New mechanisms in diisocyanate-mediated allergy/toxicity: are microRNAs in play?

PURPOSE OF REVIEW: To describe recent findings of diisocyanate-mediated mechanisms in allergy and toxicology by addressing the role of microRNA (miR) in immune responses that may contribute to the development of occupational asthma (OA). RECENT FINDINGS: Studies of diisocyanate asthma have traditionally focused on the immune and inflammatory patterns associated with diisocyanate exposures; however, recognized knowledge gaps exist regarding the detailed molecular mechanism(s) of pathogenesis. Recent studies demonstrate the critical role endogenous microRNAs play as gene regulators in maintaining homeostasis of the human body, and in the pathophysiology of many diseases including asthma. Given that diisocyanate-OA shares many pathophysiological characteristics with asthma, it is likely that miR-mediated mechanisms are involved in the pathophysiology of diisocyanate-OA. Recent reports have shown that changes in expression of endogenous miRs are associated with exposure to the occupationally relevant diisocyanates, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). Continued mechanistic study of these relevant miRs may lead to the development of novel biomarkers of occupational exposure and/or provide efficacious targets for therapeutic strategies in diisocyanate asthma. SUMMARY: The molecular mechanisms underlying diisocyanate-OA pathophysiology are heterogeneous and complicated. In this review, we highlight recent research into the roles and potential regulation of miRs in diisocyanate-OA.

Circular RNA hsa_circ_0008726 Targets the hsa-miR-206-3p/KLF4 Axis to Modulate 4,4'-Methylene Diphenyl Diisocyanate-Glutathione Conjugate-Induced Chemokine Transcription in Macrophages.

Exposure to 4,4'-methylene diphenyl diisocyanate (MDI) in the workplace may lead to the development of occupational asthma (OA). However, the specific mechanism(s) by which MDI induces OA are poorly understood. Previous reports have demonstrated that MDI and MDI-glutathione (GSH) conjugate exposure downregulates endogenous human/murine (hsa/mmu)-microRNA(miR)-206-3p, resulting in the activation of mmu/hsa-miR-206-3p-regulated signaling pathways in macrophages. Circular RNAs (circRNAs) regulate many important biological processes by targeting endogenous miRs; however, whether MDI/MDI-GSH exposure may influence circRNA expressions is unknown. Several circRNAs have been identified that regulate hsa-miR-206-3p. We hypothesize that MDI-GSH conjugate exposure induces endogenous circRNA(s) to regulate hsa-miR-206-3p in macrophages. The expression of candidate hsa-miR-206-3p-binding circRNAs was determined from MDI-GSH conjugate-treated differentiated THP-1 macrophages using RT-qPCR. MDI-GSH exposures induced hsa_circ_0008726 and its host gene transcript DNAJB6, whereas other circRNA(s) examined were either not detected or unchanged. RNA-induced silencing complex-immunoprecipitation (RISC-IP) experiments confirm that hsa-miR-206-3p can bind to hsa_circ_0008726. The expressions of endogenous hsa-miR-206-3p, hsa-miR-206-3p-regulated KLF4, and KLF4-activated M2 macrophage-associated markers and chemokines were up-/down-regulated by transfection of hsa_circ_0008726 siRNAs or hsa_circ_0008726 overexpression plasmid in macrophages, respectively. These results suggest MDI-GSH exposure downregulates hsa-miR-206-3p via induction of endogenous hsa_circ_0008726/DNAJB6, resulting in the upregulation of hsa-miR-206-3p-mediated regulations in macrophages.

Human keratinocyte response to 4,4'-methylene diphenyl diisocyanate-glutathione conjugate exposure.

Workplace exposure to diisocyanates like 4,4'-methylene diphenyl diisocyanate can cause occupational asthma (MDI-OA), and the underlying biological pathways are still being researched.Although uncertainty remains, evidence supports the hypothesis that dermal exposure to MDI plays an important role in the development of MDI-OA.Gene expression, proteomics, and informatics tools were utilised to characterise changes in expression of RNA and protein in cultured human HEKa keratinocyte cells following exposure to conjugates of MDI with glutathione (MDI-GSH).RT-qPCR analysis using a panel of 39 candidate primers demonstrated 9 candidate genes upregulated and 30 unchanged.HPLC-MS/MS analysis of HEKa cell lysate identified 18 540 proteins across all samples 60 proteins demonstrate statistically significant differential expression in exposed cells, some of which suggest activation of immune and inflammatory pathways.The results support the hypothesis that dermal exposures have the potential to play an important role in the development of MDI-OA. Furthermore, proteomic and gene expression data suggest multiple immune (adaptive and innate) and inflammatory pathways may be involved in the development of MDI-OA.

