Autophagy Modulates Inflammatory Response in Zebrafish Heart and Caudal Fin Regeneration

細胞自噬在斑馬魚心臟和尾鰭再生中對炎癥反應的調控

Student thesis: Doctoral Thesis

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Award date9 Dec 2022

Abstract

Inflammation plays a critical role in tissue repair and regeneration. The inflammatory response following injury can be classified into the pro- and anti-inflammatory phases. In the earlier, pro-inflammatory phase, damaged cells initiate a pro-inflammatory response, which includes damage-associated molecular patterns (DAMPs), activation of the complement cascade and production of reactive oxygen species (ROS). These induce the release of pro-inflammatory cytokines and the recruitment of inflammatory cells. The subsequent anti-inflammatory phase suppresses inflammation by the action of reparative mediators, which alter the roles of infiltrating leukocytes and stimulate repair of the injured area. An imbalance between the pro- and anti-inflammatory phases causes detrimental effects on tissue repair and regeneration. In the past, many therapeutic approaches have been designed, that attempt to inhibit the pro-inflammatory response, but most failed to improve the outcome. Therefore, novel strategies are required to regulate the inflammatory response after injury.

Autophagy is a conserved and fundamental process, which contributes to the cytoplasmic and metabolic quality control. A dysfunction of autophagy induces a variety of human diseases, such as cancer, neurodegeneration, infection, and cardiovascular diseases. It has been reported that autophagy can affect inflammation via the regulation of immune cell function and immune molecular systems including DAMPs and ROS. Although autophagy is known to play a role in tissue repair and regeneration, its role in the immune response during tissue repair and regeneration remains unclear. Therefore, in the present study, I explored the relationship between autophagy and inflammation during heart and caudal fin regeneration using zebrafish as an animal model.

I investigated how impaired autophagy influences the immune response during zebrafish heart regeneration. My results showed that after heart cryoinjury in the unc-51-like autophagy activating kinase (ULK) mutant zebrafish lines, ulk1b-/- and ulk2-/-, the recruitment of leukocytes and regeneration of the heart were both delayed, compared to their wild type ulk1b/2+/+. To exclude the possibility that this was due to other functions of ulk1b and ulk2, in some experiments, I treated AB wild-type zebrafish with chloroquine (CQ), a classical autophagy inhibitor, after heart cryoinjury. My results showed that shortly following CQ treatment, the numbers of leukocytes were not significantly different from the untreated controls. However, by 7 days post cryoinjury (dpc), the number of leukocytes was reduced in the CQ treated group compared with the untreated control group. These results confirmed that autophagy plays an important role in the modulation of the immune response during zebrafish heart regeneration. The differences observed between the ulk1b-/- and ulk2-/- mutants, and the CQ treatment might be because the drug takes time to work or its effects on immune cells are different. However, there was no significant difference in the numbers of neutrophils between the ulk mutants and ulk1b/2+/+ from 1 dpc to 7 dpc, and at 14 dpc, ulk2-/- mutant had more neutrophils in the injured area compared with the ulk1b/2+/+. Similar phenomenon was observed in CQ treated group. At 14 dpc, the number of neutrophils was significantly higher in the CQ treated group when compared with the untreated control group. As the leukocytes that are recruited to an injured area are mainly neutrophils and macrophages, these results indicate that the decreased leukocytes might be macrophages. Moreover, my results showed that the percentage of apoptotic cells was higher in the ulk1b-/- mutant when compared with the ulk1b/2+/+ at 1 dpc, which might be induced by the delayed clearance of the dying cells due to the loss of macrophages. Also, at 7 dpc, the percentages of apoptotic cells were higher in the ulk1b-/- and ulk2-/- mutants, which might be induced by the prolonged inflammation in zebrafish.

To further explore how autophagy affects the recruitment of leukocytes during tissue repair and regeneration, I also used a zebrafish caudal fin regeneration model to detect the accumulation of macrophages and neutrophils after fin amputation in real time. My results showed that blocking autophagy by treatment with either CQ or its derivative, hydroxychloroquine (HCQ), inhibited fin regeneration. The accumulation of macrophages was no different at the start, but by 24 hours post amputation (hpa), the numbers of macrophages were lower in the CQ and HCQ treated groups compared to the untreated controls. Conversely, the numbers of neutrophils were higher in the HCQ treated groups from 6 hpa to 96 hpa. These results were consistent with my inference in the heart regeneration that the blocking of autophagy inhibited the recruitment of macrophages after injury. Furthermore, the numbers of cells which expressed pro-inflammatory cytokine tumor necrosis factor-alpha (Tnfα) were higher in CQ or HCQ treated groups when compared with the untreated controls, indicating that the impairment of autophagy might influence the resolution of the pro-inflammatory phase. Moreover, as Tnfα is expressed mainly by pro-inflammatory macrophages, these findings indicate that the blockage of autophagy might prevent the polarization of macrophages in the tissue regeneration process.

Finally, I found that in some experiments, a high dose of HCQ induced epithelial hyperproliferation and inflammation in the zebrafish larvae. It has been reported that a side effect of HCQ treatment is the onset of psoriasis, but the reason for this remains unknown. However, the main characteristics of psoriasis include epithelial hyperproliferation and inflammation, which are coincident with my findings in zebrafish. Thus, I compared the differentially expressed genes (DEGs) in HCQ treated and untreated (control) larvae and identified 768 DEGs. In addition to genes related to inflammation, I also found that the genes of several proteases and their inhibitors were dysregulated in the HCQ treated group. Among these proteases, matriptase (ST14a) and matrix metalloproteinases (MMPs) have been reported to play important roles in maintaining homeostasis of the epithelium and their loss of inhibition might cause psoriasis. Thus, I used a morpholino oligonucleotide to knock down the translation of matriptase (st14a MO) and inhibited MMPs function pharmacologically. My results showed that all these methods helped alleviate the symptoms of epidermal hyperplasia and inflammation but only the st14a MO was able to rescue the epidermal integrity. These results indicate that it is important to regulate the action of proteases to avoid the epidermal side effects caused by HCQ and it provides an exciting new model for studying psoriasis in zebrafish.

In summary, although previous studies have described the important roles of autophagy and inflammation during tissue repair or regeneration. The crosstalk between these two signaling pathways in wound healing is rarely mentioned. Thus, in my study, I investigated the effects of impaired autophagy on the inflammatory response during zebrafish heart and caudal fin regeneration. The data indicate that blockage of autophagy can inhibit the recruitment of leukocytes, and thus delayed the clearance of the dying cells caused by the injury. Interestingly, my results showed that compared to neutrophils, the blockage of autophagy had a greater impact on macrophages. In addition, the results of the expression of Tnfα indicates that blockage of autophagy might also inhibit the polarization of macrophages from pro-inflammatory phenotype to anti-inflammatory phenotype, which might prolong the inflammation after tissue damage and impair regeneration. Together, these results illustrate about how autophagy modulates the inflammatory response after tissue injury, which might provide new therapeutic opportunities for the tissue or organ repair. Surprisingly, I also observed that a high dose of HCQ induced epithelial hyperproliferation and inflammation in the zebrafish larvae, which mimicked one of the side effects HCQ caused on humans. Through transcriptome analysis, I found that the genes of several proteases and their inhibitors were dysregulated in the HCQ treated group, and many of them have been reported to be related with the skin diseases. By knocking down the translation of matriptase (st14a MO) and inhibited MMPs function pharmacologically, the epithelial disorders were significantly alleviated. These findings provided a possible explanation of how HCQ induces the exacerbation of psoriasis in patients.

    Research areas

  • autophagy, inflammatory response, regeneration, zebrafish