Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

Induction of a memory-like CD4+ T-cell phenotype by airway smooth muscle cells.

In asthma, CD4+ T-cell interaction with airway smooth muscle (ASM) may enhance its contractile properties and promote its proliferation. However, less is known about the effects of this interaction on T cells. To explore the consequences of interaction of CD4+ T cells with ASM we placed the cells in co-culture and analyzed the phenotypic and functional changes in the T cells. Effector status as well as cytokine expression was assessed by flow cytometry. An increase in CD45RA-CD45RO+ memory T cells was observed after co-culture; however, these cells were not more responsive to CD3/28 restimulation. A reduction in mitochondrial coupling and an increase in the production of mitochondrial reactive oxygen species by CD4+ T cells post-restimulation suggested altered mitochondrial metabolism after co-culture. RNA sequencing analysis of the T cells revealed characteristic downregulation of effector T-cell-associated genes, but a lack of upregulation of memory T-cell-associated genes. The results of this study demonstrate that ASM cells can induce a phenotypic shift in CD4+ T cells into memory-like T cells but with reduced capacity for activation.

CD4+ T cell-derived IFN-γ and LIGHT synergistically upregulate chemokine production from airway smooth muscle cells.

Airway smooth muscle (ASM) remodeling in asthmatic airways may contribute to persistent airflow limitation and airway hyperresponsiveness. CD4+ T cells infiltrate the ASM layer where they may induce a proliferative and secretory ASM cell phenotype. We studied the interaction between activated CD4+ T cells and ASM cells in co-culture in vitro and investigated the effects of CD4+ T cells on chemokine production by ASM cells. CD4+ T cells induced marked upregulation of C-X-C motif chemokine ligands (CXCL) 9, 10, and 11 in ASM cells. Blockade of the IFN-γ receptor on ASM cells prevented this upregulation. Furthermore, T cell-derived IFN-γ and LIGHT (lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) synergize in a dose-dependent manner to coordinately enhance CXCL9, 10, and 11 expression. The synergistic property of LIGHT was mediated exclusively through the lymphotoxin-ß receptor (LTBR), but not herpes virus entry mediator (HVEM). Disruption of LTBR signaling in ASM cells reduced CXCL9, 10, and 11 production and ASM cell-mediated CD4+ T cell chemotaxis. We conclude that the LIGHT-LTBR signaling axis acts together with IFN-γ to regulate chemokines that mediate lymphocyte infiltration in asthmatics.

Serum Response Factor Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

T cell and airway smooth muscle interaction: a key driver of asthmatic airway inflammation and remodelling.

Crosstalk between T cells and airway smooth muscle (ASM) may play a role in modulating asthmatic airway inflammation and remodelling. Infiltrating T cells have been observed within the ASM bundles of asthmatics, and a wide range of direct and indirect interactions between T cells and ASM have been demonstrated using various in vitro and in vivo model systems. Contact-dependent mechanisms such as ligation and activation of cellular adhesion and costimulatory molecules, as well as the formation of lymphocyte-derived membrane conduits, facilitate the adhesion, bidirectional communication and transfer of materials between T and ASM cells. T cell-derived cytokines, particularly of the Th1, Th2 and Th17 subsets, modulate the secretome, proliferation and contractility of ASM cells. This review summarizes the mechanisms governing T cell-ASM crosstalk in the context of asthma. Understanding the underlying mechanistic basis is important for directing future research and developing therapeutic interventions targeted towards this complex interaction.

Mucus plugs in the airways of asthmatic subjects and smoking status.