MicroRNA-mediated Krüppel-like factor 4 upregulation induces alternatively activated macrophage-associated marker and chemokine transcription in 4,4'-methylene diphenyl diisocyanate exposed macrophages.

1. Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI) is associated with occupational asthma (OA) development. Alveolar macrophage-induced recruitment of immune cells to the lung microenvironment plays an important role during asthma pathogenesis. Previous studies identified that MDI/MDI-glutathione (GSH)-exposure downregulates endogenous hsa-miR-206-3p/hsa-miR-381-3p. Our prior report shows that alternatively activated (M2) macrophage-associated markers/chemokines are induced by MDI/MDI-GSH-mediated Krüppel-Like Factor 4 (KLF4) upregulation in macrophages and stimulates immune cell chemotaxis. However, the underlying molecular mechanism(s) by which MDI/MDI-GSH upregulates KLF4 remain unclear.2. Following MDI-GSH exposure, microRNA(miR)-inhibitors/mimics or plasmid transfection, endogenous hsa-miR-206-3p/hsa-miR-381-3p, KLF4, or M2 macrophage-associated markers (CD206, TGM2), and chemokines (CCL17, CCL22, CCL24) were measured by either RT-qPCR, western blot, or luciferase assay.3. MDI-GSH exposure downregulates hsa-miR-206-3p/hsa-miR-381-3p by 1.46- to 9.75-fold whereas upregulates KLF4 by 1.68- to 1.99-fold, respectively. In silico analysis predicts binding between hsa-miR-206-3p/hsa-miR-381-3p and KLF4. Gain- and loss-of-function, luciferase reporter assays and RNA-induced silencing complex-immunoprecipitation (RISC-IP) studies confirm the posttranscriptional regulatory roles of hsa-miR-206-3p/hsa-miR-381-3p and KLF4 in macrophages. Furthermore, hsa-miR-206-3p/hsa-miR-381-3p regulate the expression of M2 macrophage-associated markers and chemokines via KLF4.4. In conclusion, hsa-miR-206-3p/hsa-miR-381-3p play a major role in regulation of MDI/MDI-GSH-induced M2 macrophage-associated markers and chemokines by targeting the KLF4 transcript, and KLF4-mediated regulation in macrophages.

4,4'-Methylene diphenyl diisocyanate exposure induces expression of alternatively activated macrophage-associated markers and chemokines partially through Krüppel-like factor 4 mediated signaling in macrophages.

Occupational exposure to the most widely used monomeric diisocyanate (dNCO), 4,4'-methylene diphenyl diisocyanate (MDI), may lead to the development of occupational asthma (OA). Alveolar macrophages with alternatively activated (M2) phenotype have been implicated in allergic airway responses and the pathogenesis of asthma. Recent in vivo studies demonstrate that M2 macrophage-associated markers and chemokines are induced by MDI-exposure, however, the underlying molecular mechanism(s) by which this proceeds is unclear.Following MDI exposure (in vivo and in vitro) M2 macrophage-associated transcription factors (TFs), markers, and chemokines were determined by RT-qPCR, western blots, and ELISA.Expression of M2 macrophage-associated TFs and markers including Klf4/KLF4, Cd206/CD206, Tgm2/TGM2, Ccl17/CCL17, Ccl22/CCL22, and CCL24 were induced by MDI/MDI-GSH exposure in bronchoalveolar lavage cells (BALCs)/THP-1 macrophages. The expression of CD206, TGM2, CCL17, CCL22, and CCL24 are upregulated by 3.83-, 7.69-, 6.22-, 6.08-, and 1.90-fold in KLF4-overexpressed macrophages, respectively. Endogenous CD206 and TGM2 were downregulated by 1.65-5.17-fold, and 1.15-1.78-fold, whereas CCL17, CCL22, and CCL24 remain unchanged in KLF4-knockdown macrophages. Finally, MDI-glutathione (GSH) conjugate-treated macrophages show increased chemotactic ability to T-cells and eosinophils, which may be attenuated by KLF4 knockdown.Our data suggest that MDI exposure may induce M2 macrophage-associated markers partially through induction of KLF4.

