Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis

Abstract

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Keywords: Streptococcus uberis; antioxidative pathway; autophagy; bovine mastitis; inflammation; mMECs; murine mammary glands.

Figure 1

Infecting mMECs with S. uberis enhanced intracellular accumulation of ROS and induced autophagy. (A) The mMECs were challenged with S. uberis and ROS production was determined by measuring the intensity of green fluorescence generated by DCFH-DA and captured with a confocal laser scanning microscope. (B) Autophagy induction was confirmed based on morphological and subcellular (TEM) images. Note the nuclei (N), mitochondria (M), autophagosomes (A), lysosomes (L), autolysosome (AL) and S. uberis (black arrowheads). Scale bar = 1.0 μm. Magnified figure It comes from the yellow box.

Figure 2

Infecting mMECs with S. uberis induced antioxidant and autophagy marker proteins and reduced inflammatory cytokines. (A) Western blot analyses of antioxidative Sirt1, Keap1 and Nrf2 isolated from S. uberis-challenged mMECs. Proteins were collected up to 24 h post challenge. (B) Inflammatory IL-6 and TNF-α, and autophagy of LC3II/LC3I, were also analyzed following S. uberis challenge. Mean and standard deviation (three independent experiments) of proteins (quantified with ImageJ 1.49v, http://imagej.nih.gov/ij, accessed on 18 December 2023). * p < 0.05, ** p < 0.01 (compared to the Sham).

Figure 3

NAC pretreatment in S. uberis-challenged mMECs mitigated antioxidative responses. (A) Levels of Sirt1, Nrf2 and HO-1 in mMECs pretreated with 30 μM NAC and 1 h later challenged with S. uberis. (B) Levels of Sirt1, Keap1 and Nrf2 in mMECs pretreated with 40 μM Cambinol (siSirt1) and 6 h later challenged with S. uberis. Data were derived from assessments of Western blots and are mean ± SD of three independent trials. * p < 0.05 and ** p < 0.01 (compared to the Sham); # p < 0.05 and ## p < 0.01 (compared to the S. uberis group).

Figure 4

Pretreatment with NAC attenuated inflammatory and cellular damage in S. uberis-challenged mMECs and mammary glands by reducing oxidative stress. (A) Antioxidative Nrf2 and HO-1 and inflammatory IL-6 and TNF-α were determined in mMECs pretreated with NAC (30 μM) and 1 h later challenged with S. uberis. (B) Levels of total SOD and MDA activities at 6, 12 and 24 h post S. uberis challenge and pretreatment with NAC. Data represent mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01 (compared to the Sham); # p < 0.05 and ## p < 0.01 (compared to the S. uberis group).

Figure 5

NAC treatment alleviated autophagy in S. uberis-challenged mMECs. (A) Protein concentrations of Keap1, Atg5 and LC3II/LC3I in mMECs pretreated with NAC (30 μM) 1 h prior to S. uberis challenge. (B) Formation of acidified lysosomes was quantified by measuring red fluorescence intensity. In total, 30 cells were selected for each group and 10 cells per sample with three repeats were quantified for statistical analyses. Data are representative means of three independent experiments. * p < 0.05 and ** p < 0.01 (compared to the Sham); # p < 0.05 and ## p < 0.01 (compared to the S. uberis group). Magnified figure It comes from the yellow box.

Figure 6

Silencing of Sirt1 in mMECs inhibited autophagy by inducing inflammation. (A) Protein expressions of HO-1, IL-6, TNF-α and Atg5 in mMECs pretreated with Cambinol (siSirt1) (40 μM) 6 h prior to S. uberis challenge. (B) Transfection of mMECs with Ad-mCherry-GFP-LC3B to determine autophagy inhibition in Cambinol treatment. Quantification of red, green and yellow fluorescence conducted by ImageJ 1.49v software with JaCoP plugin. Statistical data analyses were conducted on 10 cells per sample. Data represent mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01 (compared to the Sham); # p < 0.05 and ## p < 0.01 (compared to the S. uberis group). Magnified figure It comes from the yellow box.

Figure 7

Activation of Nrf2 triggered autophagy by attenuating IL-6 and TNF-α. (A) Expression of Nrf2, HO-1, IL-6, TNF-α and LC3II/LC3I in mMECs pretreated with 10 μM of NK-252 (Nrf2 activator) 6 h prior to S. uberis challenge. (B) Nrf2 protein expressions in S. uberis-challenged mMECs analyzed by immunofluorescence assay. Nrf2 green fluorescence intensity was quantified using ImageJ software with JaCoP plugin. For statistical analyses, 10 cells were selected for each sample. The given data are representative of the mean of three independent experiments. * p < 0.05 and ** p < 0.01 (compared to the Sham); # p < 0.05 and ## p < 0.01 (compared to the S. uberis group). Magnified figure It comes from the yellow box.

Figure 8

Suppression of antioxidative Nrf2 inhibited autophagy mechanism by increasing inflammation. (A) Nrf2 marker proteins were silenced by using 10 μM of ML385 in mMECs and Nrf2, HO-1, IL-6, TNF-α and Atg5 protein expression was analyzed at 12 and 24 h post S. uberis challenge. (B) Immunofluorescence confocal assay of Nrf2 in S. uberis-challenged mMECs. Quantitative image analysis of green Nrf2 intensity was conducted by ImageJ with JaCoP plugin in 10 cells/sample. Represented data are mean ± SD of three independent trials. * p < 0.05 and ** p < 0.01 (compared to the Sham); # p < 0.05 and ## p < 0.01 (compared to the S. uberis group). Magnified figure It comes from the yellow box.

Figure 9

Mammary glands and mMECs activated antioxidative factors in response to oxidative stress provoked by S. uberis. (A) Expression of antioxidants Sirt1, Keap1, Nrf2, HO-1 and Atg5 in mMECs silenced in Sirt1 and Nrf2 previous to S. uberis at 3 h and 6 h challenge. (B) Mammary gland morphology after S. uberis challenge for 6 h in mice pretreated with NAC, Cambinol or NK-252. * p < 0.05 and ** p < 0.01 (compared to the Sham); # p < 0.05 and ## p < 0.01 (compared to the S. uberis group).

Figure 10

Theoretical scheme of antioxidative factors that regulate autophagy mechanism in mastitis.