BACKGROUND: Mucus plugs have been described in the airways of asthmatic subjects, particularly those with associated with type 2 inflammation and sputum eosinophilia. In the current study we addressed the question of whether smoking, neutrophilic inflammation and airway dimensions affected the prevalence of mucus plugs. METHODS: In a cohort of moderate to severe asthmatics (n = 50), including a group of ex-smokers and current smokers, the prevalence of mucus plugs was quantified using a semi-quantitative score based on thoracic computerized tomography. The relationships between mucus score, sputum inflammatory profile and airway architecture were tested according to patient's smoking status. RESULTS: Among the asthmatics (37% former or active smokers), 74% had at least one mucus plug. The median score was 3 and was unrelated to smoking status. A significant but weak correlation was found between mucus score, FEV1 and FEV1/FVC. Mucus score was significantly correlated with sputum eosinophils. Among former and active smokers, mucus score was correlated with sputum neutrophils. Mucus score was positively associated with FeNO in non-smoking subjects. The lumen dimensions of the main and lobar bronchi were significantly inversely correlated with mucus score. CONCLUSION: Airway mucus plugs could define an asthma phenotype with altered airway architecture and can occur in asthmatic subjects with either neutrophilic or eosinophilic sputum according to their smoking status.

Interferon-γ amplifies airway smooth muscle-mediated CD4+ T cell recruitment by promoting the secretion of C-X-C-motif chemokine receptor 3 ligands.

Asthmatic airways feature increased ASM mass that is largely attributable to hyperplasia, and which potentially contributes to excessive airway narrowing. T cells induce ASMC proliferation via contact-dependent mechanisms in vitro that may have importance for asthmatic ASM growth, as CD4+ T cells infiltrate ASM bundles in asthmatic human airways. In this study, we used an in vitro migration assay to investigate the pathways responsible for the trafficking of human CD4+ T cells to ASM. ASMCs induced chemotaxis of activated CD4+ T cells, which was inhibited by the CXCR3 antagonist AMG487 and neutralizing antibodies against its ligands CXCL10 and 11, but not CCR3 or CCR5 antagonists. CXCR3 expression was upregulated among all T cells following anti-CD3/CD28-activation. CD4+ T cells upregulated CXCL9, 10, and 11 expression in ASMCs in an IFN-γ/STAT1-dependent manner. Disruption of IFN-γ-signaling resulted in reduced T cell migration, along with the inhibition of CD4+ T cell-mediated STAT1 activation and CXCR3 ligand secretion by ASMCs. ASMCs derived from healthy and asthmatic donors demonstrated similar T cell-recruiting capacities. In vivo CXCL10 and 11 expression by asthmatic ASM was confirmed by immunostaining. We conclude that the CXCL10/11-CXCR3 axis causes CD4+ T cell recruitment to ASM that is amplified by T cell-derived IFN-γ.

Airway compliance measurements in mouse models of respiratory diseases.

The quantification of airway compliance (Caw) is essential to the study of airway alterations in disease models. However, the required measurements of airway pressure and volume are difficult to acquire in mice. We hypothesized that the inflation limb of full-range pressure-volume (PV) curves could be used to quantify Caw, as it contains a segment where only the airway tree is distended. The study objective was to assess the feasibility of the approach by analysis of full-range PV curves previously collected in three mouse models: an elastase model of emphysema, a genetic model spontaneously developing emphysema (leukotriene C4 synthase knockout; LTC4S-KO), and a bleomycin model of lung fibrosis. Attempts to validate results included Caw change relative to respiratory system compliance (ΔCaw/ΔC), the minute work of breathing (mWOB), and the elastance at 20.5 Hz (Ers_20.5) from prior respiratory mechanics measurements in the same subjects. Caw was estimated at 3% of total compliance in healthy mice or 2.3 ± 1 µL/cmH2O (n = 17). The technique detected changes in models of respiratory obstructive and restrictive diseases relative to control mice as well as differences in the two emphysema models studied. The changes in Caw were consistent with those seen in ΔCaw/ΔC, mWOB, or Ers_20.5, with some variations according to the model, as well as with results reported in the literature in humans and mice. Direct Caw measurements in subjects as small as mice could prove useful to further characterize other respiratory disease models associated with airway remodeling or to assess treatment effects.

Asthma and fixed airflow obstruction: Long-term trajectories suggest distinct endotypes.

