Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality

Abstract

The circadian clock is important for cellular and organ function. However, its function in sickle cell disease (SCD), a life-threatening hemolytic disorder, remains unknown. Here, we performed an unbiased microarray screen, which revealed significantly altered expression of circadian rhythmic genes, inflammatory response genes, and iron metabolic genes in SCD Berkeley transgenic mouse lungs compared with controls. Given the vital role of period 2 (Per2) in the core clock and the unrecognized role of Per2 in SCD, we transplanted the bone marrow (BM) of SCD mice to Per2^Luciferase mice, which revealed that Per2 expression was up-regulated in SCD mouse lung. Next, we transplanted the BM of SCD mice to period 1 (Per1)/Per2 double deficient [Per1/Per2 double knockout (dKO)] and wild-type mice, respectively. We discovered that Per1/Per2 dKO mice transplanted with SCD BM (SCD → Per1/Per2 dKO) displayed severe irradiation sensitivity and were more susceptible to an early death. Although we observed an increase of peripheral inflammatory cells, we did not detect differences in erythrocyte sickling. However, there was further lung damage due to elevated pulmonary congestion, inflammatory cell infiltration, iron overload, and secretion of IL-6 in lavage fluid. Overall, we demonstrate that Per1/Per2 is beneficial to counteract elevated systemic inflammation, lung tissue inflammation, and iron overload in SCD.-Adebiyi, M. G., Zhao, Z., Ye, Y., Manalo, J., Hong, Y., Lee, C. C., Xian, W., McKeon, F., Culp-Hill, R., D' Alessandro, A., Kellems, R. E., Yoo, S.-H., Han, L., Xia, Y. Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality.

Keywords: circadian clock; heme-iron metabolism; lung.

Figure 1

Unbiased microarray screen identifies circadian clock genes, iron-hemostatic genes, and inflammatory genes in SCD lung. Total RNA was extracted from SCD and WT lung for microarray gene expression analysis. A) Pathway analysis demonstrating up-regulated gene expression. B) Heat map shows 3 distinct categories of genes identified in our analysis. Expression analysis using log₂ transformations was used. Bar plots were generated to show relative fold change of gene expression in SCD lung normalized to WT. C, D) Semiquantitative RT-PCR was performed to detect circadian genes at environmental light and dark conditions, which correspond to 12 h of light and 12 h of dark, also known as ZT. Lung tissue was collected at ZT 1, 7, 13, 19, and 26 to detect Arntl1 (C) and Per2 (D) gene expression. Values are represented as means ± sem; n = 2–-3 samples/group. *P < 0.05, fold change represented as SCD normalized to WT.

Figure 2

Pulmonary Per2 rhythmic amplitude is further induced in SCD. A) Schematic of Per2^Luc bioluminescence reporter mice generated by BMT of SCD or WT BM. B) Workflow for Per2 luminescence detection in tissue explants. C) Bioluminescence recordings of lung Per2 oscillations in culture; n = 3 samples/group. *P < 0.05, SCD → Per2^Luc compared with WT→ Per2^Luc mice.

Figure 3

Genetic deletion of Per1/Per2 enhances pulmonary leukocyte infiltration and lung tissue damage, which contribute to an early mortality in SCD. A) Schematic of SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. B) Images of irradiated male mice ∼16 wk post-BMT. C) Increased mortality was identified beginning 42 d and up to 112 d after irradiation and hematopoietic adoptive transfer in female SCD BM transplanted to Per1/Per2 dKO mice. D) Panel of histologic stains, hematoxylin and eosin (H&E), and Ly6-positive leukocytes as indicated with red arrows in SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. E) Semiquantification of histologic assessment of lung isolated from SCD or WT phenotypic mice with or without Per1/Per2. F) Quantification of leukocytes in ×20 field in whole lung sections isolated from SCD and WT BM transplant mice; n = 5 mice/group. ND, not determined. *P < 0.05, SCD → WT compared with WT → WT mice, **P < 0.01, SCD → WT compared with SCD → Per1/Per2 dKO mice.

Figure 4

Further elevated inflammatory response genes detected in lungs of SCD BMT mice with Per1/Per2 deficiency. A–C) Semiquantitative real-time PCR detection of lung mRNA levels of Tlr4 (A), Cxcl2 (B), and IL-6 (C) in whole lung tissue. D) IL-6 levels in lavage fluid collected from transplant mice groups; n = 5 mice/group. *P < 0.05, SCD → WT compared with WT → WT mice, **P < 0.01, SCD → WT compared with SCD → Per1/Per2 dKO mice.

Figure 5

Per1/Per2 genetic deficiency in SCD BMT mice affects heme and iron metabolism in the lung. A) Histologic Perl’s Prussian blue iron staining in WT or SCD BM transplanted to WT or Per1/Per2 dKO mice. B) Quantification of iron deposit in lung, which is reflected as a percentage of blue stain compared with the rest of the lung tissue. C) Heme content levels measured in SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. D) Working model demonstrating that deficient Per1Per/2 gene expression in SCD contributes to multiple factors that promote disease progression; n = 5 mice/group. *P < 0.05, SCD → WT compared with WT → WT mice, **P < 0.01, SCD → WT compared with SCD → Per1/Per2 dKO mice.