Lung Dendritic Cells Research Articles

Differential inflammatory profile in the lungs of mice exposed to cannabis smoke with varying THC:CBD ratio.

Cannabis contains cannabinoids including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC causes the psychoactive effects of cannabis, and both THC and CBD are thought to be anti-inflammatory. Cannabis is typically consumed by inhaling smoke that contains thousands of combustion products that may damage the lungs. However, the relationship between cannabis smoke exposure and alterations in respiratory health is poorly defined. To address this gap in knowledge, we first developed a mouse model of cannabis smoke exposure using a nose-only rodent inhalation exposure system. We then tested the acute effects of two dried cannabis products that differ substantially in their THC-CBD ratio: Indica-THC dominant (I-THC; 16-22% THC) and Sativa-CBD dominant (S-CBD; 13-19% CBD). We demonstrate that this smoke exposure regime not only delivers physiologically relevant levels of THC to the bloodstream, but that acute inhalation of cannabis smoke modulates the pulmonary immune response. Cannabis smoke decreased the percentage of lung alveolar macrophages but increased lung interstitial macrophages (IMs). There was also a decrease in lung dendritic cells as well as Ly6Cintermediate and Ly6Clow monocytes, but an increase in lung neutrophils and CD8+ T cells. These immune cell changes were paralleled with changes in several immune mediators. These immunological modifications were more pronounced when mice were exposed to S-CBD compared to the I-THC variety. Thus, we show that acute cannabis smoke differentially affects lung immunity based on the THC:CBD ratio, thereby providing a foundation to further explore the effect of chronic cannabis smoke exposures on pulmonary health.

Investigating the role of BHLHE40 in host responses to Mycobacterium tuberculosisinfection and myeloid cell development.

Abstract We currently lack a complete understanding of the immune responses required to control Mycobacterium tuberculosis (Mtb) infection. Our lab has shown that the transcription factor BHLHE40 is essential during Mtb infection. Deletion of Bhlhe40 in CD11c +lung macrophages and dendritic cells (DCs) is sufficient to increase mouse susceptibility to Mtb infection, though the role of BHLHE40 in these cells during infection is unknown. Murine bone marrow cultured in the presence of GM-CSF, an abundant cytokine in the lung, develop into populations of CD11c +macrophages, DCs, and granulocytes, all of which express Bhlhe40. Using this system, we examined cytokine and transcriptional responses before and after exposure to heat-killed Mtb to understand how BHLHE40 impacts cell development and inflammatory responses. We found that in WT mice, GM-CSF-cultured cells develop into 9 distinct populations and expression of Bhlhe40 was required for GM-CSF-mediated pro-inflammatory cytokine and transcriptional programs. Additionally, loss of Bhlhe40 led to decreased DCs accompanied by upregulation of the transcription factor C/EBPβ in DC progenitor populations, which negatively impacts DC development. ChIP-seq analysis revealed BHLHE40 binds directly to C/EBPβ’s gene locus, indicating that BHLHE40 may directly repress Cebpb. In contrast, loss of Bhlhe40 did not affect macrophage development, but led to a transcriptional shift towards an anti-inflammatory state. These findings demonstrate that in the presence of GM-CSF, BHLHE40 is required for DC development and inflammatory macrophage polarization. Defects in either of these aspects could contribute to the essential role for BHLHE40 in lung macrophages and DCs during Mtb infection in vivo.

Current research into A20 mediation of allergic respiratory diseases and its potential usefulness as a therapeutic target.

Allergic airway diseases are characterized by excessive and prolonged type 2 immune responses to inhaled allergens. Nuclear factor κB (NF-κB) is a master regulator of the immune and inflammatory response, which has been implicated to play a prominent role in the pathogenesis of allergic airway diseases. The potent anti-inflammatory protein A20, termed tumor necrosis factor-α-inducible protein 3 (TNFAIP3), exerts its effects by inhibiting NF-κB signaling. The ubiquitin editing abilities of A20 have attracted much attention, resulting in its identification as a susceptibility gene in various autoimmune and inflammatory disorders. According to the results of genome-wide association studies, several TNFAIP3 gene locus nucleotide polymorphisms have been correlated to allergic airway diseases. In addition, A20 has been found to play a pivotal role in immune regulation in childhood asthma, particularly in the protection against environmentally mediated allergic diseases. The protective effects of A20 against allergy were observed in conditional A20-knockout mice in which A20 was depleted in the lung epithelial cells, dendritic cells, or mast cells. Furthermore, A20 administration significantly decreased inflammatory responses in mouse models of allergic airway diseases. Here, we review emerging findings elucidating the cellular and molecular mechanisms by which A20 regulates inflammatory signaling in allergic airway diseases, as well as discuss its potential as a therapeutic target.