MicroRNA-mediated calcineurin signaling activation induces CCL2, CCL3, CCL5, IL8, and chemotactic activities in 4,4'-methylene diphenyl diisocyanate exposed macrophages.

Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI), the most widely used monomeric diisocyanate, is one of the leading causes of occupational asthma (OA). Previously, we identified microRNA (miR)-206-3p/miR-381-3p-mediated PPP3CA/calcineurin signalling regulated iNOS transcription in macrophages and bronchoalveolar lavage cells (BALCs) after acute MDI exposure; however, whether PPP3CA/calcineurin signalling participates in regulation of other asthma-associated mediators secreted by macrophages/BALCs after MDI exposure is unknown.Several asthma-associated, macrophage-secreted mediator mRNAs from MDI exposed murine BALCs and MDI-glutathione (GSH) conjugate treated differentiated THP-1 macrophages were analysed using RT-qPCR.Endogenous IL1B, TNF, CCL2, CCL3, CCL5, and TGFB1 were upregulated in MDI or MDI-GSH conjugate exposed BALCs and macrophages, respectively. Calcineurin inhibitor tacrolimus (FK506) attenuated the MDI-GSH conjugate-mediated induction of CCL2, CCL3, CCL5, and CXCL8/IL8 but not others. Transfection of either miR-inhibitor-206-3p or miR-inhibitor-381-3p in macrophages induced chemokine CCL2, CCL3, CCL5, and CXCL8 transcription, whereas FK506 attenuated the miR-inhibitor-206-3p or miR-inhibitor-381-3p-mediated effects. Finally, MDI-GSH conjugate treated macrophages showed increased chemotactic ability to various immune cells, which may be attenuated by FK506.In conclusion, these results indicate that MDI exposure to macrophages/BALCs may recruit immune cells into the airway via induction of chemokines by miR-206-3p and miR-381-3p-mediated calcineurin signalling activation.

A comparison of performance metrics for cloth face masks as source control devices for simulated cough and exhalation aerosols.

Universal mask wearing is recommended by the Centers for Disease Control and Prevention to help control the spread of COVID-19. Masks reduce the expulsion of respiratory aerosols (called source control) and offer some protection to the wearer. However, masks vary greatly in their designs and construction materials, and it is not clear which are most effective. Our study tested 15 reusable cloth masks (which included face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators as source control devices for aerosols ≤ 7 µm produced during simulated coughing and exhalation. These measurements were compared with the mask filtration efficiencies, airflow resistances, and fit factors. The source control collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. The filtration efficiencies of the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on an elastomeric manikin headform and 1.0 to 4.0 on human test subjects. The correlation coefficients between the source control efficacies and the other performance metrics ranged from 0.31 to 0.66 and were significant in all but one case. However, none of the alternative metrics were strong predictors of the source control performance of cloth masks. Our results suggest that a better understanding of the relationships between source control performance and metrics like filtration efficiency, airflow resistance, and fit factor are needed to develop simple methods to estimate the effectiveness of masks as source control devices for respiratory aerosols.

A comparison of performance metrics for cloth masks as source control devices for simulated cough and exhalation aerosols.

Universal mask wearing is recommended to help control the spread of COVID-19. Masks reduce the expulsion of aerosols of respiratory fluids into the environment (called source control) and offer some protection to the wearer. Masks are often characterized using filtration efficiency, airflow resistance, and manikin or human fit factors, which are standard metrics used for personal protective devices. However, none of these metrics are direct measurements of how effectively a mask blocks coughed and exhaled aerosols. We studied the source control performance of 15 cloth masks (face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators by measuring their ability to block aerosols ≤ 7 µm expelled during simulated coughing and exhalation (called source control collection efficiency). These measurements were compared with filtration efficiencies, airflow resistances, and fit factors measured on manikin headforms and humans. Collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. Filtration efficiencies for the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on headforms and 1.0 to 4.0 on human subjects. The Spearman's rank correlation coefficients between the source control collection efficiencies and the standard metrics ranged from 0.03 to 0.68 and were significant in all but two cases. However, none of the standard metrics were strongly correlated with source control performance. A better understanding of the relationships between source control collection efficiency, filtration efficiency, airflow resistance, and fit factor is needed.