BACKGROUND: Long-term trajectories of asthma with fixed airflow obstruction (FAO) may reveal links to inflammatory endotypes. OBJECTIVE: We investigated whether measures of asthma control and airway inflammation and remodelling differed by long-term FAO status in moderate-to-severe asthma. METHODS: Adults enrolled in the Difficult Asthma Study assessed initially using serial Asthma Control Questionnaire (ACQ), exacerbation history, spirometry and sputum cytology over 12 months, as well as endoscopic bronchial biopsy with airway smooth muscle (ASM) quantification, were revaluated three or more years later with questionnaires and spirometry. FAO was defined as a persistent post-bronchodilator forced expired volume in one second (FEV1 )-to-forced vital capacity ratio below 0.70. RESULTS: Sixty-two participants (mean ± SD age 48 ± 11 years; 50% female; 75% atopic; asthma duration 24 ± 14 years) returned for follow-up assessment (median interval 7.9 years; IQR: 5.4-8.8 years). Compared to participants without FAO (n = 28), those with FAO at baseline and long-term follow-up (n = 18) had higher baseline sputum neutrophil content and ASM, and a higher exacerbation frequency that persisted at long-term follow-up. Sputum eosinophils, ACQ and long-term FEV1 decline did not differ. Participants with incident FAO at long-term follow-up (n = 16) had higher baseline exacerbation frequency, sputum eosinophil content, higher ACQ scores and greater decline in FEV1 , whereas baseline ASM was similar to those without FAO. CONCLUSION: In moderate-to-severe asthma, long-term FAO is characterized by neutrophilic sputum inflammation and airway remodelling, but FEV1 decline is similar to those without FAO. Long-term incident FAO is preceded by higher exacerbation frequency, higher sputum eosinophil content and significant FEV1 decline.

HB-EGF Synthesized by CD4 T Cells Modulates Allergic Airway Eosinophilia by Regulating IL-5 Synthesis.

CD4 T cells express the epidermal growth factor (EGF) receptor ligand, heparin-binding EGF (HB-EGF), with no defined immuno-pathophysiological function. Therefore, we wished to elucidate the function of HB-EGF synthesized by CD4 T cells in the context of allergic pulmonary inflammation and the asthma surrogate, airway hyperresponsiveness, in a murine acute model of asthma. In this study, we show how knocking out HB-EGF expression in CD4 T cells in vivo attenuates IL-5 synthesis in the lung that is accompanied by diminished eosinophilic inflammation and airway hyperresponsiveness. HB-EGF coimmunoprecipitates with the transcriptional repressor B cell lymphoma 6 (Bcl-6) in CD4 T cells. Knocking out HB-EGF in CD4 T cells resulted in increased Bcl-6 binding to the IL-5 gene and decreased IL-5 mRNA expression. Thus, these findings suggest an immunoregulatory function for intrinsic HB-EGF expressed by CD4 T cells in TH2 inflammation and airway dysfunction by modulating IL-5 expression via binding to and inhibiting the repressive function of Bcl-6.

Cellular Source of Cysteinyl Leukotrienes Following Chlorine Exposure.

Exposure of mice to high concentrations of chlorine leads to the synthesis of cysteinyl leukotrienes (cysLTs). CysLTs contribute to chlorine-induced airway hyperresponsiveness. The aim of the current study was to determine the cellular source of the cysLTs. To achieve this aim, we exposed mice to 100 ppm of chlorine for 5 minutes. Intranasal instillation of clodronate in liposomes and of diphtheria toxin in CD11c-DTR mice was used to deplete macrophages. CCR2-/- mice were used to assess the contribution of recruited macrophages. Eosinophils and neutrophils were depleted with specific antibodies. Platelet-neutrophil aggregation was prevented with an antibody against P-selectin. The potential roles of phagocytosis of neutrophils by macrophages and of transcellular metabolism between epithelial cells and neutrophils were explored in coculture systems. We found that depletion of neutrophils was the only intervention that inhibited the synthesis of cysLTs at 24 hours after chlorine exposure. Although macrophages did synthesize cysLTs in response to phagocytosis of neutrophils, depletion of macrophages did not reduce the increment in cysLTs triggered by chlorine exposure. However, coculture of airway epithelial cells with neutrophils resulted in a significant increase in the synthesis of cysLTs, dependent on the expression of 5-lipoxygenase by neutrophils. We conclude that cysLT synthesis following chlorine exposure may be dependent on transcellular metabolism by neutrophil-epithelial interactions.

Suboptimal treatment response to anti-IL-5 monoclonal antibodies in severe eosinophilic asthmatics with airway autoimmune phenomena.

BACKGROUND: In clinical trials, the two anti-interleukin (IL)-5 monoclonal antibodies (mAbs: mepolizumab and reslizumab) approved to treat severe eosinophilic asthma reduce exacerbations by ∼50-60%. OBJECTIVE: To observe response to anti-IL-5 mAbs in a real-life clinical setting, and to evaluate predictors of suboptimal response. METHODS: In four Canadian academic centres, predefined clinical end-points in 250 carefully characterised moderate-to-severe asthmatic patients were collected prospectively to assess response to the two anti-IL-5 mAbs. Suboptimal response was determined based on failure to reduce maintenance corticosteroid (MCS) or asthma symptoms scores (Asthma Control Questionnaire (ACQ)) or exacerbations, in addition to persistence of sputum/blood eosinophils. Worsening in suboptimal responders was assessed based on reduced lung function by 25% or increase in MCS/ACQ. A representative subset of 39 patients was evaluated for inflammatory mediators, autoantibodies and complement activation in sputum (by ELISA) and for immune-complex deposition by immunostaining formalin-fixed paraffin-embedded sputum plugs. RESULTS: Suboptimal responses were observed in 42.8% (107 out of 250) patients treated with either mepolizumab or reslizumab. Daily prednisone requirement, sinus disease and late-onset asthma diagnoses were the strongest predictors of suboptimal response. Asthma worsened in 13.6% (34 out of 250) of these patients. The majority (79%) of them were prednisone-dependent. Presence of sputum anti-eosinophil peroxidase immunoglobulin (Ig)G was a predictor of suboptimal response to an anti-IL-5 mAb. An increase in sputum C3c (marker of complement activation) and deposition of C1q-bound/IL-5-bound IgG were observed in the sputa of those patients who worsened on therapy, suggesting an underlying autoimmune-mediated pathology. CONCLUSION: A significant number of patients who meet currently approved indications for anti-IL5 mAbs show suboptimal response to them in real-life clinical practice, particularly if they are on high doses of prednisone. Monitoring blood eosinophil count is not helpful to identify these patients. The concern of worsening of symptoms associated with immune-complex mediated complement activation in a small proportion of these patients highlights the relevance of recognising airway autoimmune phenomena and this requires further evaluation.

Tolerogenic signaling of alveolar macrophages induces lung adaptation to oxidative injury.

BACKGROUND: Inhaled oxidative toxicants present in ambient air cause airway epithelial injury, inflammation, and airway hyperresponsiveness. Effective adaptation to such environmental insults is essential for the preservation of pulmonary function, whereas failure or incomplete adaptation to oxidative injury can render the host susceptible to the development of airway disease. OBJECTIVE: We sought to explore the mechanisms of airway adaptation to oxidative injury. METHODS: For a model to study pulmonary adaptation to oxidative stress-induced lung injury, we exposed mice to repeated nose-only chlorine gas exposures. Outcome measures were evaluated 24 hours after the last chlorine exposure. Lung mechanics and airway responsiveness to methacholine were assessed by using the flexiVent. Inflammation and antioxidant responses were assessed in both bronchoalveolar lavage fluid and lung tissue. Using both loss or gain of function and genomic approaches, we further dissected the cellular and molecular mechanisms involved in pulmonary adaptation. RESULTS: Repeated exposures to oxidative stress resulted in pulmonary adaptation evidenced by abrogation of neutrophilic inflammation and airway hyperresponsiveness. This adaptation was independent of antioxidant mechanisms and regulatory T cells but dependent on residential alveolar macrophages (AMs). Interestingly, 5% of AMs expressed forkhead box P3, and depletion of these cells abolished adaptation. Results from transcriptomic profiling and loss and gain of function suggest that adaptation might be dependent on TGF-ß and prostaglandin E2. CONCLUSION: Pulmonary adaptation during oxidative stress-induced lung injury is mediated by a novel subset of forkhead box P3-positive AMs that limits inflammation, favoring airway adaptation and host fitness through TGF-ß and prostaglandin E2.

