Chemokine CCL19 promotes type 2 T-cell differentiation and allergic airway inflammation.

BACKGROUND: Allergic asthma is driven largely by allergen-specific TH2 cells, which develop in regional lymph nodes on the interaction of naive CD4+ T cells with allergen-bearing dendritic cells that migrate from the lung. This migration event is dependent on CCR7 and its chemokine ligand, CCL21. However, is has been unclear whether the other CCR7 ligand, CCL19, has a role in allergic airway disease. OBJECTIVE: This study sought to define the role of CCL19 in TH2 differentiation and allergic airway disease. METHODS: Ccl19-deficient mice were studied in an animal model of allergic asthma. Dendritic cells or fibroblastic reticular cells from wild-type and Ccl19-deficient mice were cultured with naive CD4+ T cells, and cytokine production was measured by ELISA. Recombinant CCL19 was added to CD4+ T-cell cultures, and gene expression was assessed by RNA-sequencing and quantitative PCR. Transcription factor activation was assessed by flow cytometry. RESULTS: Lungs of Ccl19-deficient mice had less allergic airway inflammation, reduced airway hyperresponsiveness, and less IL-4 and IL-13 production compared with lungs of Ccl19-sufficient animals. Naive CD4+ T cells cocultured with Ccl19-deficient dendritic cells or fibroblastic reticular cells produced lower amounts of type 2 cytokines than did T cells cocultured with their wild-type counterparts. Recombinant CCL19 increased phosphorylation of STAT5 and induced expression of genes associated with TH2 cell and IL-2 signaling pathways. CONCLUSIONS: These results reveal a novel, TH2 cell-inducing function of CCL19 in allergic airway disease and suggest that strategies to block this pathway might help to reduce the incidence or severity of allergic asthma.

Pathogenic TH17 inflammation is sustained in the lungs by conventional dendritic cells and Toll-like receptor 4 signaling.

BACKGROUND: Mechanisms that elicit mucosal TH17 cell responses have been described, yet how these cells are sustained in chronically inflamed tissues remains unclear. OBJECTIVE: We sought to understand whether maintenance of lung TH17 inflammation requires environmental agents in addition to antigen and to identify the lung antigen-presenting cell (APC) types that sustain the self-renewal of TH17 cells. METHODS: Animals were exposed repeatedly to aspiration of ovalbumin alone or together with environmental adjuvants, including common house dust extract (HDE), to test their role in maintaining lung inflammation. Alternatively, antigen-specific effector/memory TH17 cells, generated in culture with CD4+ T cells from Il17a fate-mapping mice, were adoptively transferred to assess their persistence in genetically modified animals lacking distinct lung APC subsets or cell-specific Toll-like receptor (TLR) 4 signaling. TH17 cells were also cocultured with lung APC subsets to determine which of these could revive their expansion and activation. RESULTS: TH17 cells and the consequent neutrophilic inflammation were poorly sustained by inhaled antigen alone but were augmented by inhalation of antigen together with HDE. This was associated with weight loss and changes in lung physiology consistent with interstitial lung disease. The effect of HDE required TLR4 signaling predominantly in lung hematopoietic cells, including CD11c+ cells. CD103+ and CD11b+ conventional dendritic cells interacted directly with TH17 cells in situ and revived the clonal expansion of TH17 cells both ex vivo and in vivo, whereas lung macrophages and B cells could not. CONCLUSION: TH17-dependent inflammation in the lungs can be sustained by persistent TLR4-mediated activation of lung conventional dendritic cells.

Inhaled house dust programs pulmonary dendritic cells to promote type 2 T-cell responses by an indirect mechanism.

