Selective production of IL-33-neutralizing autoantibody ameliorates asthma responses and severity.

BACKGROUND: Natural anti-cytokine autoantibodies can regulate homeostasis of infectious and inflammatory diseases. The anti-cytokine autoantibody profile and relevance to the pathogenesis of asthma are unknown. We aim to identify key anti-cytokine autoantibodies in asthma patients, and reveal their immunological function and clinical significance. METHODS: A Luciferase Immunoprecipitation System was used to screen serum autoantibodies against 11 key cytokines in patients with allergic asthma and healthy donors. The antigen-specificity, immunomodulatory functions and clinical significance of anti-cytokine autoantibodies were determined by ELISA, qPCR, neutralization assays and statistical analysis, respectively. Potential conditions for autoantibody induction were revealed by in vitro immunization. RESULTS: Of 11 cytokines tested, only anti-IL-33 autoantibody was significantly increased in asthma, compare to healthy controls, and the proportion positive was higher in patients with mild-to-moderate than severe allergic asthma. In allergic asthma patients, the anti-IL-33 autoantibody level correlated negatively with serum concentration of pathogenic cytokines (e.g., IL-4, IL-13, IL-25 and IL-33), IgE, and blood eosinophil count, but positively with mid-expiratory flow FEF25-75%. The autoantibodies were predominantly IgG isotype, polyclonal and could neutralize IL-33-induced pathogenic responses in vitro and in vivo. The induction of the anti-IL-33 autoantibody in blood B-cells in vitro required peptide IL-33 antigen along with a stimulation cocktail of TLR9 agonist and cytokines IL-2, IL-4 or IL-21. CONCLUSIONS: Serum natural anti-IL-33 autoantibodies are selectively induced in some asthma patients. They ameliorate key asthma inflammatory responses, and may improve lung function of allergic asthma.

Vanadium exposure exacerbates allergic airway inflammation and remodeling through triggering reactive oxidative stress.

Background: Metal components of environmental PM2.5 are associated with the exacerbation of allergic diseases like asthma. In our recent hospital-based population study, exposure to vanadium is shown to pose a significant risk for current asthma, but the causal relationship and its underlying molecular mechanisms remain unclear. Objective: We sought to determine whether vanadium co-exposure can aggravate house dust mite (HDM)-induced allergic airway inflammation and remodeling, as well as investigate its related mechanisms. Methods: Asthma mouse model was generated by using either vanadium pentoxide (V2O5) or HDM alone or in combination, in which the airway inflammation and remodeling was investigated. The effect of V2O5 co-exposure on HDM-induced epithelial-derived cytokine release and oxidative stress (ROS) generation was also examined by in vitro analyses. The role of ROS in V2O5 co-exposure-induced cytokine release and airway inflammation and remodeling was examined by using inhibitors or antioxidant. Results: Compared to HDM alone, V2O5 co-exposure exacerbated HDM-induced airway inflammation with increased infiltration of inflammatory cells and elevated levels of Th1/Th2/Th17 and epithelial-derived (IL-25, TSLP) cytokines in the bronchoalveolar lavage fluids (BALFs). Intriguingly, V2O5 co-exposure also potentiated HDM-induced airway remodeling. Increased cytokine release was further supported by in vitro analysis in human bronchial epithelial cells (HBECs). Mechanistically, ROS, particularly mitochondrial-derived ROS, was significantly enhanced in HBECs after V2O5 co-exposure as compared to HDM challenge alone. Inhibition of ROS with its inhibitor N-acetyl-L-cysteine (NAC) and mitochondrial-targeted antioxidant MitoTEMPO blocked the increased epithelial release caused by V2O5 co-exposure. Furthermore, vitamin D3 as an antioxidant was found to inhibit V2O5 co-exposure-induced increased airway epithelial cytokine release and airway remodeling. Conclusions: Our findings suggest that vanadium co-exposure exacerbates epithelial ROS generation that contribute to increased allergic airway inflammation and remodeling.

Benzo(a)pyrene Enhanced Dermatophagoides Group 1 (Der f 1)-Induced TGFß1 Signaling Activation Through the Aryl Hydrocarbon Receptor-RhoA Axis in Asthma.

