Gene-metabolite expression in blood can discriminate allergen-induced isolated early from dual asthmatic responses

Amrit Singh, Masatsugu Yamamoto, Sarah H Y Kam, Jian Ruan, Gail M Gauvreau, Paul M O'Byrne, J Mark FitzGerald, Robert Schellenberg, Louis-Philippe Boulet, Gabriella Wojewodka, Cynthia Kanagaratham, Juan B De Sanctis, Danuta Radzioch, Scott J Tebbutt, Amrit Singh, Masatsugu Yamamoto, Sarah H Y Kam, Jian Ruan, Gail M Gauvreau, Paul M O'Byrne, J Mark FitzGerald, Robert Schellenberg, Louis-Philippe Boulet, Gabriella Wojewodka, Cynthia Kanagaratham, Juan B De Sanctis, Danuta Radzioch, Scott J Tebbutt

Abstract

Some asthmatic individuals undergoing allergen inhalation challenge develop an isolated early response whereas others develop a dual response (early plus late response). In the present study we have used transcriptomics (microarrays) and metabolomics (mass spectrometry) of peripheral blood to identify molecular patterns that can discriminate allergen-induced isolated early from dual asthmatic responses. Peripheral blood was obtained prior to (pre-) and 2 hours post allergen inhalation challenge from 33 study participants. In an initial cohort of 14 participants, complete blood counts indicated significant differences in neutrophil and lymphocyte counts at pre-challenge between early and dual responders. At post-challenge, significant genes (ALOX15, FADS2 and LPCAT2) and metabolites (lysolipids) were enriched in lipid metabolism pathways. Enzymes encoding for these genes are involved in membrane biogenesis and metabolism of fatty acids into pro-inflammatory and anti-inflammatory mediators. Correlation analysis indicated a strong negative correlation between ALOX15, FADS2, and IL5RA expression with 2-arachidonoylglycerophosphocholine levels in dual responders. However, measuring arachidonic acid and docosahexaenoic acid levels in a validation cohort of 19 participants indicated that the free form of DHA (nmoles/µg of protein) was significantly (p = 0.03) different between early and dual responders after allergen challenge. Collectively these results may suggest an imbalance in lipid metabolism which dictates pro- (anti-) inflammatory and pro-resolving mechanisms. Future studies with larger sample sizes may reveal novel mechanisms and therapeutic targets of the late phase asthmatic response.

Conflict of interest statement

Competing Interests: The authors have the following interests. This study was funded by AllerGen NCE Inc. Metabolon Incorporated (Durham, North Carolina, United States) performed the metabolite profiling and data processing for this study. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Lung function during allergen inhalation…
Figure 1. Lung function during allergen inhalation challenge.
Forced expiratory volume in 1 second (FEV1) measurements at 0.2 h, 0.3 h, 0.5 h, 0.8 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h and 7 h for eight early and six dual responders. FEV1 measurements are statistically different (*p<0.05) between early and dual responders at each of the time points between three and seven hours inclusive. The p-value for each time point comparing ERs and DRs was computed using a robust linear model (see Methods).
Figure 2. Complete blood counts.
Figure 2. Complete blood counts.
Relative frequencies of various leukocytes obtained at pre and post-challenge. The relative neutrophil counts were significantly (p = 0.01) elevated in ERs compared to DRs, whereas the relative lymphocyte counts were significantly reduced (p = 0.02) in ERs compared to DRs at pre-challenge. The p-value was computed using a robust linear model (see Methods); * denotes p

Figure 3. Gene network.

Ingenuity Pathway Analysis…

Figure 3. Gene network.

Ingenuity Pathway Analysis network of differentially expressed genes at post-challenge comparing…

Figure 3. Gene network.
Ingenuity Pathway Analysis network of differentially expressed genes at post-challenge comparing ERs and DRs. Dash lines indicate indirect relationships, whereas solid lines indicate direct relationships. The red colour indicates significant genes at an FDR of 10%. The grey colour indicates non-significant genes at an FDR of 10%.

Figure 4. Network plots highlighting the correlation…

Figure 4. Network plots highlighting the correlation between ΔG and ΔM for early and dual…

Figure 4. Network plots highlighting the correlation between ΔG and ΔM for early and dual responders.
A. Gene-metabolite clusters for early responders (ΔG25,8 and ΔM11,8). B. Gene-metabolite clusters for dual responders (ΔG25,6 and ΔM11,6). A correlation coefficient cut-off of 0.5 is applied to both networks.

Figure 5. Levels of free docosahexaenoic acid…

Figure 5. Levels of free docosahexaenoic acid in the plasma of early and dual responders…

Figure 5. Levels of free docosahexaenoic acid in the plasma of early and dual responders undergoing allergen inhalation challenge.
Levels of DHA (left) decrease in early responders from pre to post-challenge whereas no consistent change is observed in dual responders following allergen challenge. At post-challenge (scaled to pre-challenge levels), DHA was differentially expressed (p = 0.033) between ERs and DRs (right). The p-value was computed using a robust linear model (see Methods).
Figure 3. Gene network.
Figure 3. Gene network.
Ingenuity Pathway Analysis network of differentially expressed genes at post-challenge comparing ERs and DRs. Dash lines indicate indirect relationships, whereas solid lines indicate direct relationships. The red colour indicates significant genes at an FDR of 10%. The grey colour indicates non-significant genes at an FDR of 10%.
Figure 4. Network plots highlighting the correlation…
Figure 4. Network plots highlighting the correlation between ΔG and ΔM for early and dual responders.
A. Gene-metabolite clusters for early responders (ΔG25,8 and ΔM11,8). B. Gene-metabolite clusters for dual responders (ΔG25,6 and ΔM11,6). A correlation coefficient cut-off of 0.5 is applied to both networks.
Figure 5. Levels of free docosahexaenoic acid…
Figure 5. Levels of free docosahexaenoic acid in the plasma of early and dual responders undergoing allergen inhalation challenge.
Levels of DHA (left) decrease in early responders from pre to post-challenge whereas no consistent change is observed in dual responders following allergen challenge. At post-challenge (scaled to pre-challenge levels), DHA was differentially expressed (p = 0.033) between ERs and DRs (right). The p-value was computed using a robust linear model (see Methods).

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