The induction of allergen-specific T helper 2 (Th2) cells by lung dendritic cells (DCs) is a critical step in allergic asthma development. Airway delivery of purified allergens or microbial products can promote Th2 priming by lung DCs, but how environmentally relevant quantities and combinations of these factors affect lung DC function is unclear. Here, we investigated the ability of house dust extract (HDE), which contains a mixture of environmental adjuvants, to prime Th2 responses against an innocuous inhaled antigen. Inhalational exposure to HDE conditioned lung conventional DCs, but not monocyte-derived DCs, to induce antigen-specific Th2 differentiation. Conditioning of DCs by HDE was independent of Toll-like receptor 4 signaling, indicating that environmental endotoxin is dispensable for programming DCs to induce Th2 responses. DCs directly treated with HDE underwent maturation but were poor stimulators of Th2 differentiation. In contrast, DCs treated with bronchoalveolar lavage fluid (BALF) from HDE-exposed mice induced robust Th2 differentiation. DC conditioning by BALF was independent of the proallergic cytokines IL-25, IL-33, and thymic stromal lymphopoietin. BALF treatment of DCs resulted in upregulation of CD80 but low expression of CD40, CD86, and IL-12p40, which was associated with Th2 induction. These findings support a model whereby environmental adjuvants in house dust indirectly program DCs to prime Th2 responses by triggering the release of endogenous soluble factor(s) by airway cells. Identifying these factors could lead to novel therapeutic targets for allergic asthma.

Susceptibility-based positive contrast MRI of brachytherapy seeds.

PURPOSE: To provide visualization of the brachytherapy seeds and differentiation with natural structures in MRI by taking advantage of their high magnetic susceptibility to generate positive-contrast images. METHODS: The method is based on mapping the susceptibility using an equivalent short-TE sequence and a kernel deconvolution algorithm with a regularized L1 minimization. An appealing aspect of the method is that signals from the surrounding areas where signal to noise ratio (SNR) is sufficiently high are used to derive the susceptibility of the seeds, even though the SNR in the immediate vicinity of the seeds can be extremely low due to rapid signal decay. RESULTS: The method is tested using computer simulations and experimental data. Comparing to conventional methods, the proposed method improves seed definition by a factor of >70% in the experiments. It produces the enhanced contrast at the exact seed location, whereas methods based on susceptibility gradient mapping produce highlighted regions surrounding the seeds. The proposed method is capable to perform the function for a wide range of resolutions and SNRs. CONCLUSION: The results show that the proposed method provides positive contrast for the seeds and correctly differentiates them from other structures that appear similar to the seeds on conventional magnitude images.

ATP binding cassette transporter G1 deletion induces IL-17-dependent dysregulation of pulmonary adaptive immunity.

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1(+/+) and Abcg1(-/-) mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1(-/-) mice displayed increased B cells, CD4(+) T cells, CD8(+) T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b(+) DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1(-/-) airway, compared with Abcg1(+/+), displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1(-/-) mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1(-/-) CD4(+) T cells displayed normal ex vivo differentiation, whereas Abcg1(-/-) DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17(+) γδT cells, and IL-17(+) neutrophils were all increased in Abcg1(-/-) lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1(-/-) airway. We conclude that Abcg1(-/-) mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.

GM-CSF-dependent CD301b+ lung dendritic cells confer tolerance to inhaled allergens.

The severity of allergic asthma is driven by the balance between allergen-specific T regulatory (Treg) and T helper (Th)2 cells. However, it is unclear whether specific subsets of conventional dendritic cells (cDCs) promote the differentiation of these two T cell lineaeges. We have identified a subset of lung resident type 2 cDCs (cDC2s) that display high levels of CD301b and have potent Treg-inducing activity ex vivo. Single cell RNA sequencing and adoptive transfer experiments show that during allergic sensitization, many CD301b+ cDC2s transition in a stepwise manner to CD200+ cDC2s that selectively promote Th2 differentiation. GM-CSF augments the development and maintenance of CD301b+ cDC2s in vivo, and also selectively expands Treg-inducing CD301b+ cDC2s derived from bone marrow. Upon their adoptive transfer to recipient mice, lung-derived CD301b+ cDC2s confer immunological tolerance to inhaled allergens. Thus, GM-CSF maintains lung homeostasis by increasing numbers of Treg-inducing CD301b+ cDC2s.

IL-35 production by inducible costimulator (ICOS)-positive regulatory T cells reverses established IL-17-dependent allergic airways disease.

BACKGROUND: Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined. OBJECTIVE: We sought to identify the regulatory cells and molecules that suppress IL-17-dependent allergic airways disease. METHODS: Mice were sensitized by means of airway instillations of ovalbumin together with low levels of LPS. Leukocyte recruitment to the lung and AHR were assessed after daily challenges with aerosolized ovalbumin. Flow cytometry, quantitative PCR, and gene-targeted mice were used to identify naturally arising subsets of regulatory T (Treg) cells and their cytokines required for the suppression of established allergic airway disease. RESULTS: Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR decreased, even though pulmonary levels of T(H)17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg cell subset expressing both forkhead box protein 3 and inducible costimulator. These Treg cells also expressed the regulatory cytokines IL-10, TGF-ß, and IL-35. Whereas IL-10 and TGF-ß were dispensable for suppression of AHR, IL-35 was required. CONCLUSION: IL-35 production by inducible costimulator-positive Treg cells can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. Targeting this pathway might therefore be of therapeutic value for treating allergic asthma in human subjects.

