MAPK members regulated chemotaxis, but not the migratory speed, of DCs. CCR7 induced activation of PI3K/Akt; however, these enzymes did not regulate either chemotaxis or the speed of DCs. CCR7 also induced activation of the GTPase Rho, the tyrosine kinase Pyk2, and inactivation of cofilin. Pyk2 activation was independent of G(i) and Src and was dependent on Rho. Interference with Rho or Pyk2 inhibited cofilin inactivation and the migratory speed of DCs, but did not affect chemotaxis. Interference with Rho/Pyk2/cofilin inhibited DC migratory speed even in the absence of chemokines, suggesting that this module controls the speed of DCs and that CCR7, by activating its components, induces an increase in migratory speed. Therefore, CCR7 activates two independent signaling modules, one involving G(i) and a hierarchy of MAPK family members and another involving Rho/Pyk2/cofilin, which control, respectively, chemotaxis and the migratory speed of DCs. The use of independent signaling modules to control chemotaxis and speed can contribute to regulate the chemotactic effects of CCR7.