We have previously demonstrated that benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced airway inflammation. The underlying mechanism, however, remains undetermined. Here we investigated the molecular mechanisms underlying the potentiation of BaP exposure on Der f 1-induced airway inflammation in asthma. We found that BaP co-exposure potentiated Der f 1-induced TGFß1 secretion and signaling activation in human bronchial epithelial cells (HBECs) and the airways of asthma mouse model. Moreover, BaP exposure alone or co-exposure with Der f 1-induced aryl hydrocarbon receptor (AhR) activity was determined by using an AhR-dioxin-responsive element reporter plasmid. The BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation were attenuated by either AhR antagonist CH223191 or AhR knockdown in HBECs. Furthermore, AhR knockdown led to the reduction of BaP and Der f 1 co-exposure-induced active RhoA. Inhibition of RhoA signaling with fasudil, a RhoA/ROCK inhibitor, suppressed BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation. This was further confirmed in HBECs expressing constitutively active RhoA (RhoA-L63) or dominant-negative RhoA (RhoA-N19). Luciferase reporter assays showed prominently increased promoter activities for the AhR binding sites in the promoter region of RhoA. Inhibition of RhoA suppressed BaP and Der f 1 co-exposure-induced airway hyper-responsiveness, Th2-associated airway inflammation, and TGFß1 signaling activation in asthma. Our studies reveal a previously unidentified functional axis of AhR-RhoA in regulating TGFß1 expression and signaling activation, representing a potential therapeutic target for allergic asthma.

Serum levels of the IgA isotype switch factor TGF-ß1 are elevated in patients with COVID-19.

We previously observed enhanced immunoglobulin A (IgA) responses in severe COVID-19, which might confer damaging effects. Given the important role of IgA in immune and inflammatory responses, the aim of this study was to investigate the dynamic response of the IgA isotype switch factor TGF-ß1 in COVID-19 patients. We observed, in a total of 153 COVID-19 patients, that the serum levels of TGF-ß1 were increased significantly at the early and middle stages of COVID-19, and correlated with the levels of SARS-CoV-2-specific IgA, as well as with the APACHE II score in patients with severe disease. In view of the genetic association of the TGF-ß1 activator THBS3 with severe COVID-19 identified by the COVID-19 Host Genetics Initiative, this study suggests TGF-ß1 may play a key role in COVID-19.

Benzo(a)pyrene facilitates dermatophagoides group 1 (Der f 1)-induced epithelial cytokine release through aryl hydrocarbon receptor in asthma.

BACKGROUND: Environmental pollutants, which coexist with allergens, have been associated with the exacerbation of asthma. However, the underlying molecular mechanisms remain elusive. We sought to determine whether benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced asthma and its underlying mechanisms. METHODS: The effect of BaP was investigated in Der f 1-induced mouse model of asthma, including airway hyper-responsiveness, allergic inflammation, and epithelial-derived cytokines. The impact of BaP on Der f 1-induced airway epithelial cell oxidative stress (ROS) and cytokine release was further analyzed. The role of aryl hydrocarbon receptor (AhR) signaling in BaP-promoted Der f 1-induced ROS, cytokine production, and allergic inflammation was also investigated. RESULTS: Compared with Der f 1, BaP co-exposure with Der f 1 led to airway hyper-responsiveness and increased lung inflammation in mouse model of asthma. Increased expression of TSLP, IL-33, and IL-25 was also found in the airways of these mice. Moreover, BaP co-exposure with Der f 1 activated AhR signaling with increased expression of AhR and CYP1A1 and promoted airway epithelial ROS generation and TSLP and IL-33, but not IL-25, expression. Interestingly, AhR antagonist CH223191 or cells with AhR knockdown abrogated the increased expression of ROS, TSLP, and IL-33. Furthermore, ROS inhibitor N-acetyl-L-cysteine (NAC) also suppressed BaP co-exposure-induced expression of epithelial TSLP, IL-33, and IL-25. Finally, AhR antagonist CH223191 and NAC inhibited BaP co-exposure with Der f 1-induced lung inflammation. CONCLUSIONS: Our findings suggest that BaP facilitates Der f 1-induced epithelial cytokine release through the AhR-ROS axis.