Alicyclic Ring Size Variation of 4-Phenyl-2-naphthoic Acid Derivatives as P2Y14 Receptor Antagonists.

P2Y14 receptor (P2Y14R) is activated by extracellular UDP-glucose, a damage-associated molecular pattern that promotes inflammation in the kidney, lung, fat tissue, and elsewhere. Thus, selective P2Y14R antagonists are potentially useful for inflammatory and metabolic diseases. The piperidine ring size of potent, competitive P2Y14R antagonist (4-phenyl-2-naphthoic acid derivative) PPTN 1 was varied from 4- to 8-membered rings, with bridging/functional substitution. Conformationally and sterically modified isosteres included N-containing spirocyclic (6-9), fused (11-13), and bridged (14, 15) or large (16-20) ring systems, either saturated or containing alkene or hydroxy/methoxy groups. The alicyclic amines displayed structural preference. An α-hydroxyl group increased the affinity of 4-(4-((1R,5S,6r)-6-hydroxy-3-azabicyclo[3.1.1]heptan-6-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic acid 15 (MRS4833) compared to 14 by 89-fold. 15 but not its double prodrug 50 reduced airway eosinophilia in a protease-mediated asthma model, and orally administered 15 and prodrugs reversed chronic neuropathic pain (mouse CCI model). Thus, we identified novel drug leads having in vivo efficacy.

A neutrophil/TGF-ß axis limits the pathogenicity of allergen-specific CD4+ T cells.

The intensity and longevity of inflammatory responses to inhaled allergens is determined largely by the balance between effector and regulatory immune responses, but the mechanisms that determine the relative magnitudes of these opposing forces remain poorly understood. We have found that the type of adjuvant used during allergic sensitization has a profound effect on both the nature and longevity of the pulmonary inflammation triggered by subsequent reexposure to that same provoking allergen. TLR ligand adjuvants and house dust extracts primed immune responses characterized by a mixed neutrophilic and eosinophilic inflammation that was suppressed by multiple daily allergen challenges. During TLR ligand-mediated allergic sensitization, mice displayed transient airway neutrophilia, which triggered the release of TGF-ß into the airway. This neutrophil-dependent production of TGF-ß during sensitization had a delayed, suppressive effect on eosinophilic responses to subsequent allergen challenge. Neutrophil depletion during sensitization did not affect numbers of Foxp3+ Tregs but increased proportions of Gata3+CD4+ T cells, which, upon their transfer to recipient mice, triggered stronger eosinophilic inflammation. Thus, a neutrophil/TGF-ß axis acts during TLR-mediated allergic sensitization to fine-tune the phenotype of developing allergen-specific CD4+ T cells and limit their pathogenicity, suggesting a novel immunotherapeutic approach to control eosinophilia in asthma.

Effects of Purinergic Receptor Deletion or Pharmacologic Modulation on Pulmonary Inflammation in Mice.

COVID-19 disease is associated with progressive accumulation of SARS-CoV-2-specific mRNA, which is recognized by innate immune receptors, such as TLR3. This in turn leads to dysregulated production of multiple cytokines, including IL-6, IFN-γ, CXCL1, and TNF-α. Excessive production of these cytokines leads to acute lung injury (ALI), which consequently compromises alveolar exchange of O2 and CO2. It is therefore of considerable interest to develop novel therapies that reduce pulmonary inflammation and stem production of pro-inflammatory cytokines, potentially for COVID-19 patients that are at high risk of developing severe disease. Purinergic signaling has a central role in fine-tuning the innate immune system, with P2 (nucleotide) receptor antagonists and adenosine receptor agonists having anti-inflammatory effects. Accordingly, we focused here on the potential role of purinergic receptors in driving neutrophilic inflammation and cytokine production in a mouse model of pulmonary inflammation. To mimic the effects of SARS-CoV-2-specific RNA accumulation in mice, we administered progressively increasing daily doses of a viral mimetic, polyinosinic:polycytidylic acid [poly(I:C)] into the airways of mice over the course of 1 week. Some mice also received increasing daily doses of ovalbumin to mimic virus-encoded protein accumulation. Animals receiving both poly(I:C) and ovalbumin displayed particularly high cytokine levels and neutrophilia, suggestive of both innate and antigen-specific, adaptive immune responses. The extent of these responses was diminished by genetic deletion (P2Y14R, P2X7R) or pharmacologic modulation (P2Y14R antagonists, A3AR agonists) of purinergic receptors. These results suggest that pharmacologic modulation of select purinergic receptors might be therapeutically useful in treating COVID-19 and other pulmonary infections.