Physiological estrogen levels are dispensable for the sex difference in immune responses during allergen-induced airway inflammation

Women show an increased prevalence of adult-onset asthma compared to men and previous studies have shown that testosterone inhibits while estrogen worsens allergen-induced airway inflammation. However, detailed knowledge about the aggravating effects of estrogen on immune responses remain unclear. Defining the effects of physiological levels of estrogen on immune responses in asthma would aid in the development of improved treatment strategies.In this study, the importance of estrogen for the sex difference in asthma was determined using a murine model of house dust mite (HDM)-induced airway inflammation on intact female and male mice, as well as on ovariectomized (OVX) female mice treated with a physiological dose of 17β-estradiol (E2). Innate and adaptive immune responses were defined in bronchoalveolar lavage fluid, mediastinal lymph node (mLN) and lung tissue.The results reveal increased numbers of lung eosinophils, macrophages, and dendritic cells in female but not in male mice after HDM challenge. Females also exhibit higher numbers of Th17 cells in both mLN and lung in response to HDM. However, treatment of OVX mice with physiological levels of E2 does not influence any of the analyzed cell populations.Together, this study confirms the previously reported sex difference in allergen-induced airway inflammation and show that female mice mount stronger innate and adaptive immune responses to HDM challenge, but these effects are not mediated by physiological levels of E2.

Early-life hyperoxia-induced Flt3L drives neonatal lung dendritic cell expansion and proinflammatory responses.

Premature infants with chronic lung disease, bronchopulmonary dysplasia (BPD), develop recurrent cough and wheezing following respiratory viral infections. The mechanisms driving the chronic respiratory symptoms are ill-defined. We have shown that hyperoxic exposure of neonatal mice (a model of BPD) increases the activated lung CD103+ dendritic cells (DCs) and these DCs are required for exaggerated proinflammatory responses to rhinovirus (RV) infection. Since CD103+ DC are essential for specific antiviral responses and their development depends on the growth factor Flt3L, we hypothesized that early-life hyperoxia stimulates Flt3L expression leading to expansion and activation of lung CD103+ DCs and this mediates inflammation. We found that hyperoxia numerically increased and induced proinflammatory transcriptional signatures in neonatal lung CD103+ DCs, as well as CD11bhi DCs. Hyperoxia also increased Flt3L expression. Anti-Flt3L antibody blocked CD103+ DC development in normoxic and hyperoxic conditions, and while it did not affect the baseline number of CD11bhi DCs, it neutralized the effect of hyperoxia on these cells. Anti-Flt3L also inhibited hyperoxia-induced proinflammatory responses to RV. In tracheal aspirates from preterm infants mechanically-ventilated for respiratory distress in the first week of life levels of FLT3L, IL-12p40, IL-12p70 and IFN-γ were higher in infants who went on to develop BPD and FLT3L levels positively correlated with proinflammatory cytokines levels. This work highlights the priming effect of early-life hyperoxia on lung DC development and function and the contribution of Flt3L in driving these effects.

NCoR1 controls immune tolerance in conventional dendritic cells by fine-tuning glycolysis and fatty acid oxidation