ß-Lactam hypersensitivity involves expansion of circulating and skin-resident TH22 cells.

BACKGROUND: ß-Lactam hypersensitivity has been classified according to the phenotype and function of drug-specific T cells. However, new T-cell subsets have not been considered. OBJECTIVE: The objective of this study was to use piperacillin as a model of ß-lactam hypersensitivity to study the nature of the drug-specific T-cell response induced in the blood and skin of hypersensitive patients and healthy volunteers. METHODS: Drug-specific T cells were cloned from blood and inflamed skin, and cellular phenotype and function were explored. Naive T cells from healthy volunteers were primed to piperacillin, cloned, and subjected to the similar analyses. RESULTS: PBMC and T-cell clones (n = 570, 84% CD4+) from blood of piperacillin-hypersensitive patients proliferated and secreted TH1/TH2 cytokines alongside IL-22 after drug stimulation. IL-17A secretion was not detected. Drug-specific clones from inflamed skin (n = 96, 83% CD4+) secreted a similar profile of cytokines but displayed greater cytolytic activity, secreting perforin, granzyme B, and Fas ligand when activated. Blood- and skin-derived clones expressed high levels of skin-homing chemokine receptors and migrated in the presence of the ligands CCL17 and CCL27. Piperacillin-primed naive T cells from healthy volunteers also secreted IFN-γ, IL-13, IL-22, and cytolytic molecules. Aryl hydrocarbon receptor blockade prevented differentiation of the naive T cells into antigen-specific IL-22-secreting cells. CONCLUSION: Together, our results reveal that circulating and skin-resident, antigen-specific, IL-22-secreting T cells are detectable in patients with ß-lactam hypersensitivity. Furthermore, differentiation of naive T cells into antigen-specific TH22 cells is dependent on aryl hydrocarbon receptor signaling.

Identification of a novel cofilin-related molecule (Der f 31) as an allergen from Dermatophagoides farinae.

House dust mite (HDM) allergen is a major cause of allergic disease. In this study, two-dimensional immunoblot and Matrix-Assisted Laser Desorption Ionization tandem Time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify Der f 31. After Der f 31 was cloned, expressed and purified, skin prick test (SPT), Immune inhibitory assays, Western blot, ELISA and asthmatic mouse model were employed to examine the allergenicity of recombinant Der f 31. The gene of Der f 31 includes 447 bps, and encoded 148 amino acids. Positive responses of SPT to r-Der f 31 were 32.5% in 43 HDM-allergic patients. r-Der f 31 can induce allergic pulmonary inflammation in the mouse model. In conclusion, Der f 31 is a novel subtype of dust mite allergens.

The role of IL-25 in the reduction of oxidative stress and the apoptosis of airway epithelial cells with specific immunotherapy in an asthma mouse model.

Oxidative stress and cell apoptosis play important roles in the pathogenesis of asthma. Specific immunotherapy (SIT) is the only curative approach for asthma and is effective at decreasing asthmatic oxidation and cell apoptosis, but the mechanisms remain unclear. In this study, by using in vivo and in vitro models, we indirectly demonstrated that SIT alleviated the apoptosis and oxidative stress of bronchial epithelial cells in an asthma model through regulating interleukin (IL)-25. Female BALB/c mice were used for an asthma model induced by exposure to house dust mite (HDM) extracts as allergens. Prior to the challenge, the mice were either given the SIT vaccine or N-Acetyl-L-cysteine (NAC). RESULTS: Compared with that in asthma models, SIT administration decreased (1) airway hyper-responsiveness; (2) the production of cytokines, including IL-4, IL-5, IL-13, and IL-25, as well as serum HDM-specific IgE and IgG1, as shown by ELISA; and (3) lipid oxidative species, such as reactive oxidative species (ROS) and malondialdehyde (MDA), in the lung tissue. Moreover, TUNEL staining showed that SIT alleviated pulmonary cell apoptosis. In vitro, flow cytometry showed that human recombinant IL-25 (rIL-25) led to increased cell apoptosis and ROS in the human epithelial cell line 16HBE in a dose and time-dependent fashion. In conclusion, in vivo, SIT reduced asthmatic Th2 cytokine levels and the production of IL-25 and alleviated oxidative stress and cell apoptosis in the lung tissue. In vitro, IL-25 increased the number of apoptotic cells and the production of ROS in16HBE cells.

Interleukin-33 promoting Th1 lymphocyte differentiation dependents on IL-12.

The pro-Th2 cytokine IL-33 is now emerging as an important Th1 cytokine-IFN-γ inducer in murine CD4(+) T cells that is essential for protective cell-mediated immunity against viral infection in mice. However, whether IL-33 can promote human Th1 cell differentiation and how IL-33 polarizes Th1 cells is less understood. We assessed the ability of IL-33 to induce Th1 cell differentiation and IFN-γ production in vitro and in vivo. We report here that IL-33 alone had no ability in Th1 cell polarization. However it potentiated IL-12-mediated Th1 cell differentiation and IFN-γ production in TCR-stimulated murine and human CD4(+) T cells in vitro and in vivo. IL-33 promoted Th1 cell development via MyD88 and synergized with IL-12 to enhance St2 and IL-12R expression in CD4(+) T cells. These data therefore provide a novel mechanism for Th1 cell differentiation and optimal induction of a Type 1 response. Thus, IL-33 is capable of inducing IL-12-dependent Th1 cell differentiation in human and mouse CD4(+) T cells.

Anti-Toll-like receptor 2 and 4 antibodies suppress inflammatory response in mice.

Toll-like receptors (TLRs) 2 and 4 recognize different endogenous and exogenous agonists and play a distinct role in infection and inflammation. However, their ultimate effect in a given infectious and inflammatory disease is less understood. We produced polyclonal anti-murine TLR2 and TLR4 antibodies and investigated their effect on cutaneous leishmaniasis and inflammatory arthritis. Administration of these antibodies to susceptible BALB/c mice, infected in the footpad with Leishmania major, reduced footpad swelling but not the parasite load compared with mice treated with control IgG. The antibodies synergistically reduced leishmanial-specific T-cell proliferation, T helper type 1 and type 2 cytokine production, specific IgG1 and IgG2a antibody synthesis, and T-cell receptor and co-stimulatory molecule expression on dendritic cells in infected mice. We then tested the effect of these antibodies on collagen-induced arthritis (CIA) in DBA/1 mice, a classic model of chronic inflammation. Both antibodies markedly suppressed the development of clinical parameters with concomitant reduction of pro-inflammatory cytokine production. These data therefore suggest that anti-TLR2 and 4 antibodies may have a synergistic therapeutic effect on inflammatory disease in vivo.

Negative regulation by PD-L1 during drug-specific priming of IL-22-secreting T cells and the influence of PD-1 on effector T cell function.

Activation of PD-1 on T cells is thought to inhibit Ag-specific T cell priming and regulate T cell differentiation. Thus, we sought to measure the drug-specific activation of naive T cells after perturbation of PD-L1/2/PD-1 binding and investigate whether PD-1 signaling influences the differentiation of T cells. Priming of naive CD4(+) and CD8(+) T cells against drug Ags was found to be more effective when PD-L1 signaling was blocked. Upon restimulation, T cells proliferated more vigorously and secreted increased levels of IFN-γ, IL-13, and IL-22 but not IL-17. Naive T cells expressed low levels of PD-1; however, a transient increase in PD-1 expression was observed during drug-specific T cell priming. Next, drug-specific responses from in vitro primed T cell clones and clones from hypersensitive patients were measured and correlated with PD-1 expression. All clones were found to secrete IFN-γ, IL-5, and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL-22-secreting clones expressed the skin-homing receptors CCR4, CCR10, and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the Ag-specific proliferative response or the secretion of cytokines/cytolytic molecules. This study shows that PD-L1/PD-1 binding negatively regulates the priming of drug-specific T cells. ELISPOT analysis uncovered an Ag-specific FasL/IL-22-secreting T cell subset with skin-homing properties.