Bridged Piperidine Analogues of a High Affinity Naphthalene-Based P2Y14R Antagonist.

High affinity phenyl-piperidine P2Y14R antagonist 1 (PPTN) was modified with piperidine bridging moieties to probe receptor affinity and hydrophobicity. Various 2-azanorbornane, nortropane, isonortropane, isoquinuclidine, and ring-opened cyclopentylamino derivatives preserved human P2Y14R affinity (fluorescence binding assay), and their pharmacophoric overlay was compared. Enantiomeric 2-azabicyclo[2.2.1]hept-5-en-3-one precursors assured stereochemically unambiguous, diverse products. Pure (S,S,S) 2-azanorbornane enantiomer 15 (MRS4738) displayed higher affinity than 1 (3-fold higher affinity than enantiomer 16) and in vivo antihyperallodynic and antiasthmatic activity. Its double prodrug 143 (MRS4815) dramatically reduced lung inflammation in a mouse asthma model. Related lactams 21-24 and dicarboxylate 42 displayed intermediate affinity and enhanced aqueous solubility. Isoquinuclidine 34 (IC50 15.6 nM) and isonortropanol 30 (IC50 21.3 nM) had lower lipophilicity than 1. In general, rigidified piperidine derivatives did not lower lipophilicity dramatically, except those rings with multiple polar groups. P2Y14R molecular modeling based on a P2Y12R structure showed stable and persistent key interactions for compound 15.

In utero supplementation with methyl donors enhances allergic airway disease in mice.

Asthma is a complex heritable disease that is increasing in prevalence and severity, particularly in developed countries such as the United States, where 11% of the population is affected. The contribution of environmental and genetic factors to this growing epidemic is currently not well understood. We developed the hypothesis, based on previous literature, that changes in DNA methylation resulting in aberrant gene transcription may enhance the risk of developing allergic airway disease. Our findings indicate that in mice, a maternal diet supplemented with methyl donors enhanced the severity of allergic airway disease that was inherited transgenerationally. Using a genomic approach, we discovered 82 gene-associated loci that were differentially methylated after in utero supplementation with a methyl-rich diet. These methylation changes were associated with decreased transcriptional activity and increased disease severity. Runt-related transcription factor 3 (Runx3), a gene known to negatively regulate allergic airway disease, was found to be excessively methylated, and Runx3 mRNA and protein levels were suppressed in progeny exposed in utero to a high-methylation diet. Moreover, treatment with a demethylating agent increased Runx3 gene transcription, further supporting our claim that a methyl-rich diet can affect methylation status and consequent transcriptional regulation. Our findings indicate that dietary factors can modify the heritable risk of allergic airway disease through epigenetic mechanisms during a vulnerable period of fetal development in mice.

CD11b+ lung dendritic cells at different stages of maturation induce Th17 or Th2 differentiation.

Dendritic cells (DC) in the lung that induce Th17 differentiation remain incompletely understood, in part because conventional CD11b+ DCs (cDC2) are heterogeneous. Here, we report a population of cDCs that rapidly accumulates in lungs of mice following house dust extract inhalation. These cells are Ly-6C+, are developmentally and phenotypically similar to cDC2, and strongly promote Th17 differentiation ex vivo. Single cell RNA-sequencing (scRNA-Seq) of lung cDC2 indicates 5 distinct clusters. Pseudotime analysis of scRNA-Seq data and adoptive transfer experiments with purified cDC2 subpopulations suggest stepwise developmental progression of immature Ly-6C+Ly-6A/E+ cDC2 to mature Ly-6C-CD301b+ lung resident cDC2 lacking Ccr7 expression, which then further mature into CD200+ migratory cDC2 expressing Ccr7. Partially mature Ly-6C+Ly-6A/E-CD301b- cDC2, which express Il1b, promote Th17 differentiation. By contrast, CD200+ mature cDC2 strongly induce Th2, but not Th17, differentiation. Thus, Th17 and Th2 differentiation are promoted by lung cDC2 at distinct stages of maturation.