Acute 4,4'-Methylene Diphenyl Diisocyanate Exposure-Mediated Downregulation of miR-206-3p and miR-381-3p Activates Inducible Nitric Oxide Synthase Transcription by Targeting Calcineurin/NFAT Signaling in Macrophages.

Exposure to 4,4'-methylene diphenyl diisocyanate (MDI) in the occupational setting may lead to development of occupational asthma (OA), and the underlying molecular mechanisms of MDI-induced disease pathogenesis remain an active area of research. Using a nose-only mouse inhalation model, we find that circulating microRNA (miR)-206-3p and miR-381-3p are downregulated after MDI exposure; however, cellular miR-206-3p and miR-381-3p responses after MDI aerosol exposure and their pathophysiological roles in MDI-OA are unknown. We hypothesize that miR-206-3p and miR-381-3p-regulated mechanisms cause increased expression of the inducible nitric oxide synthase (iNOS) after MDI aerosol exposure. We examined cellular miR-206-3p and miR-381-3p, calcineurins, nuclear factors of activated T cells (NFATs), and iNOS levels from both nose-only exposed murine bronchoalveolar lavage cells (BALCs) and differentiated THP-1 macrophages treated with MDI-glutathione (GSH) conjugates. Both in vivo murine MDI aerosol exposure and in vitro MDI-GSH exposures in THP-1 macrophages result in downregulation of endogenous miR-206-3p and miR-381-3p and upregulation of PPP3CA and iNOS expression. Transfection of THP-1 macrophages with miR-inhibitor-206-3p and miR-inhibitor-381-3p resulted in the upregulation of PPP3CA and iNOS. Using RNA-induced silencing complex immunoprecipitation and translational reporter assays, we verified that PPP3CA, but not iNOS, is directly targeted by both miR-206-3p and miR-381-3p. Downregulation of miR-206-3p and miR-381-3p following by MDI exposure induces calcineurin/NFAT signaling-mediated iNOS transcription in macrophages and BALCs.

Circulating miRs-183-5p, -206-3p and -381-3p may serve as novel biomarkers for 4,4'-methylene diphenyl diisocyanate exposure.

BACKGROUND: Occupational exposure to the most widely used diisocyanate, 4,4'-methylene diphenyl diisocyanate (MDI), is a cause of occupational asthma (OA). Early recognition of MDI exposure and sensitization is essential for the prevention of MDI-OA. OBJECTIVE: Identify circulating microRNAs (miRs) as novel biomarkers for early detection of MDI exposure and prevention of MDI-OA. MATERIALS AND METHODS: Female BALB/c mice were exposed to one of three exposure regimens: dermal exposure to 1% MDI in acetone; nose-only exposure to 4580 ± 1497 µg/m3 MDI-aerosol for 60 minutes; or MDI dermal exposure/sensitization followed by MDI-aerosol inhalation challenge. Blood was collected and miRCURY™ miRs qPCR Profiling Service was used to profile circulate miRs from dermally exposed mice. Candidate miRs were identified and verified from mice exposed to three MDI-exposure regimens by TaqMan® miR assays. RESULTS: Up/down-regulation patterns of circulating mmu-miRs-183-5p, -206-3p and -381-3p were identified and verified. Circulating mmu-miR-183-5p was upregulated whereas mmu-miRs-206-3p and -381-3p were downregulated in mice exposed via all three MDI exposure regimens. DISCUSSION AND CONCLUSION: Upregulation of circulating miR-183-5p along with downregulation of circulating miRs-206-3p and -381-3p may serve as putative biomarkers of MDI exposure and may be considered as potential candidates for validation in exposed human worker populations.

Mass spectrometry-based analysis of murine bronchoalveolar lavage fluid following respiratory exposure to 4,4'-methylene diphenyl diisocyanate aerosol.

