Exposure to isocyanates predicts atopic dermatitis prevalence and disrupts therapeutic pathways in commensal bacteria

Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin condition increasing in industrial nations at a pace that suggests environmental drivers. We hypothesize that the dysbiosis associated with AD may signal microbial adaptations to modern pollutants. Having previously modeled the benefits of health-associated Roseomonas mucosa, we now show that R. mucosa fixes nitrogen in the production of protective glycerolipids and their ceramide by-products. Screening EPA databases against the clinical visit rates identified diisocyanates as the strongest predictor of AD. Diisocyanates disrupted the production of beneficial lipids and therapeutic modeling for isolates of R. mucosa as well as commensal Staphylococcus. Last, while topical R. mucosa failed to meet commercial end points in a placebo-controlled trial, the subgroup who completed the full protocol demonstrated sustained, clinically modest, but statistically significant clinical improvements that differed by study site diisocyanate levels. Therefore, diisocyanates show temporospatial and epidemiological association with AD while also inducing eczematous dysbiosis.

Figures

Fig. 1.. R. mucosa from healthy controls shows differential carbon and nitrogen metabolism.

(A to J) Three different isolates of Roseomonas from healthy volunteers (RmHV1-3) and three different isolates of Roseomonas from patients with AD (RmAD1-3) were grown in the BioLog system, fold change (FC) for each challenge condition versus control was calculated, the log2FC for RmHV divided by the log2FC for RmAD is shown (A), and then the ratio of log2FC was superimposed onto the urea cycle (arginine biosynthesis) and citrate cycle (alanine, aspartate, and glutamate metabolism; H). (B and C) Individual BioLog components were ranked by significance and analyzed for pathways by MetaboAnalyst. (D) RmHV1-3 and RmAD1-3 were cultured with known amounts of LL-37 for 24 hours; supernatants were collected and assessed for remaining LL-37 by ELISA and the percentage of input LL-37 signal calculated and displayed. (E) Absorbance for cultures of RmHV1-3 or RmAD1-3 in medium with no energy source [minimal medium (MM)], with only carbohydrate energy sources (Nitrogen free), with only amino acid energy sources (Carb free), or with both (Replete). (F) Picture of experimental setup where anaerobic chambers were evacuated before filling with custom gas mixtures. (G) Total change in 13C to 12C ratio for all R. mucosa isolates after 48 hours in culture supplemented with 1% 13CO2. (H) MetaboAnalyst pathway analysis for RmHV1-3 and RmAD1-3 derived from metabolites with significant differences in 13C to 12C ratio after culture in 1% 13CO2 or metabolites differentially affected in RmHV by culture in ambient conditions or in CO2 deprivation (80% N2 and 20% O2). (I and J) Representative image (1.25×) of culture morphology (I) and statistical enumeration (J) of area covered per imaging field by culture of RmHV and RmAD in ambient or CO2 deprivation conditions. Data shown represent two or more independent experiments and are displayed as means ± SEM. For (C) and (H), only −log10P values of >1 were considered significant.

Fig. 2.. Roseomonas isolates differ in nitrogen fixation.

(A) Total change in 15N to 14N ratio for three isolates of Roseomonas from healthy volunteers (RmHV1-3), three isolates of Roseomonas from patients with AD (RmAD1-3), or one isolate of A. brasilense (A. bras) cultured with 15N2 in head space. (B) Flux analysis was performed on metabolites with increase in 15N ratios; metabolites with greatest 15N change were ranked by P value and analyzed by MetaboAnalyst for pathways in lipidomic and metabolic acquisition conditions. (C) Representative images (1.25×) from spiral plated culture on agar for bacteria cultured in ambient air (Amb) or in head space devoid of N2 [79% argon (Ar), 20% oxygen (O2), and 1% carbon dioxide (CO2)]. (D and E) R. mucosa metabolites most affected by N2 deprivation were assessed by MALDI–time-of-flight (TOF) MS and analyzed for pathways affected by MetaboAnalyst. (F) Total intensity values for annotated ceramide containing compounds assessed by MALDI. (G) Mice treated in the MC903 model of AD for 8 days of dermatitis induction, 3 days of treatment with either RmHV cultured in ambient conditions of N2 deprivation (−N2), and then 4 days of observation; change in mouse ear thickness between days 8 and 15 is shown. Reduced thickness indicates reduced swelling and improved outcomes. (H) MALDI-MSI performed on mouse ears; segmentation performed by SCiLS Lab. (I) Pathway analysis derived from metabolites that best distinguish mice ears treated with RmHV1 from ambient conditions versus treatment with RmHV1 cultured in N2 deprivation. (J and K) Distribution of indicated metabolites as indicated by MALDI-MSI. GPL, glycerophospholipids; GPI, glycerophosphatidylinositol; deg., degradation; αLa, α-linolenic acid. Data shown represent two (G to K) or three or more (A to F) independent experiments. *P < 0.05; ns, not significant as determined by ANOVA.

Fig. 3.. Diisocyanates associated with AD diagnoses.

(A) Summary of published environmental factors influencing AD (, , –32, 41, 42). Migration from developing to United States or European Union nations (US/EU); PM, particulate matter; ETS, environmental tobacco smoke; OC, organic carbon; OR, odds ratio; CI, confidence interval. (B) RmHV was cultured in sealed chamber with trace CO, CO + PM, SO2, or gasoline in agar dish lid and compared to pathways modified by N2 deprivation; green indicates that the MetaboAnalyst identified pathway was altered by chemical challenge; dark green indicates significant alteration; black indicates that no growth was evident. (C) Raw values and diagnosis rate for AD by zip code. (D) Cluster analysis for relative risk of having above-average AD diagnosis rate. Regression was performed for 1500+ pollution variables from the EPA TRI, AD diagnosis rates, obesity, deprivation index, and access to pediatricians, allergists, or dermatologist. (E to G) Linear regression using lasso with 1 SE limitation (E), random forest (F), or partial dependence plots (G) for total release values from 2014 to 2019 versus 2019 visit rates. (H) Random forest as in (F) but restricting to only billings by pediatricians. (I) Lasso linear regression with 1 SE for total 2019 pediatric visit rates versus 2014–2019 release values from the RSEI EPA database. (J) Aggregate AD-associated pollutant scores were summated by zip code for each pollutant multiplied by its β correlation value; mean values for each SD section were indicated.

Fig. 4.. TDI disrupts beneficial pathways in R. mucosa.

(A) Total U.S. production of TDI and MDI since 1940. (B) The 2019 TDI production for nations with available data in linear regression with 95% confidence intervals against reported rates of AD in children under 14 years of age. (C) Detected levels of diisocyanates (DIC) including TDI released during in vitro experiments compared to reported factory release. (D) Representative image (1.25×) of RmHV1-3 cultured with TDI in ambient or N2 deprivation conditions. (E) TDI was measured at serial dilutions so that values at higher dilutions below the lower limit of detection (LLD) could be extrapolated. Percent change in growth and total intensity of annotated ceramides by MALDI at 48 hours are shown. (F) Change in total intensity for annotated ceramides as compared to RmHV1-3 cultured in ambient conditions or RmAD1-3 in ambient conditions (AD) or RmHV under N2 deprivation (−N2), or with TDI, toluene (TOL), or HNCO. (G) MetaboAnalyst indicated affected pathways in RmHV1-3 by culture with TDI or toluene. (H) Lower explosive limit (LEL) detection for RmHV1-3 with and without TDI exposure in ambient or N2 deprivation. (I) Mice treated in the MC903 model of AD before treatment with either diluent or RmHV1 grown with toluene or with TDI; change in mouse ear thickness is shown. Data shown are representative of three or more independent experiments and shown as means ± SEM. *P < 0.05, **P < 0.01 as determined by ANOVA.

Fig. 5.. HNCO disrupts beneficial pathways in R. mucosa.

(A) Representative image of RmHV1 cultured with 1 μg of HNCO placed into the lid of the agar dish and cultured for 48 hours in ambient or N2 deprivation conditions. (B) Lower explosive limit (LEL) detection by gas monitoring for RmHV with and without HNCO exposure in ambient or N2 deprivation culture conditions. (C) Change in mouse ear thickness during MC903 model of AD for mice treated with RmHV1 in ambient conditions or exposed to HNCO. (D) MetaboAnalyst pathway analysis for metabolites affected in RmHV1-3 or RmAD1-3 cultured with HNCO in the lid of agar dish. (E) Representative image of RmHV1-3 cultured with diisocyanate-containing glue or corn starch–based diisocyanate-negative glue placed into the lid of the agar dish and cultured for 48 hours. (F) Representative image of RmHV1-3 cultured with diisocyanate-containing glue placed into the lid of the agar dish and cultured for 48 hours in ambient or N2 deprivation conditions. (G and H) MetaboAnalyst pathway analysis for metabolites affected in RmHV1-3 cultured with diisocyanate-containing glue (G) or polyurethane-based glue (H) in the lid of agar dish. (I) Representative image of RmHV1-3 and RmAD1-3 cultured with polyurethane-based glue placed into the lid of the agar dish and cultured for 48 hours. (J) Summary table of RmHV1-3 pathways affected by N2 deprivation compared to pathways affected by indicated challenges along with fold change of total annotated ceramide containing metabolites; green indicates pathway affected per MetaboAnalyst, while dark green indicates significant impact and gray indicates pathway not affected. All images in (A), (E), (F), and (I) were taken at ×1.25 magnification. Data shown are representative of three or more independent experiments and shown as means ± SEM. *P < 0.05 and **P < 0.01 as determined by ANOVA.

Fig. 6.. Clinical results from R. mucosa treatment were influenced by TDI exposure.

Patients were treated with topical R. mucosa (FB-401) or placebo (sucrose in water) thrice weekly for 16 weeks before a 4-week washout. Two weeks before treatment, topical corticosteroids (TCS) were stopped and a minimally dysbiotic moisturizer regiment was initiated. (A) Example images from participants. (B) ITT analysis for those achieving an IGA of 0 and 1 (C) and percent change in EASI during active treatment. (D) For those completing the washout phase, percent change between week 16 and week 20 as a percentage of enrollment values is shown for EASI, BSA, and pruritis NRS (itch); each dot represents one participant. (E to L) For those completing the entire 20-week trial without topical steroids, raw EASI change (E), the proportion achieving 90% improvement in EASI (EASI90; F), and EASI75 (G). (H to L) Post hoc stratification of the completer population results by study sites’ historical TDI exposure as above the mean (>avg) or below for IGA0-1 (H), raw EASI (I), EASI90 (J), EASI75 (K), and pruritus (L); significance indicated for FB-401 in above or below average TDI zip codes against matched control unless indicated. Data are shown as proportion (B, F to H, J, and K) and means + SD (C, D, I, and L). ns, not significant, +P < 0.1; *P < 0.05; **P < 0.01 as determined by ANOVA (C and D), rANOVA (E, I, and L), or chi-square (B, F to H, J, and K).

Fig. 7.. S. cohnii improves outcomes in mouse models of AD via steroid metabolism.

(A) Mice treated in the MC903 model of AD before treatment with either diluent or S. cohnii. (B) Segmented MALDI-MSI performed on mouse ears. (C) MetaboAnalyst pathways affected on mouse ears treated with S. cohnii. (D) Summary table of MetaboAnalyst pathways differentiating isolates of Staphylococcus spp. from healthy volunteers (Staph HV) versus isolates of S. aureus from patients with AD (Staph AD); green indicates that the pathway was affected, dark green indicates significant impact, and gray indicates not affected.

Fig. 8.. TDI and HNCO disrupt beneficial pathways in S. cohnii.

(A) Percent change in growth area for RmHV1-3, RmAD1-3, Staph HV1-3, and Staph AD1-3 as quantified from images shown in Figs. 1I, 2C, 4D, 5 (A, E, F, and I), and 8 (B to D and G). Significance is indicated versus Staph AD group. Impacts of NOx on Staph taken from (62). (B to D) Representative image of Staph HV1-3 and Staph AD1-3 cultured with TDI (B), HNCO (C), or polyurethane-containing glue (D). (E) Change in mouse ear thickness in MC903 mouse model after treatment with S. cohnii grown under ambient conditions or with exposure to TDI or HNCO. (F) Intensity levels of LysPG identified by mass/charge ratio and collisional cross section for the three isolates of StaphHV and three isolates from StaphAD. (G and H) Representative images (G) and non-metric multi-dimensional scaling (NMDS) plots (H) for S. cohnii and RmHV1 exposed to TDI with and without pretreatment with lysine (Lys) supplementation. (I) Change in total annotated ceramides with exposure to TDI versus ambient for RmHV with and without Lys supplementation pretreatment. All images were taken at ×1.25 magnification. Data are representative of two or more independent experiments and are displayed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by ANOVA.