UDP-glucose and P2Y14 receptor amplify allergen-induced airway eosinophilia.

Airway eosinophilia is a hallmark of allergic asthma and is associated with mucus production, airway hyperresponsiveness, and shortness of breath. Although glucocorticoids are widely used to treat asthma, their prolonged use is associated with several side effects. Furthermore, many individuals with eosinophilic asthma are resistant to glucocorticoid treatment, and they have an unmet need for novel therapies. Here, we show that UDP-glucose (UDP-G), a nucleotide sugar, is selectively released into the airways of allergen-sensitized mice upon their subsequent challenge with that same allergen. Mice lacking P2Y14R, the receptor for UDP-G, had decreased airway eosinophilia and airway hyperresponsiveness compared with wild-type mice in a protease-mediated model of asthma. P2Y14R was dispensable for allergic sensitization and for the production of type 2 cytokines in the lung after challenge. However, UDP-G increased chemokinesis in eosinophils and enhanced their response to the eosinophil chemoattractant, CCL24. In turn, eosinophils triggered the release of UDP-G into the airway, thereby amplifying eosinophilic recruitment. This positive feedback loop was sensitive to therapeutic intervention, as a small molecule antagonist of P2Y14R inhibited airway eosinophilia. These findings thus reveal a pathway that can be therapeutically targeted to treat asthma exacerbations and glucocorticoid-resistant forms of this disease.

Allergic sensitization through the airway primes Th17-dependent neutrophilia and airway hyperresponsiveness.

RATIONALE: In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant. OBJECTIVES: We investigated whether allergic sensitization through the airway elicits immune responses qualitatively different than those arising in the peritoneum. METHODS: Mice were sensitized to allergen through the airway using low-dose LPS as an adjuvant, or through the peritoneum using aluminum hydroxide as an adjuvant. After a single allergen challenge, ELISA and flow cytometry were used to measure cytokines and leukocyte subsets. Invasive measurements of airway resistance were used to measure allergen-induced airway hyperreactivity (AHR). MEASUREMENTS AND MAIN RESULTS: Sensitization through the peritoneum primed strong Th2 responses and eosinophilia, but not AHR, after a single allergen challenge. By contrast, allergic sensitization through the airway primed only modest Th2 responses, but strong Th17 responses. Th17 cells homed to the lung and released IL-17 into the airway on subsequent encounter with inhaled allergen. As a result, these mice developed IL-17-dependent airway neutrophilia and AHR. This AHR was neutrophil-dependent because it was abrogated in CXCR2-deficient mice and also in wild-type mice receiving a neutrophil-depleting antibody. Individually, neither IL-17 nor ongoing Th2 responses were sufficient to confer AHR, but together they acted synergistically to promote neutrophil recruitment, eosinophil recruitment and AHR. CONCLUSIONS: Allergic sensitization through the airway primes modest Th2 responses but strong Th17 responses that promote airway neutrophilia and acute AHR. These findings support a causal role for neutrophils in severe asthma.

Therapeutic suppression of pulmonary neutrophilia and allergic airway hyperresponsiveness by a RORγt inverse agonist.

Airway neutrophilia occurs in approximately 50% of patients with asthma and is associated with particularly severe disease. Unfortunately, this form of asthma is usually refractory to corticosteroid treatment, and there is an unmet need for new therapies. Pulmonary neutrophilic inflammation is associated with Th17 cells, whose differentiation is controlled by the nuclear receptor, RORγt. Here, we tested whether VTP-938, a selective inverse agonist of this receptor, can reduce disease parameters in animal models of neutrophilic asthma. When administered prior to allergic sensitization through the airway, the RORγt inverse agonist blunted allergen-specific Th17 cell development in lung-draining lymph nodes and attenuated allergen-induced production of IL-17. VTP-938 also reduced pulmonary production of IL-17 and airway neutrophilia when given during the allergen challenge of the model. Finally, in an environmentally relevant model of allergic responses to house dust extracts, VTP-938 suppressed production of IL-17 and neutrophilic inflammation, and also markedly diminished airway hyperresponsiveness. Together, these findings suggest that orally available inverse agonists of RORγt might provide an effective therapy to treat glucocorticoid-resistant neutrophilic asthma.