1. Diisocyanates are highly reactive electrophiles utilized in the manufacture of a wide range of polyurethane products and have been identified as causative agents of occupational allergic respiratory disease. However, in spite of the significant occupational health burden associated with diisocyanate-induced asthma, the mechanism of disease pathogenesis remains largely unknown. 2. To better understand the fate of inhaled diisocyanates, a nose-only aerosol exposure system was constructed and utilized to expose a BALB/c mouse model to an aerosol generated from 4,4'-methylene diphenyl diisocyanate (MDI). Tissue and bronchoalveolar lavage samples were evaluated 4 and 24 h post-exposure for evidence of diisocyanate-protein haptenation, and a label-free quantitative proteomics strategy was employed to evaluate relative changes to the protein content of the cellular fraction of the lavage fluid. 3. Following MDI aerosol exposure, expression of the number of proteins with immunological or xenobiotic metabolism relevance is increased, including endoplasmin, cytochrome P450 and argininosuccinate synthase. Western blot analysis indicated MDI-conjugated protein in the lavage fluid, which was identified as serum albumin. 4. Tandem mass spectrometry analysis of MDI-albumin revealed MDI conjugation occurs at a dilysine motif at Lys525, as well as at a glutamine-lysine motif at Lys414, in good agreement with previously published in vitro data on diisocyanate-conjugated serum albumin.

Topical application of the anti-microbial chemical triclosan induces immunomodulatory responses through the S100A8/A9-TLR4 pathway.

The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfα, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (TH2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy.

Spirochaete flagella hook proteins self-catalyse a lysinoalanine covalent crosslink for motility.

Spirochaetes are bacteria responsible for several serious diseases, including Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum) and leptospirosis (Leptospira interrogans), and contribute to periodontal diseases (Treponema denticola)(1). These spirochaetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochaete flagella (periplasmic flagella) reside and rotate within the periplasmic space(2-11). The universal joint or hook that links the rotary motor to the filament is composed of ∼120-130 FlgE proteins, which in spirochaetes form an unusually stable, high-molecular-weight complex(9,12-17). In other bacteria, the hook can be readily dissociated by treatments such as heat(18). In contrast, spirochaete hooks are resistant to these treatments, and several lines of evidence indicate that the high-molecular-weight complex is the consequence of covalent crosslinking(12,13,17). Here, we show that T. denticola FlgE self-catalyses an interpeptide crosslinking reaction between conserved lysine and cysteine, resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine crosslinks are not needed for flagellar assembly, but they are required for cell motility and hence infection. The self-catalytic nature of FlgE crosslinking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochaetes.

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition.

Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences.

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin.

Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.

Production of a Chaetomium globosum enolase monoclonal antibody.

Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species.

A murine inhalation model to characterize pulmonary exposure to dry Aspergillus fumigatus conidia.

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.

Toluene diisocyanate (TDI) disposition and co-localization of immune cells in hair follicles.

Diisocyanates (dNCOs) are potent chemical allergens utilized in various industries. It has been proposed that skin exposure to dNCOs produces immune sensitization leading to work-related asthma and allergic disease. We examined dNCOs sensitization by using a dermal murine model of toluene diisocyanate (TDI) exposure to characterize the disposition of TDI in the skin, identify the predominant haptenated proteins, and discern the associated antigen uptake by dendritic cells. Ears of BALB/c mice were dosed once with TDI (0.1% or 4% v/v acetone). Ears and draining lymph nodes (DLNs) were excised at selected time points between 1 h and 15 days post-exposure and were processed for histological, immunohistochemical, and proteomic analyses. Monoclonal antibodies specific for TDI-haptenated protein (TDI-hp) and antibodies to various cell markers were utilized with confocal microscopy to determine co-localization patterns. Histopathological changes were observed following exposure in ear tissue of mice dosed with 4% TDI/acetone. Immunohistochemical staining demonstrated TDI-hp localization in the stratum corneum, hair follicles, and sebaceous glands. TDI-hp were co-localized with CD11b(+) (integrin αM/Mac-1), CD207(+) (langerin), and CD103(+) (integrin αE) cells in the hair follicles and in sebaceous glands. TDI-hp were also identified in the DLN 1 h post-exposure. Cytoskeletal and cuticular keratins along with mouse serum albumin were identified as major haptenated species in the skin. The results of this study demonstrate that the stratum corneum, hair follicles, and associated sebaceous glands in mice are dendritic cell accessible reservoirs for TDI-hp and thus identify a mechanism for immune recognition following epicutaneous exposure to TDI.

A murine monoclonal antibody with broad specificity for occupationally relevant diisocyanates.

Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 µg of 4,4'-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4'-MDI- human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4'-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4'-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers.

Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures.

Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8(+) IL-17(+) (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia.