Aerosolized Harmful Algal Bloom Toxin Microcystin-LR Induces Type 1/Type 17 Inflammation of Murine Airways

Abstract

Harmful algal blooms are increasing globally and pose serious health concerns releasing cyanotoxins. Microcystin-LR (MC-LR), one of the most frequently produced cyanotoxins, has recently been detected in aerosols generated by the normal motions of affected bodies of water. MC-LR aerosol exposure has been linked to a pro-inflammatory influence on the airways of mice; however, little is understood about the underlying mechanism or the potential consequences. This study aimed to investigate the pro-inflammatory effects of aerosolized MC-LR on murine airways. C57BL/6 and BALB/c mice were exposed to MC-LR aerosols, as these strains are predisposed to type 1/type 17 and type 2 immune responses, respectively. Exposure to MC-LR induced granulocytic inflammation in C57BL/6 but not BALB/c mice, as observed by increased expression of cytokines MIP-1α, CXCL1, CCL2, and GM-CSF compared with their respective vehicle controls. Furthermore, the upregulation of interleukins IL-17A and IL-12 is consistent with Th1- and Th17-driven type 1/type 17 inflammation. Histological analysis confirmed inflammation in the C57BL/6 lungs, with elevated neutrophils and macrophages in the bronchoalveolar lavage fluid and increased pro-inflammatory and pro-resolving oxidized lipids. In contrast, BALB/c mice showed no significant airway inflammation. These results highlight the ability of aerosolized MC-LR to trigger harmful airway inflammation, requiring further research, particularly into populations with predispositions to type 1/type 17 inflammation.

Keywords: MC-LR; aerosol; harmful algal bloom; inflammation; microcystin.

Figure 1

Overview of MC-LR aerosol exposures. (A) Timeline of the exposure study. (B) Body weight changes over the course of the 14-day study displayed as grams compared with the initial body weights of each animal. n = 4; Student’s t-test between MC-LR-exposed (red) and respective vehicle (blue), * indicates p ≤ 0.05.

Figure 2

Molecular markers of inflammation in the lung tissue. Concentration of cytokine and chemokine proteins from lung lysate of male and female C57BL/6 (A) and BALB/c (B) mice. Values indicate fold change (FC) in concentrations from MC-LR-exposed mice compared with vehicle-exposed mice. Red dashed line indicates unchanged (FC of 1). Type of immunity associated with protein analyte: type 1 (blue), type 2 (yellow), mixed (green). *, **, **** indicates p ≤ 0.05, 0.01, 0.0001, respectively. Statistics by Student’s t-test between MC-LR-exposed and respective vehicle controls. n = 4 in all measurements.

Figure 3

Histological evaluation of the lung. (A) Representative images of H&E-stained sections of male and female C57BL/6 mouse lung after MC-LR or vehicle aerosol exposure. (B) Pathologist’s scoring of inflammation severity in the H&E-stained sections. (C) Representative images of sections stained by IHC for F4/80 (a murine macrophage marker) in male and female C57BL/6 mouse lung after MC-LR or vehicle aerosol exposure. Red arrows indicate positively stained cells. (D) Results of an image analysis software-assisted enumeration of F4/80+ cells, normalized to the tissue area of each section. *, **, **** indicates p ≤ 0.05, 0.01, 0.0001, respectively. Statistics by Student’s t-test between MC-LR-exposed and respective vehicle controls.

Figure 4

Bronchoalveolar lavage fluid analysis. Cellular constituents of BAL fluid normalized to the recovered volume. (A) total cells, (B) neutrophils, (C) eosinophils, (D) lymphocytes, and (E) macrophages. * and ** indicate p ≤ 0.05 and 0.01, respectively. Statistics by Student’s t-test between MC-LR-exposed and respective vehicle controls.

Figure 5

Lipid markers of inflammation in lung tissue. Concentration of arachidonic acid metabolites from lung lysate of male C57BL/6 mice. Values indicate log2FC in concentrations from MC-LR-exposed mice compared with vehicle. Dashed lines indicate Log2FC of 1 or −1, which correspond with an FC of 2 or −2, respectively. *, **, *** indicate p ≤ 0.05, 0.01, 0.001, respectively. Statistics by Student’s t-test between MC-LR-exposed and respective vehicle controls. n = 4 in all measurements.