Dendritic cells (DCs) undergo rapid metabolic reprogramming to generate signal-specific immune responses. The fine control of cellular metabolism underlying DC immune tolerance remains elusive. We have recently reported that NCoR1 ablation generates immune-tolerant DCs through enhanced IL-10, IL-27 and SOCS3 expression. In this study, we did comprehensive metabolic profiling of these tolerogenic DCs and identified that they meet their energy requirements through enhanced glycolysis and oxidative phosphorylation (OXPHOS), supported by fatty acid oxidation-driven oxygen consumption. In addition, the reduced pyruvate and glutamine oxidation with a broken TCA cycle maintains the tolerogenic state of the cells. Mechanistically, the AKT-mTOR–HIF–1α-axis mediated glycolysis and CPT1a-driven β-oxidation were enhanced in these tolerogenic DCs. To confirm these observations, we used synthetic metabolic inhibitors and found that the combined inhibition of HIF-1α and CPT1a using KC7F2 and etomoxir, respectively, compromised the overall transcriptional signature of immunological tolerance including the regulatory cytokines IL-10 and IL-27. Functionally, treatment of tolerogenic DCs with dual KC7F2 and etomoxir treatment perturbed the polarization of co-cultured naïve CD4+ T helper (Th) cells towards Th1 than Tregs, ex vivo and in vivo. Physiologically, the Mycobacterium tuberculosis (Mtb) infection model depicted significantly reduced bacterial burden in BMcDC1 ex vivo and in CD103+ lung DCs in Mtb infected NCoR1DC−/−mice. The spleen of these infected animals also showed increased Th1-mediated responses in the inhibitor-treated group. These findings suggested strong involvement of NCoR1 in immune tolerance. Our validation in primary human monocyte-derived DCs (moDCs) showed diminished NCOR1 expression in dexamethasone-derived tolerogenic moDCs along with suppression of CD4+T cell proliferation and Th1 polarization. Furthermore, the combined KC7F2 and etomoxir treatment rescued the decreased T cell proliferative capacity and the Th1 phenotype. Overall, for the first time, we demonstrated here that NCoR1 mediated control of glycolysis and fatty acid oxidation fine-tunes immune tolerance versus inflammation balance in murine and human DCs.

Excipient-Free Ionizable Polyester Nanoparticles for Lung-Selective and Innate Immune Cell Plasmid DNA and mRNA Transfection.

Extrahepatic nucleic acid delivery using polymers typically requires the synthesis and purification of custom monomers, post-synthetic modifications, and incorporation of additional excipients to augment their stability, endosomal escape, and in vivo effectiveness. Here, we report the development of a single-component and excipient-free, polyester-based nucleic acid delivery nanoparticle platform comprising ionizable N-methyldiethanolamine (MDET) and various hydrophobic alkyl diols (Cp) that achieves lung-selective nucleic acid transfection in vivo. PolyMDET and polyMDET-Cp polyplexes displayed high serum and enzymatic stability, while delivering pDNA or mRNA to "hard-to-transfect" innate immune cells. PolyMDET-C4 and polyMDET-C6 mediated high protein expression in lung alveolar macrophages and dendritic cells without inducing tissue damage or systemic inflammatory responses. Improved strategies using readily available starting materials to produce a simple, excipient-free, non-viral nucleic acid delivery platform with lung-selective and innate immune cell tropism has the potential to expedite clinical deployment of polymer-based genetic medicines.

β-Glucosylceramides and Tocopherols Regulate Development and Function of Dendritic Cells.

In humans and mice, offspring of allergic mothers are predisposed to development of allergy. In mice, allergic mothers have elevated β-glucosylceramides (βGlcCers) that are transported to the fetus via the placenta and to offspring via milk. The elevated βGlcCers increase the number of fetal liver CD11c+CD11b+ dendritic cells (DCs) and offspring allergen-induced lung eosinophilia. These effects are modifiable by maternal dietary supplementation with the plant-derived lipids α-tocopherol and γ-tocopherol. It is not known whether βGlcCers and tocopherols directly regulate development of DCs. In this study, we demonstrated that βGlcCers increased development of GM-CSF-stimulated mouse bone marrow-derived DCs (BMDCs) in vitro without altering expression of costimulatory molecules. This increase in BMDC numbers was blocked by α-tocopherol and potentiated by γ-tocopherol. Furthermore, βGlcCers increased protein kinase Cα (PKCα) and PKCδ activation in BMDCs that was blocked by α-tocopherol. In contrast, γ-tocopherol increased BMDC PKCα and PKCδ activation and enhanced the βGlcCer-induced increase in PKCδ activation in a DC subset. Ag processing per DC was minimally enhanced in βGlcCer-treated BMDCs and not altered ex vivo in lung DCs from pups of allergic mothers. Pups of allergic mothers had an increased proportion of CD11b+CD11c+ subsets of DCs, contributing to enhanced stimulation of T cell proliferation ex vivo. Thus, βGlcCer, which is both necessary and sufficient for development of allergic predisposition in offspring of allergic mothers, directly increased development and PKC activation in BMDCs. Furthermore, this was modifiable by dietary tocopherols. This may inform design of future studies for the prevention or intervention in asthma and allergic disease.

IRF4 expression by lung dendritic cells drives acute but not Trm cell-dependent memory Th2 responses.

Expression of the transcription factor interferon regulatory factor 4 (IRF4) is required for the development of lung conventional DCs type 2 (cDC2s) that elicit Th2 responses, yet how IRF4 functions in lung cDC2s throughout the acute and memory allergic response is not clear. Here, we used a mouse model that loses IRF4 expression after lung cDC2 development to demonstrate that mice with IRF4-deficient DCs display impaired memory responses to allergen. This defect in the memory response was a direct result of ineffective Th2 induction and impaired recruitment of activated effector T cells to the lung after sensitization. IRF4-deficient DCs demonstrated defects in their migration to the draining lymph node and in T cell priming. Finally, T cells primed by IRF4-competent DCs mediated potent memory responses independently of IRF4-expressing DCs, demonstrating that IRF4-expressing DCs are not necessary during the memory response. Thus, IRF4 controlled a program in mature DCs governing Th2 priming and effector responses, but IRF4-expressing DCs were dispensable during tissue-resident memory T cell-dependent memory responses.

Expansion of Phenotypically Altered Dendritic Cell Populations in the Small Airways and Alveolar Parenchyma in Patients with Chronic Obstructive Pulmonary Disease

Contrasting the antigen-presenting dendritic cells (DCs) in the conducting airways, the alveolar DC populations in human lungs have remained poorly investigated. Consequently, little is known about how alveolar DCs are altered in diseases such as chronic obstructive pulmonary disease (COPD). This study maps multiple tissue DC categories in the distal lung across COPD severities. Specifically, single-multiplex immunohistochemistry was applied to quantify langerin/CD207+, CD1a+, BDCA2+, and CD11c+ subsets in distal lung compartments from patients with COPD (GOLD stage I–IV) and never-smoking and smoking controls. In the alveolar parenchyma, increased numbers of CD1a+langerin− (p < 0.05) and BDCA-2+ DCs (p < 0.001) were observed in advanced COPD compared with controls. Alveolar CD11c+ DCs also increased in advanced COPD (p < 0.01). In small airways, langerin+ and BDCA-2+ DCs were also significantly increased. Contrasting the small airway DCs, most alveolar DC subsets frequently extended luminal protrusions. Importantly, alveolar and small airway langerin+ DCs in COPD lungs displayed site-specific marker profiles. Further, multiplex immunohistochemistry with single-cell quantification was used to specifically profile langerin DCs and reveal site-specific expression patterns of the maturation and activation markers S100, fascin, MHC2, and B7. Taken together, our results show that clinically advanced COPD is associated with increased levels of multiple alveolar DC populations exhibiting features of both adaptive and innate immunity phenotypes. This expansion is likely to contribute to the distal lung immunopathology in COPD patients.

TOLLIP Optimizes Dendritic Cell Maturation to Lipopolysaccharide and Mycobacterium tuberculosis.

TOLLIP is a central regulator of multiple innate immune signaling pathways, including TLR2, TLR4, IL-1R, and STING. Human TOLLIP deficiency, regulated by single-nucleotide polymorphism rs5743854, is associated with increased tuberculosis risk and diminished frequency of bacillus Calmette-Guérin vaccine-specific CD4+ T cells in infants. How TOLLIP influences adaptive immune responses remains poorly understood. To understand the mechanistic relationship between TOLLIP and adaptive immune responses, we used human genetic and murine models to evaluate the role of TOLLIP in dendritic cell (DC) function. In healthy volunteers, TOLLIP single-nucleotide polymorphism rs5743854 G allele was associated with decreased TOLLIP mRNA and protein expression in DCs, along with LPS-induced IL-12 secretion in peripheral blood DCs. As in human cells, LPS-stimulated Tollip -/- bone marrow-derived murine DCs secreted less IL-12 and expressed less CD40. Tollip was required in lung and lymph node-resident DCs for optimal induction of MHC class II and CD40 expression during the first 28 d of Mycobacterium tuberculosis infection in mixed bone marrow chimeric mice. Tollip -/- mice developed fewer M. tuberculosis-specific CD4+ T cells after 28 d of infection and diminished responses to bacillus Calmette-Guérin vaccination. Furthermore, Tollip -/- DCs were unable to optimally induce T cell proliferation. Taken together, these data support a model where TOLLIP-deficient DCs undergo suboptimal maturation after M. tuberculosis infection, impairing T cell activation and contributing to tuberculosis susceptibility.

Pulmonary Conventional Type 1 Langerin-Expressing Dendritic Cells Play a Role in Impairing Early Protective Immune Response against Cryptococcus neoformans Infection in Mice.

Lung dendritic cells (DC) are powerful antigen-presenting cells constituted by various subpopulations that differ in terms of their function and origin and differentially regulate cell-mediated antifungal immunity. The lung is the primary target organ of Cryptococcus neoformans and C. gattii infections, which makes it essential in the establishment of the first line of anti-cryptococcal defense. However, the lung-specific dynamics and function of DC subsets are poorly understood in cryptococcosis. In this study, we provide evidence for the in vivo function of a conventional langerin-expressing DC1 dendritic cell (LangDC1) population during the first week of intratracheal C. neoformans infection in mice. By using conditional depletion of LangDC1 after diphtheria toxin treatment of LangDTREGFP mice, we demonstrate that these animals better control the fungal infection and produce type 1 and 17 cytokines in the context of a type 2 immune response, favoring a predominance of iNOS over arginase-1 expression by pulmonary cells. Our results suggest that LangDC1 cells play a role in impairing immune response for the clearance of C. neoformans in the early stage of pulmonary infection.

Suppression of Allergic Asthma by Loss of Function of Miz1-mediated Th1 Skewing.

Asthma is the most prevalent chronic respiratory disease worldwide. There is currently no cure, and it remains an important cause of morbidity and mortality. Here we report that lung-specific loss of function of the transcription factor Miz1 (c-Myc-interacting zinc finger protein-1) upregulates the pro-T-helper cell type 1 cytokine IL-12. Upregulation of IL-12 in turn stimulates a Th1 response, thereby counteracting T-helper cell type 2 response and preventing the allergic response in mouse models of house dust mite- and OVA (ovalbumin)-induced asthma. Using transgenic mice expressing Cre under a cell-specific promoter, we demonstrate that Miz1 acts in lung epithelial cells and dendritic cells in asthma. Chromatin immunoprecipitation coupled with high-throughput DNA sequencing or quantitative PCR reveals the binding of Miz1 on the Il12 promoter indicating direct repression of IL-12 by Miz1. In addition, HDAC1 (histone deacetylase 1) is recruited to the Il12 promoter in a Miz1-depdenent manner, suggesting epigenetic repression of Il12 by Miz1. Furthermore, Miz1 is upregulated in the lungs of asthmatic mice. Our data together suggest that Miz1 is upregulated during asthma, which in turn promotes asthma pathogenesis by preventing Th1 skewing through the transcriptional repression of IL-12.

Intranasal Delivery of RIG-I Agonist Drives Pulmonary Myeloid Cell Activation in Mice.

Viral respiratory infections cause substantial health and economic burden. There is a pressing demand for efficacious vaccination strategies and, therefore, a need for a better understanding of the mechanisms of action of novel potential adjuvants. Here we investigated the effect of a synthetic RIG-I agonist, the dsRNA hairpin 3p10LA9, on the activation of pulmonary myeloid cells. Analysis of early innate immune responses revealed that a single intranasal 3p10LA9 dose induces a transient pulmonary interferon-stimulated gene (ISG) and pro-inflammatory cytokine/chemokine response, which leads to the maturation of three distinct dendritic cell subpopulations in the lungs. While lung resident dendritic cell decrease shortly after 3p10LA9 delivery, their numbers increase in the draining mediastinal lymph node, where they have migrated, maintaining their activated phenotype. At the same time, dsRNA hairpin-induced chemokines attract transiently infiltrating monocytes into the lungs, which causes a short temporary pulmonary inflammation. However, these monocytes are dispensable in controlling influenza infection since in CCR2 deficient mice, lacking these infiltrating cells, the virus load was similar to the wild type mice when infected with the influenza virus at a sublethal dose. In summary, our data suggest that intranasal delivery of dsRNA hairpins, used as a RIG-I targeting adjuvant, represents an attractive strategy to boost type I inteferon-mediated lung dendritic cell maturation, which supports viral reduction in the lungs during infection.

ADAMTS7 Attenuates House Dust Mite-Induced Airway Inflammation and Th2 Immune Responses

PurposeADAMTS7 is a secreted metalloproteinase enzyme and proteoglycan associated with the early progression of coronary artery disease. However, there is limited information regarding the role of ADAMTS7 in lung adaptive immunity and inflammation. Thus, we sought to assess whether ADAMTS7 expression in the lung modulates house dust mite (HDM)-induced airway inflammation and Th2 immune response.MethodsThe role of ADAMTS7 in HDM-induced airway disease was assessed in ADAMTS7-deficient (ADAMTS7−/−) mice and compared with the wild-type control mice by flow cytometry, ELISA, and histopathology. Furthermore, the antigen priming capability of dendritic cells (DC) was determined ex vivo by employing coculture with CD4+ OT-II cells.ResultsADAMTS7−/− mice develop an augmented eosinophilic airway inflammation, mucous cell metaplasia, and increased Th2 immune response to inhaled HDM. In addition, allergen uptake by lung DC and migration to draining mediastinal lymph node were significantly increased in ADAMTS7−/− mice, which shows an enhanced capacity to mount allergen-specific T-cell proliferation and effector Th2 cytokine productions. We propose that the mechanism by which ADAMTS7 negatively regulates DC function involves attenuated antigen uptake and presentation capabilities, which reduces allergic sensitization and Th2 immune responses in the lung.ConclusionIn aggregate, we provide compelling evidence that ADAMTS7 plays a pivotal role in allergic airway disease and Th2 immunity and would be an attractive target for asthma.

β-Glucosylceramides and Tocopherols Regulate Development and Function of Dendritic Cells

Abstract In humans and mice, offspring of allergic mothers are predisposed to development of allergy. In mice, allergic mothers have elevated β-glucosylceramides (βGlcCers) that are transported to the fetus via the placenta and to offspring via milk. Elevated βGlcCers increase numbers of fetal liver CD11c+CD11b+ dendritic cells (DCs) and offspring allergen-induced lung eosinophilia. These effects are modifiable by maternal dietary supplementation with the plant-derived lipids α-tocopherol and γ-tocopherol. It is not known whether βGlcCers and tocopherols directly regulate development of DCs. In this study, we demonstrated that βGlcCers (C16:0, C18:0, C18:1, and C24:1) increased development of GM-CSF-stimulated bone marrow-derived DCs (BMDCs) in vitro. This increase in BMDC numbers was blocked by α-tocopherol and potentiated by γ-tocopherol. Furthermore, βGlcCer increased PKCα and PKCδ activation in BMDCs that was blocked by α-tocopherol. In contrast, γ-tocopherol increased BMDC PKCα and PKCδ activation and enhanced the βGlcCer-induced increase in PKCδ activation in a DC subset. Antigen processing per DC was minimally enhanced in βGlcCer-treated BMDCs and not altered ex vivo in lung DCs from pups of allergic mothers. However, lung DCs from pups of allergic mothers had an increased proportion of CD11b+CD11c+ DCs and exhibited enhanced stimulation of T cells ex vivo. Thus, βGlcCer, which is both necessary and sufficient for development of allergic predisposition in offspring of allergic mothers, directly increased development and PKC activation in BMDCs. Furthermore, this was modifiable by dietary tocopherols. This may inform design of future studies for approaches in the prevention or intervention in asthma and allergic disease. Supported by grants from the NIH (U01 AI131337 (Cook-Mills), R01AI127695 (Cook-Mills)) as well as the Pediatric Scientist Development Program (Lajiness) and Marshall Klaus Perinatal Research Award (Lajiness)

Exploring CCRL2 chemerin binding using accelerated molecular dynamics.

Chemokine (C–C motif) receptor‐like 2 (CCRL2), is a seven transmembrane receptor closely related to the chemokine receptors CCR1, CCR2, CCR3, and CCR5. Nevertheless, CCRL2 is unable to activate conventional G‐protein dependent signaling and to induce cell directional migration. The only commonly accepted CCRL2 ligand is the nonchemokine chemotactic protein chemerin (RARRES2). The chemerin binding to CCLR2 does induce leukocyte chemotaxis, yet, genetic targeting of CCRL2 was shown to modulate the inflammatory response in different experimental models. This mechanism was shown to be crucial for lung dendritic cell migration, neutrophil recruitment, and Natural Killer cell‐dependent immune surveillance in lung cancer. To gain more insight in the interactions involved in the CCRL2‐chemerin, the binding complexes were generated by protein–protein docking, then submitted to accelerated molecular dynamics. The obtained trajectories were inspected by principal component analyses followed by kernel density estimation to identify the ligand‐receptor regions most frequently involved in the binding. To conclude, the reported analyses led to the identification of the putative hot‐spot residues involved in CCRL2‐chemerin binding.

Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th17 polarization during infection

SummaryEarly Th17 responses are necessary to provide protection against Mycobacterium tuberculosis (Mtb). Mtb impedes Th17 polarization by restricting CD40 co-stimulatory pathway on dendritic cells (DCs). We previously demonstrated that engaging CD40 on DCs increased Th17 responses. However, the molecular mechanisms that contributed to Th17 polarization were unknown. Here, we identify the Notch ligand DLL4 as necessary for Th17 polarization and demonstrate that Mtb limits DLL4 on DCs to prevent optimal Th17 responses. Although Mtb infection induced only low levels of DLL4, engaging CD40 on DCs increased DLL4 expression. Antibody blockade of DLL4 on DCs reduced Th17 polarization in vitro and in vivo. In addition, we show that the Mtb Hip1 protease attenuates DLL4 expression on lung DCs by impeding CD40 signaling. Overall, our results demonstrate that Mtb impedes CD40-dependent DLL4 expression to restrict Th17 responses and identify the CD40-DLL4 pathways as targets for developing new Th17-inducing vaccines and adjuvants for tuberculosis.

OASL1-Mediated Inhibition of Type I IFN Reduces Influenza A Infection-Induced Airway Inflammation by Regulating ILC2s.

PurposeThree observations drove this study. First, 2′-5′-oligoadenylate synthetase-like protein (OASL) is a negative regulator of type I interferon (IFN). Second, type I IFN plays a central role during virus infections and the pathogenesis of various diseases, including asthma. Third, influenza A virus (IAV) causes non-eosinophilic asthma. To evaluate the potential relationships between OASL, type I IFN, and pulmonary innate immune cells in IAV-induced acute airway inflammation by using Oasl1-/- mice.MethodsAsthma was induced in wild-type (WT) and Oasl1-/- mice with IAV or ovalbumin (OVA). Airway hyperreactivity (AHR) and immune cell infiltration in the bronchoalveolar lavage (BAL) fluids were measured. The immune cells in the lungs were analyzed by flow cytometry. To investigate the ability of type I IFN to shape the response of lung type 2 innate lymphoid cells (ILC2s), IFN-α was treated intratracheally. Plasmacytoid dendritic cells (pDCs) sorted from bone marrow and ILC2s sorted from lungs of naive mice were co-cultured with/without interferon-alpha receptor subunit 1 (IFNAR-1)-blocking antibodies.ResultsIn the IAV-induced asthma model, Oasl1-/- mice developed greater AHR and immune cell infiltration in the BAL fluids than WT mice. This was not observed in OVA-induced asthma, a standard model of allergen-induced asthma. The lungs of infected Oasl1-/- mice also had elevated DC numbers and Ifna expression and depressed IAV-induced ILC2 responses, namely, proliferation and type 2 cytokine and amphiregulin production. Intratracheal administration of type I IFN in naïve mice suppressed lung ILC2 production of type 2 cytokines and amphiregulin. Co-culture of ILC2s with pDCs showed that pDCs inhibit the function of ILC2s by secreting type I IFN.ConclusionsOASL1 may impede the IAV-induced acute airway inflammation that drives AHR by inhibiting IAV-induced type I IFN production from lung DCs, thereby preserving the functions of lung ILC2s, including their amphiregulin production.

Isolation of Conventional Murine Lung Dendritic Cell Subsets.

In the lungs, immune cells make contact with different antigens every day. This requires an adequate immune response. Dendritic cells (DCs) form a dense network in the respiratory mucosa and continuously sample inhaled allergens. They play an important role in bridging innate and adaptive immunity. DCs are classically divided into plasmacytoid DCs (pDCs) and conventional DCs (cDCs). cDCs in the steady-state are further subdivided into cDC1s and cDC2s based on their ontogeny and distinct non-redundant functions. Recently, a hyperactivated state of cDC2s has been described that arises during inflammation, coined inflammatory cDC2s (inf-cDC2s) that phenotypically mimics monocyte-derived cells and has a hybrid cDC1/macrophage functional identity. This chapter describes different enrichment methods and a fluorescence-activated cell sorting protocol that in combination allow for discrimination and isolation of pure DC subsets from the murine lung. The chapter represents an up-to-date, universal framework that can be adapted to other tissues and species which is an added value in intra- and interspecies comparative research.