An automated intensity-weighted brachytherapy seed localization algorithm.

Brachytherapy has proven to be an effective treatment for various forms of cancer, whereby radioactive material is inserted directly into the body to maximize dosage to malignant tumors while preserving healthy tissue. In order to validate the preoperative or intraoperative dosimetric model, a postimplant evaluation procedure is needed to ensure that the locations of the implanted seeds are consistent with the planning stage. Moreover, development of an automated algorithm for seed detection and localization is necessary to expedite the postimplant evaluation process and reduce human error. Most previously reported algorithms have performed binary transforms on images before attempting to localize seeds. Furthermore, traditional approaches based upon three-dimensional seed shape parameterization and matching require high resolution imaging. The authors propose a new computationally efficient algorithm for automatic seed localization for full three-dimensional, low-resolution data sets that directly applies voxel intensity to the estimation of both seed centroid location and angular seed orientation. Computer simulations, phantom studies, and in vivo computed tomography prostate seed imaging results show that the proposed algorithm can produce reliable results even for low-resolution images.

Spontaneous mutations in recombinant inbred mice: mutant toll-like receptor 4 (Tlr4) in BXD29 mice.

Recombinant inbred (RI) mice are frequently used to identify QTL that underlie differences in measurable phenotypes between two inbred strains of mice. Here we show that one RI strain, C57BL/6J x DBA/2J (BXD29), does not develop an inflammatory response following inhalation of LPS. Approximately 25% of F2 mice [F1(BXD29 x DBA/2J) x F1] are also unresponsive to inhaled LPS, suggesting the presence of a recessive mutation in the BXD29 strain. A genomic scan of these F2 mice revealed that unresponsive animals, but not responsive animals, are homozygous for C57BL/6J DNA at a single locus on chromosome 4 close to the genomic location of Tlr4. All progeny between BXD29 and gene-targeted Tlr4-deficient mice are unresponsive to inhaled LPS, suggesting that the mutation in the BXD29 strain is allelic with Tlr4. Moreover, the intact Tlr4 receptor is not displayed on the cell surface of BXD29 macrophages. Finally, a molecular analysis of the Tlr4 gene in BXD29 mice revealed that it is interrupted by a large insertion of repetitive DNA. These findings explain the unresponsiveness of BXD29 mice to LPS and suggest that data from BXD29 mice should not be included when using BXD mice to study phenotypes affected by Tlr4 function. Our results also suggest that the frequency of such unidentified, spontaneously occurring mutations is an issue that should be considered when RI strains are used to identify QTL.

Distinct functions of CXCR4, CCR2, and CX3CR1 direct dendritic cell precursors from the bone marrow to the lung.

Precursors of dendritic cells (pre-DCs) arise in the bone marrow (BM), egress to the blood, and finally migrate to peripheral tissue, where they differentiate to conventional dendritic cells (cDCs). Upon their activation, antigen-bearing cDCs migrate from peripheral tissue to regional lymph nodes (LNs) in a manner dependent on the chemokine receptor, CCR7. To maintain immune homeostasis, these departing cDCs must be replenished by new cDCs that develop from pre-DCs, but the molecular signals that direct pre-DC trafficking from the BM to the blood and peripheral tissues remain poorly understood. In the present study, we found that pre-DCs express the chemokine receptors CXCR4, CCR2, and CX3CR1, and that each of these receptors has a distinct role in pre-DC trafficking. Flow cytometric analysis of pre-DCs lacking CXCR4 revealed that this receptor is required for the retention of pre-DCs in the BM. Analyses of mice lacking CCR2 or CX3CR1, or both, revealed that they promote pre-DC migration to the lung at steady state. CCR2, but not CX3CR1, was required for pre-DC migration to the inflamed lung. Thus, these multiple chemokine receptors cooperate in a step-wise fashion to coordinate the trafficking of pre-DCs from the BM to the circulation and peripheral tissues.

TNF is required for TLR ligand-mediated but not protease-mediated allergic airway inflammation.

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma.