Dietary Docosahexaenoic Acid as a Potential Treatment for Semi-acute and Chronic Particle-Induced Pulmonary Inflammation in Balb/c Mice

Abstract

Acute and chronic inflammation are vital contributing factors to pulmonary diseases which can be triggered by exposure to occupational and man-made particles; however, there are no established treatments. One potential treatment shown to have anti-inflammatory capabilities is the dietary supplement docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid found in fish oil. DHA's anti-inflammatory mechanisms are unclear for particle-induced inflammation; therefore, this study evaluated DHA as a prophylactic treatment for semi-acute and chronic particle-induced inflammation in vivo. Balb/c mice were fed a control or 1% DHA diet and exposed to dispersion media, an inflammatory multi-walled carbon nanotube (MWCNT), or crystalline silica (SiO₂) either once (semi-acute) or once a week for 4 weeks (chronic). The hypothesis was that DHA will decrease pulmonary inflammatory markers in response to particle-induced inflammation. Results indicated that DHA had a trending anti-inflammatory effect in mice exposed to MWCNT. There was a general decrease in inflammatory signals within the lung lavage fluid and upregulation of M2c macrophage gene expression in the spleen tissue. In contrast, mice exposed to SiO₂ while on the DHA diet significantly increased most inflammatory markers. However, DHA stabilized the phagolysosomal membrane upon prolonged treatment. This indicated that DHA treatment may depend upon certain inflammatory particle exposures as well as the length of the exposure.

Keywords: alveolar macrophage; crystalline silica; macrophage phenotype; multi-walled carbon nanotube; phagolysosomal membrane damage; pulmonary inflammation.

Fig. 1

Histopathology analysis of lung tissue in semi-acute model. Balb/c mice were put on a control or 1% DHA diet for 5 weeks. At week 4, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 1 week later. a Pathology was scored blinded by a board-certified veterinary pathologist, b airway wall thickness was measured blinded, and c representative images (10 ×) of Gömöri’s trichrome-stained lung sections of an airway and surrounding tissue. Data presented as mean ± SEM, n = 3. *P < 0.05, **P < 0.01 compared to DM, and (+) compared to different diet within the same exposure group. Arrows indicate particle encapsulation, arrow heads indicate increased lung airway thickness, and blue staining indicates collagen deposition.

Fig. 2

Inflammatory mediators in semi-acute model. Balb/c mice were put on a control or 1% DHA diet for 5 weeks. At week 4, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 1 week later. a LDH and b protein concentration was assessed in the LLF for lung damage. c Inflammatory cytokines IFNγ, IL-1β, TNFα, IL-33, pro-resolving cytokines IL-10 and IL-13, and IL-6 were analyzed in the LLF. Data presented as mean ± SEM, n = 7–9. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to DM, and (+) compared to different diet within the same exposure group. Analyses were done with log-transformed data.

Fig. 3

Assessment of pulmonary cells and LMP in semi-acute model. Balb/c mice were put on a control or 1% DHA diet for 5 weeks. At week 4, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 1 week later. a Total cell counts and b percentage of cell types via cytospins of cell differentials were evaluated. LMP was assessed through detection of c IL-1β release directly from AM ex vivo, d total cathepsin release within the LLF, and e cathepsin B release within the LLF. Data presented as mean ± SEM, n = 7–9 except c n = 5–7. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to DM, and (+) compared to different diet within the same exposure group. Analyses were done with log-transformed data. CTS; cathepsin.

Fig. 4

Impact of particle exposure and DHA treatment on macrophage phenotype in semi-acute model. Balb/c mice were put on a control or 1% DHA diet for 5 weeks. At week 4, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 1 week later. Relative gene expression of control diet MWCNT or SiO₂ mice were normalized to control diet DM-only mice for assessment of particle effect in a lung and c spleen tissue. Relative gene expression of 1% DHA diet mice were normalized to corresponding control diet mice for assessment of diet effect in b lung and d spleen tissue. Each bar within the heat maps consists of 5–6 mice for lung tissue and 4–5 mice for spleen tissue.

Fig. 5

Histopathology analysis of lung tissue in chronic model. Balb/c mice were put on a control or 1% DHA diet for 18 weeks. At weeks 2, 3, 4, and 5, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 13 weeks later. a Pathology was scored blinded by two observers; data presented as median ± SEM. b Airway wall thickness was measured blinded; data presented as mean ± SEM. c Representative images (10 ×) of Masson’s trichrome-stained lung sections of an airway and surrounding tissue. n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 compared to DM, and (+) compared to different diet within the same exposure group. Arrows indicate particle encapsulation, asterisks indicate lesion formation, and blue staining indicates collagen deposition.

Fig. 6

Inflammatory mediators in chronic model. Balb/c mice were put on a control or 1% DHA diet for 18 weeks. At weeks 2, 3, 4, and 5, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 13 weeks later. a LDH and b protein concentration was assessed in the LLF for lung damage. c Inflammatory cytokines IFNγ, IL-1β, TNFα, IL-33, pro-resolving cytokines IL-10 and IL-13, and IL-6 were analyzed in the LLF. Data presented as mean ± SEM, n = 4–5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to DM, and (+) compared to different diet within the same exposure group. Analyses were done with log-transformed data.

Fig. 7

Assessment of pulmonary cells and LMP in chronic model. Balb/c mice were put on a control or 1% DHA diet for 18 weeks. At weeks 2, 3, 4, and 5, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 13 weeks later. a Total cell counts and b percentage of cell types via cytospins of cell differentials were evaluated. LMP was assessed through detection of c IL-1β release directly from AM ex vivo, d total cathepsin release within the LLF, and e cathepsin B release within the LLF. Data presented as mean ± SEM, n = 4–5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to DM, and (+) compared to different diet within the same exposure group. Analyses were done with log-transformed data. CTS; cathepsin.

Fig. 8

Impact of particle exposure and DHA treatment on macrophage phenotype in chronic model. Balb/c mice were put on a control or 1% DHA diet for 18 weeks. At weeks 2, 3, 4, and 5, mice were exposed to either DM-only, MWCNT, or SiO₂ and euthanized 13 weeks later. Relative gene expression of control diet MWCNT or SiO₂ mice were normalized to control diet DM-only mice for assessment of particle effect in a lung and c spleen tissue. Relative gene expression of 1% DHA diet mice were normalized to corresponding control diet mice for assessment of diet effect in b lung and d spleen tissue. Each bar within the heat maps consists of 4–5 mice for lung tissue and 4 mice for spleen tissue.