Contractile Properties of Intrapulmonary Airway Smooth Muscle in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Many patients with CF have asthma-like symptoms and airway hyperresponsiveness, which are potentially associated with altered airway smooth muscle (ASM) contractility. Our goal in this study was to assess the contractility of the CF intrapulmonary ASM. ASM strips were dissected from human control and CF intrapulmonary airways, and assessed for methacholine-induced shortening velocity, maximal force, and stress. We also assessed isoproterenol responses in maximally methacholine-contracted ASM. ASM strips were then incubated for 16 hours with IL-13 and measurements were repeated. Myosin light chain kinase (MLCK) expression was assessed by Western blotting. Airways were immunostained for morphometry. ASM mass was increased in CF airways, which likely contributes to airway hyperresponsiveness. Although ASM contractile properties were not intrinsically different between patients with CF and control subjects, CF ASM responded differently in the presence of the inflammatory mediator IL-13, showing impairment in ß-adrenergic-induced relaxation. Indeed, the percentage of relaxation measured at maximal isoproterenol concentrations in the CF ASM was significantly lower after incubation with IL-13 (46.0% ± 6.7% relaxation) than without IL-13 (74.0% ± 7.7% relaxation, P = 0.018). It was also significantly lower than that observed in control ASM incubated with IL-13 (68.8% ± 4.9% relaxation, P = 0.048) and without IL-13 (82.4% ± 9.9%, P = 0.0035). CF ASM incubated with IL-13 also expressed greater levels of MLCK. Thus, our data suggest that the combination of an increase in ASM mass, increased MLCK expression, and inflammation-induced ß-adrenergic hyporesponsiveness may contribute to airway dysfunction in CF.

Organic dust, causing both oxidative stress and Nrf2 activation, is phagocytized by bronchial epithelial cells.

Inhalation of organic dust (OD) from swine confinement facilities leads to pulmonary inflammation, airway hyperresponsiveness, and oxidative stress. In mice, pretreatment with a hydroxyl radical scavenger prevents airway inflammation and airway hyperresponsiveness (AHR) induced by OD exposure. We sought to determine a mechanism by which OD could induce oxidative stress in bronchial epithelial cells. Human bronchial epithelial cells (BEAS-2B or NHBE) were treated with various concentrations of OD, followed by evaluation of intracellular oxidative stress using 2',7'-dichlorofluorescein diacetate (DCFDA). After stimulation with OD, gene expression of antioxidant genes was assessed by real-time quantitative PCR followed by quantification of Nrf2 nuclear translocation using a luciferase reporter assay. Phagocytic markers (CD36 and CD68) were analyzed by FACS. Cells were treated with an actin inhibitor, cytochalasin D, before OD exposure and evaluated for Nrf2 nuclear translocation and DCFDA. Mice were pretreated with sulforaphane, the Nrf2 activator, before OD exposure and evaluated for pulmonary inflammation and airway reactivity. OD induced a time- and concentration-dependent increase in DCFDA. mRNA expression levels of Nrf2-dependent genes and Nrf2 nuclear translocation were increased after OD exposure. OD exposure increased the expression of CD68 and CD36. Cytochalasin D prevented oxidative stress and Nrf2 nuclear translocation after OD. Pretreatment with sulforaphane prevented OD-induced inflammation and AHR while increasing the uptake of OD in bronchial epithelial cells. Bronchial epithelial cells can phagocytose OD, resulting in an increase in endogenous oxidative stress. Nrf2-dependent mechanisms mediate the antioxidant response to OD.

Characterization of cystic fibrosis airway smooth muscle cell proliferative and contractile activities.

Cystic fibrosis (CF) is a genetic disease that causes multiple airway abnormalities. Two major respiratory consequences of CF are airway hyperresponsiveness (AHR) and airway remodeling. Airway smooth muscle (ASM) is hypothesized to be responsible for the airway dysfunction, since their thickening is involved in remodeling, and excessive contraction by the ASM may cause AHR. It is unclear whether the ASM is intrinsically altered to favor increased contractility or proliferation or if microenvironmental influences induce pathological behavior in vivo. In this study, we examined the contractile and proliferative properties of ASM cells isolated from healthy donor and CF transplant lungs. Assays of proliferation showed that CF ASM proliferates at a higher rate than healthy cells. Through calcium analysis, no differences in contractile activation in response to histamine were found. However, CF ASM cells lagged in their reuptake of calcium in the sarcoplasmic reticulum. The combination CFTR corrector and potentiator, VX-809/770, used to restore CFTR function in CF ASM, resulted in a reduction in proliferation and in a normalization of calcium reuptake kinetics. These results show that impaired CFTR function in ASM cells causes intrinsic changes in their proliferative and contractile properties.

RIPK3 interacts with MAVS to regulate type I IFN-mediated immunity to Influenza A virus infection.

The type I interferon pathway plays a critical role in both host defense and tolerance against viral infection and thus requires refined regulatory mechanisms. RIPK3-mediated necroptosis has been shown to be involved in anti-viral immunity. However, the exact role of RIPK3 in immunity to Influenza A Virus (IAV) is poorly understood. In line with others, we, herein, show that Ripk3-/- mice are highly susceptible to IAV infection, exhibiting elevated pulmonary viral load and heightened morbidity and mortality. Unexpectedly, this susceptibility was linked to an inability of RIKP3-deficient macrophages (Mφ) to produce type I IFN in the lungs of infected mice. In Mφ infected with IAV in vitro, we found that RIPK3 regulates type I IFN both transcriptionally, by interacting with MAVS and limiting RIPK1 interaction with MAVS, and post-transcriptionally, by activating protein kinase R (PKR)-a critical regulator of IFN-ß mRNA stability. Collectively, our findings indicate a novel role for RIPK3 in regulating Mφ-mediated type I IFN anti-viral immunity, independent of its conventional role in necroptosis.

Basic Fibroblast Growth Factor 2 Is a Determinant of CD4 T Cell-Airway Smooth Muscle Cell Communication through Membrane Conduits.

Activated CD4 T cells connect to airway smooth muscle cells (ASMCs) in vitro via lymphocyte-derived membrane conduits (LMCs) structurally similar to membrane nanotubes with unknown intercellular signals triggering their formation. We examined the structure and function of CD4 T cell-derived LMCs, and we established a role for ASMC-derived basic fibroblast growth factor 2 (FGF2b) and FGF receptor (FGFR)1 in LMC formation. Blocking FGF2b's synthesis and FGFR1 function reduced LMC formation. Mitochondrial flux from ASMCs to T cells was partially FGF2b and FGFR1 dependent. LMC formation by CD4 T cells and mitochondrial transfer from ASMCs was increased in the presence of asthmatic ASMCs that expressed more mRNA for FGF2b compared with normal ASMCs. These observations identify ASMC-derived FGF2b as a factor needed for LMC formation by CD4 T cells, affecting intercellular communication.

Semaphorin 4C Protects against Allergic Inflammation: Requirement of Regulatory CD138+ Plasma Cells.

The regulatory properties of B cells have been studied in autoimmune diseases; however, their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C), an axonal guidance molecule, plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure, with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells, indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19+CD138+ cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c-/- CD19+CD138+ cells induced marked pulmonary inflammation, eosinophilia, and increased bronchoalveolar lavage fluid IL-4 and IL-5, whereas adoptive transfer of wild-type CD19+CD138+IL-10+ cells dramatically decreased allergic airway inflammation in wild-type and Sema4c-/- mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138+ B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore, we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138+ B cells.