Pink1/Parkin-Mediated Mitophagy Regulated the Apoptosis of Dendritic Cells in Sepsis

Dendritic cells (DCs) are vital antigen-presenting cells (APCs) in the immune system, whose apoptosis is closely related to the development of sepsis. Mitophagy is one of the necessary forms of selective autophagy that removes damaged or dysfunctional mitochondria to regulate immunity and inflammation. However, its effect on the apoptosis of DC in sepsis remains unknown. Here, we showed that sepsis activated the apoptosis and mitophagy of DC, and mitophagy had an anti-apoptotic effect on sepsis-induced DC apoptosis. In this study, we used cecal ligation and puncture (CLP) to simulate the pathophysiological state of sepsis. Apoptosis and mitophagy of DC were significantly enhanced in CLP mice compared with controls, and in the Pink1-KO (Pink1-knockout) mice CLP model, the level of apoptosis in DC was further increased while the level of mitophagy was decreased. In addition, more severe mitochondrial dysfunction was exhibited in DC of Pink1-KO mice CLP model compared to wild-type (WT) mice. The results suggest that Pink1/Parkin-mediated mitophagy is activated during sepsis and has an anti-apoptotic effect on DC, which regulates immune functions.


INTRODUCTION
Sepsis is one of the significant causes of death in acute and critically ill patients, which can be life-threatening due to dysregulation of the response to infection, resulting in organ dysfunction [1]. The imbalance between the proinflammatory and anti-inflammatory mechanisms in the septic state leads to a significant "immune paralysis" at the later stage of sepsis, making the infection persist, and the mortality rate of patients remains high [2,3]. Due to the massive apoptosis of immune cells, immune dysfunction is considered the link of immunosuppression in sepsis, and apoptosis of lymphocytes, including innate and adaptive immune cells, is closely related to secondary infection and poor prognosis in sepsis [4]. Dendritic cell (DC), as critical antigen-presenting cells (APCs), bridge innate and adaptive immune, whose apoptosis is closely related to the development of sepsis [5].
Mitophagy, a selective autophagic process, is specifically targeted for eliminating dysfunctional or nonessential mitochondria from the population to maintain cellular homeostasis [6,7]. Pink1 (PTEN-induced putative kinase protein1) and Parkin (E3 ubiquitin ligases) mediated mitophagy response to a dissipation of the mitochondrial membrane potential (MMP) [8]. Mitophagy is involved in developing neurodegenerative diseases, cardiac diseases, and tumors, but few studies have been conducted on its role in sepsis [9][10][11][12]. Kang et al. found that PINK1 and PARK2 knockout mice showed earlier organ dysfunction and higher mortality in a sepsis model with multiple microbial infections [13]. Piquereau et al. found that PARK2 knockout mice exhibited more severe mitochondrial and myocardial function impairment in an LPSinduced sepsis model [14]. These suggested that PINK1/ PARK2 pathway-mediated mitophagy could protect organ functions to some extent in septic mice. Mitophagy promotes antigen presentation to major histocompatibility complex (MHC) I and reduces inflammasome activation, suggesting that mitophagy plays an essential role in regulating immunity and inflammation [15].
In sepsis, the organism is in a dysregulated response, and various factors combine to cause persistent dysfunction of dendritic cells. Previous studies have identified excessive endoplasmic reticulum stress, TIPE2 protein overexpression, and low micro-RNA expression as causing dendritic cell dysfunction in sepsis [16][17][18]. Over the years, studies on autophagy and dendritic cell functions have gradually increased. It was found that autophagy regulated the immune response of dendritic cells. When the autophagyrelated genes Atg16, Atg7, and Beclin-1 were knocked down, the ability of dendritic cells to mature, activate, present antigen, mediate T cell proliferation, and polarize was reduced [19][20][21]. In contrast, induction of autophagy enhanced the antigen-presenting ability of dendritic cells and increased the expression of the surface costimulatory molecule MHC-II [22]. In addition, inhibition of autophagy in CD4 + T lymphocytes in sepsis caused increased apoptosis of immune cells and immune malfunction [23]. Apoptosis of DC was a key link in regulating immune homeostasis through multiple pathways, and its abnormality could trigger autoimmunity [24]. Previous studies in our laboratory have shown that DC apoptosis increases in the septic state and that survival in septic mice can be improved by anti-apoptotic treatment [16]. Studies have found that sepsis induced the onset of mitophagy in tissues and cells such as liver, heart, neuronal cells, and immune cells. With the development of sepsis, the autophagy flow disorder appeared, that was, the combination disorder of autophagosome and lysosome, followed by impaired autophagosome formation and the impaired induction of mitophagy [23,[25][26][27]. It has been found that Pink1/Parkin-mediated mitophagy played a protective role on LPS-induced macrophage apoptosis and inflammation. Knocking down Pink1 reduced the clearance of dysfunctional mitochondria by mitophagy and increased the concentrations of IL-1β, TNF-α, and ROS and decreased the cell viability and ATP concentration [28]. But the effect of mitophagy on dendritic cell functions and apoptosis in sepsis has not been elucidated yet. Therefore, we speculated that the dysfunction of splenic dendritic cells in sepsis might be related to reducing Pink1/Parkin-mediated mitophagy.
This study evaluated dendritic cell apoptosis and its relationship with mitophagy in CLP-induced sepsis in mice. The effect of Pink1 on DC mitophagy and apoptosis was assessed in vivo. Furthermore, we examined the functions of mitochondria in dendritic cells to investigate the underlying mechanisms.

Mice and Experimental Groups
Male wild-type C57BL/6 mice (6 to 8 weeks old, 18-22 g) were purchased from the Laboratory Animal Institute, Chinese Academy of Medical Sciences (Beijing, China). Pink1-KO mice in the C57BL/6 background were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). All mice were housed on a 12-h light-dark cycle with controlled temperature (21 to 23 °C) and provided with a standard rodent diet and water ad libitum throughout all experiments. All experimental protocols were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All methods were approved by the ethics committee of the Laboratory Animal Ethics Committee of Wenzhou Medical University. Mice were randomly divided into the sham and cecal ligation and puncture (CLP) groups.

CLP
An experimental sepsis model can be established by CLP because of its ease of reproducibility and resemblance to human sepsis progression [29]. As previously described, sepsis was induced in male C57BL/6 mice by CLP [30]. In brief, mice were anesthetized with 200 μl 5% chloral hydrate (Biotopped, Beijing, China). The abdominal skin of mice was disinfected with mice immobilized. An approximately 1-cm middle abdominal incision was made, and the cecum was exposed and ligated with 4-0 silk suture without causing intestinal obstruction. Then, the cecum was punctured entirely once with a 22-gauge needle, squeezed out a small amount of fecal matter, and returned to the peritoneal cavity. The abdomen was closed in two layers with 4-0 silk sutures, and mice were then injected with 1 ml sterile saline subcutaneously to revitalize and prevent dehydration. Animals were euthanized 6 h, 12 h, 24 h, 48 h, and 72 h later. Sham-operated controls underwent laparotomy and bowel manipulation without cecal ligation and puncture.

Generation of DC
For isolation of murine splenic DC, spleens were removed from C57BL/6 mice and digested after perfusion with 0.4 mg of collagenase/DNAse (Sigma Aldrich, St. Louis, MO) per milliliter of phosphate-buffered saline (PBS) (pH 7.2). According to the manufacturer's protocol, cells were incubated with anti-CD11c-conjugated magnetic microbeads (Miltenyi Biotec, Germany) and isolated with a magnetic-activated cell sorting MS column (Miltenyi Biotec, Germany). The selected cells were harvested, which were mostly DC by phenotypic analysis using flow cytometry.

Flow Cytometric Analysis
Annexin V-PE/7-AAD Apoptosis Assay Cells were stained with Annexin V-PE and 7-ADD (BD Biosciences, Mountain View, CA). DCs were collected and washed twice with cold PBS, and then resuspended in 200 μl 1 × binding buffer, to which were added 2.5 μl Annexin V-PE and 2.5 μl 7-ADD. After incubation for 15 min in darkness at room temperature, the cells were analyzed by flow cytometry. Three negative control tubes were set up in the control group: a double-negative tube without dye, a single-dyed 7-AAD tube, and a single-dyed Annexin V-PE tube. Dates were acquired on an LSRII (BD Biosciences Inc., San Jose, CA) and analyzed using FlowJo (Tree Star Inc., Ashland, OR).

Intracellular Calcium Measurement
We measured the intracellular calcium dynamics in DC with flow cytometry using the Ca 2+ -sensitive fluorescent dye Fluo-4/AM (Beyotime, Shanghai, China). Five micromolar of Fluo-4/AM was loaded for 30 min at 37 °C. Cells were washed by centrifugation, and incubated for another 30 min at 37 °C. The intracellular calcium was analyzed immediately for Fluo-4/AM fluorescence intensity by flow cytometry. Dates were acquired on an LSRII and analyzed using FlowJo.

TUNEL Staining
Apoptosis of DC was detected by TUNEL staining. DCs were fixed with 4% paraformaldehyde for 30 min. Moreover, cells were centrifuged with PBS at 1000 rpm for 5 min and were broken the membrane with 0.3% Triton X-100 (Solarbio, Beijing, China) for 5 min at room temperature. Cells were centrifuged with PBS at 1000 rpm for 5 min and incubated at 37 °C for 1 h with 50 μl TUNEL detection solution (Beyotime, Shanghai, China). Cells were centrifuged with PBS at 1000 rpm for 5 min, and 100 μl liquid was left on the slide. Ten microliters DAPI (Solarbio, Beijing, China) was added to stain the nucleus. Cells were stored at -20 °C and were used to detect apoptosis by fluorescent microscope (Leica, Germany).

ROS level in cells was detected using ROS Assay
Kit (Beyotime, Shanghai, China). Cells were collected and added an appropriate amount of DCFH-DA (10 μmol/l) (Beyotime, Shanghai, China), and they were incubated for 30 min at 37 °C in the dark. Cells were washed and centrifuged using RPMI1640, and 100 μl of the base solution was reserved for making slides.
The level of ROS in cells was then determined with the fluorescent microscope according to the manufacturer's instructions.
Cells were collected, and the membrane was ruptured on Digitionin (Sigma Aldrich, St. Louis, MO) for 2 min on ice fixed with 4% paraformaldehyde for 1 h. Cells were blocked with 1% BSA for 1 h. They were then incubated with LC3B primary antibody (1: 200) at 4 °C overnight. After 3 PBS washes, cells were incubated with fluorescent secondary antibody (1:500) (Abbkine, US) at room temperature for 1 h. Cells were washed with RPMI1640 and were incubated with Mito-Bright probe (Dojindo, Japan) for 10 min at room temperature. Finally, cells were washed with RPMI1640 3 times, leaving about 100 μl of the substrate drop on the slide. The nucleus was stained with 10 μl DAPI solution (Solarbio, Beijing, China), and the solution was placed in a wet box at -20 °C. The localization of LC3 on mitochondria was observed by confocal microscopy (Leica, Germany). In addition, cells were collected and incubated using Mtphagy Dye (Dojindo, Japan) at 37 °C for 30 min and centrifuged with RPMI1640. Then, cells were washed. The nucleus was stained with 10 μl DAPI solution, and the solution was stored in a humidified box at -20 °C. The level of mitophagy was observed by confocal microscopy.

Transmission Electron Microscopy
Cells were prefixed in 2.5% glutaraldehyde (Biotopped, Beijing, China) in PBS for 1 h at room temperature, then stored at 4 °C overnight before processing. This grids section was observed in a transmission electron microscope (Leica, Germany). Images were acquired with a charge-coupled device camera (AMT).

Statistical Analysis
All results are presented as the mean ± standard deviation (SD) and analyzed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). To determine the significance of the differences between the experimental conditions, one-way analysis of variance (ANOVA) was performed using the Newman-Keuls method. P < 0.05 was considered statistically significant. All experiments were repeated at least three times.

The Apoptosis of DC Was Induced During Sepsis
We isolated splenic mononuclear cells from WT and Pink1-KO mice after CLP or Sham procedure and performed a comparative phenotypic analysis of DC. Previous studies demonstrated that CD11c is a specific cell surface integrin of DC [31]. Thus, DCs were gated using a CD11c scatter plot, where CD11c negative cells were excluded from further analysis. Doublet exclusion was performed by plotting the height or width against the area for forward scatter or sideward scatter areas. The measured purity of CD11c + DC was reached more than 95% on average (Fig. 1A). We selected two points in time, 24 h and 72 h after CLP, to examine the apoptosis of DC in mice spleen based on the above experiments. To verify the sepsis-induced apoptosis level of DC, the abundance of apoptosis-associated proteins was monitored at 24 h, 72 h after CLP, and sham group (Fig. 1B). Furthermore, the Bcl-2/Bax ratio was decreased in the splenic DC of mice after CLP compared with those in the sham group (P < 0.05). And the Bcl-2/Bax ratio was lower at 72 h than 24 h after CLP (P < 0.05). The level of cleaved-caspase-3 protein was higher in the CLP 24 h and 72 h groups than in the sham group (P < 0.05). In particular, the results of TUNEL (Fig. 1C) and flow cytometry (Fig. 1D) showed that the apoptosis rate of DC at 24 h and 72 h after CLP was more than 50% higher than that of the sham group, which was consistent with the results of Tinsley's study [32].

Pink1/Parkin-Mediated Mitophagy Was Induced During Sepsis
To investigate the effect of Pink1/Parkin signaling pathway on mitophagy during sepsis, the abundance of mitophagy-associated proteins was monitored at 6 h, 12 h, 24 h, 48 h, and 72 h after CLP and sham group. The protein abundance of Tom20, Pink1, and Parkin was all upregulated in the splenic DC of mice after CLP compared with those in the sham group ( Fig. 2A). It showed that Pink1 tended to combine with the TOM complex rather than degradation, leading to enhanced Parkin activation, an essential pathway for Pink1/Parkin to activate mitophagy. Overall, the evidence indicated that sepsis could induce Pink1/Parkin-mediated mitophagy in DC.
Meanwhile, our observation by confocal microscopy showed the relationship between the location of LC3 and mitochondria after double staining with LC3 fluorescent antibody (red) and mitochondria-specific fluorescent dye Mito-Tracker (green) (Fig. 2B). It showed that the expression level of LC3 in the splenic DC of mice at 24 h after CLP was significantly increased compared with the sham group, and there was co-localization of LC3 with mitochondria. Mitophagy was also detected by confocal microscopy after staining with Mtphagy Dye (red), a fluorescent dye specific for mitophagy (Fig. 2C), which showed that mitophagy was significantly enhanced 24 h after CLP. In addition, the swelling of mitochondria, the disorder of cristae arrangement, and the fusion with autophagosomes in the splenic DC mitochondria were observed by transmission electron microscopy at 24 h after CLP (Fig. 2D).

The Mitophagy of DC Was Effectively Attenuated by Blocking the Pink1/Parkin Signaling Pathway
Western blot analysis revealed a complete deletion of Pink1 protein in Pink1-KO mice (Fig. 3C). To investigate the difference between WT mice and Pink1-KO mice during sepsis, the change of LC3 expression and mitophagy was observed by confocal microscopy (Fig. 3A, B). It showed that LC3 was co-localized with mitochondria in splenic DC of Pink1-KO mice, but its expression was significantly lower than WT mice at 24 h after CLP. Mitophagy was also observed by confocal microscopy after staining with Mtphagy Dye (red). The results showed that compared with WT mice 24 h after CLP, mitophagy in spleen DC of PinK1-KO mice was significantly weakened.
Moreover, the abundance of mitophagy-associated proteins was monitored 24 h after CLP (Fig. 3C). At 24 h after CLP, the quantity of Tom20 protein in the DC of WT mice was markedly higher than that of the sham group, while the abundance of Tom20 protein in the DC of Pink1-KO mice after CLP was significantly lower than that of WT mice (P < 0.05). At 24 h after CLP, the abundance of Parkin protein in the DC of WT mice was markedly high, while the abundance of Parkin protein in the DC of Pink1-KO mice was significantly lower than that of WT mice (P < 0.05). At 24 h after CLP, the abundance of LC3 protein and p62 protein was increased considerably in DC of WT mice, whereas the expression levels of these two proteins were decreased in Pink1-KO mice (P < 0.05).

Pink1/Parkin-Mediated Mitophagy Had Anti-apoptotic Effects During Sepsis
We speculated that Pink1/Parkin-mediated mitophagy played a vital regulatory role in sepsis-induced apoptosis of DC. Thus, we used western blot, flow cytometry, and TUNEL to investigate the effect of knockdown of the Pink1 gene on the apoptosis of DC after CLP. Western Blot was used to examine apoptosis-related proteins at 24 h after CLP (Fig. 4A), and it showed that the Bcl-2/Bax ratio was significantly low in the WT + CLP 24 h group. The Bcl-2/Bax ratio was further reduced in Pink1-KO mice compared with WT mice after CLP 24 h (P < 0.05). The abundance of cleaved-caspase-3 protein in DC of Pink1-KO septic mice was significantly lower than in WT septic mice (P < 0.05). Both flow cytometry (Fig. 4B) and TUNEL (Fig. 4C) results showed an increase in splenic DC apoptosis rate at 24 h after CLP, and the rise in DC apoptosis rate was more pronounced in Pink1-KO + CLP 24 h than in the WT + CLP 24 h group (P < 0.05).

Pink1/Parkin-Mediated Mitophagy May Contribute to Anti-apoptotic Effects by Improving Mitochondria Functions
We hypothesized that Pink1/Parkin-mediated mitophagy might contribute to the anti-apoptotic effect by improving mitochondrial functions. Thus, we examined the effects of Pink1/Parkin-mediated mitophagy on DC mitochondrial functions in sepsis in terms of cellular ATP synthesis, Ca 2+ levels, and ROS content. At 24 h after CLP, the abundance of ATP5A1 protein in DC of WT mice was significantly lower than in the sham group, whereas the abundance of ATP5A1 protein in DC of Pink1-KO + CLP 24 h group was lower than that in the WT + CLP 24 h group (P < 0.05). In addition, CLP induced a decrease in UQCRC1 protein abundance in DC, whereas Pink1-KO septic mice had a further reduction in UQCRC1 protein abundance in DC compared with WT + CLP 24 h group (P < 0.05) (Fig. 5A). The changes of Ca 2+ in DC were observed by flow cytometry with Fluo-4 AM calcium fluorescent probe incubation, and the result showed that Ca 2+ in DC increased significantly at 24 h after CLP (P < 0.05) (Fig. 5B). There was a significant difference between Pink1-KO + CLP group and WT + CLP group. After DCFH-DA incubation, the changes of ROS in DC were observed by fluorescence microscope. The result showed that at 24 h after CLP, the ROS content in DC of WT mice was markedly increased, and the content of ROS in DC of Pink1-KO mice increased compared with WT mice (P < 0.05) (Fig. 5C).

DISCUSSION
Sepsis-induced immune cell apoptosis is considered a critical factor in the state of immunosuppression. The DC apoptosis induces self-growth and maturation disorders and causes the differentiation of naïve T cells into Treg [33]. It was reported that mitophagy is utilized by immune cells, including NK cells and macrophages, to resist apoptosis [34,35]. In our present study, we investigated the apoptosis and mitophagy activity change of DC and the effect on the apoptosis of sepsis-induced splenic DC. We showed that (1) sepsis enhanced the activity of apoptosis and mitophagy of DC; (2) the Pink1/Parkin pathway mediated the mitophagy; (3) Pink1/Parkinmediated mitophagy resisted the apoptosis of DC during sepsis; (4) Pink1/Parkin-mediated mitophagy ameliorated mitochondria functions to moderate the apoptosis of DC. In conclusion, these results indicated that sepsis activated mitophagy and then played an anti-apoptosis role in splenic DC in sepsis-induced mice by improving the mitochondria functions.
Sepsis, defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, is a significant public health concern [1]. Being essential antigen-presenting cells, DCs are closely related to the occurrence and development of sepsis [5]. Recent studies have shown that the change of DC immune functions in sepsis is involved with various mechanisms, such as apoptosis, activation of Wnt signaling pathway, Toll-like receptor (TLR)-dependent signaling, and ROS generation [36]. Herein, we demonstrated that the apoptosis rate in DC was significantly higher in sepsis. Therefore, inhibition of DC apoptosis may be a novel target for the treatment of sepsis.
Depolarization of the MMP due to increased oxidative stress in sepsis is a solid signal for triggering mitophagy [37,38]. Mitophagy is caused by a decrease in MMP that activates the Pink1-Parkin signaling pathway to ubiquitinate autophagy receptors [39,40]. Our study also demonstrated the increased mitophagy at 24 h after CLP surgery in DC. It has been reported that Pink1/ Parkin-mediated mitophagy is enhanced by cardiac overexpression of Beclin-1 in response to LPS stimulation, which in turn reduces cardiac inflammation and improves cardiac functions [41]. It has been found that Pink1-KO mice have significantly increased mortality compared to WT mice after the CLP procedure, whereas HMGB1 and NLRP3 inhibitors can suppress the septic inflammatory response and organ dysfunction in Pink1-KO mice which indicates the organ-protective effect of Pink1 in the course of sepsis [13].
It has been clear that DC apoptosis is one of the primary mechanisms responsible for the considerable reduction of DC in sepsis [42]. Recent literature suggests that LPS-stimulated macrophages can induce mitochondrial injury, apoptosis, and Pink1-mediated mitophagy. Meanwhile, the Pink1-mediated mitophagy is protective in LPS-induced inflammation and apoptosis by phagocytosis of dysfunctional mitochondria [28]. It has been observed that Akt1 kinase-mediated mitophagy has an anti-apoptotic effect in alveolar macrophages [35]. In studies of other diseases, Pink1 has also been found to play a vital role in inhibiting cellular apoptosis [43][44][45]. Thus, we hypothesized that sepsis-induced DC apoptosis was regulated by Pink1/Parkin-mediated mitophagy. Our study showed that blocking the Pink1/Parkin signaling pathway led to a further increase in DC apoptosis, proving our hypothesis correct.
Mitochondrial dysfunction in sepsis is mainly manifested as ATP depletion due to inhibition of oxidative phosphorylation (OXPHOS), excessive ROS production, the disorder of Ca 2+ homeostasis, and the release of proapoptotic proteins [46]. There is increasing evidence that mitochondria themselves have become significant executors of apoptosis and that their dysfunction can trigger apoptosis and at last contribute to organ failure [47,48]. Oxidative stress injury, caused by ROS excess, can be alleviated with mitophagy by specifically removing damaged mitochondria [38]. In a recent study, it has been reported that knockdown of the Pink1 gene was found to result in mitochondrial ROS generation, NLRP3 inflammasome activation, and increased renal impairment [43,49]. Moreover, the decreased level of Parkin in idiopathic pulmonary fibrosis (IPF) lung fibroblasts contributed to the production of mitochondrial ROS [45]. To date, a significant decrease in the function of the electron transfer chain (ETC) is observed in the striatum of Pink1-KO mice [50]. Moreover, it has been shown that the mitochondrial Ca 2+ level can be increased by the mutation of Pink1 gene [51,52]. The evidence above has demonstrated that the Pink1-Parkin signaling pathway is involved in regulating ROS, ETC, and Ca 2+ . Furthermore, mitochondrial damage and increased inflammasome hyperactivation and mortality are associated with mitophagy inhibition by knocking down the SESN2 (Sestrin2) gene in mice during sepsis [27], suggesting mitophagy has anti-mitochondrial damage and anti-inflammatory effects in sepsis. Therefore, we speculated that Pink1/Parkin-mediated mitophagy might play its anti-apoptotic effect by improving mitochondrial functions. We examined the pathophysiological mechanism in terms of cellular ATP synthesis, Ca 2+ level, and ROS content, which confirmed that the Pink1/ Parkin-mediated mitophagy could delay DC mitochondrial dysfunction during sepsis.
In aggregate, our results demonstrated that apoptosisrelated Bcl2/Bax ratio was decreased and cleavedcaspase-3 expression was increased in splenic DC after CLP, and increased levels of DC apoptosis were detected by flow cytometry and TUNEL assays. The expression of mitophagy-related proteins Tom20, Pink1, Parkin, LC3, and p62 was increased, and increased levels of mitophagy after CLP were observed by confocal microscopy and electron microscopy. In the Pink1-KO mice CLP model, we found that the level of apoptosis in DC was further increased, while the level of DC mitophagy was reduced. In addition, we noticed the decreased expression of mitochondrial respiratory chain function-related proteins ATP5A1 and UQCRC1, the impairment of mitochondrial Ca 2+ transport function, and ROS level increment in DC. These verified that Pink1/Parkin was indeed involved in mitophagy. These results suggested that CLP-induced DC apoptosis might be an apoptotic pathway dependent on Pink1/Parkin-mediated mitophagy, or at least had partial involvement. Mitophagy might improve mitochondrial function by removing already damaged mitochondria, reducing the level of apoptosis in DC.
The present study suffers from certain limitations. For example, there exists a lack of exploration on detecting inflammatory factor expression levels in serum and DC, expression levels of MHC II, and PDL 1 on DC. Changes in the survival of septic mice after Pink1 knockout are also not monitored. Moreover, mitochondria are highly active organelles that can affect cells through mitochondrial biosynthesis, mitochondrial division and fusion, mitophagy, and apoptosis. Our study focuses only on Pink1/Parkin-mediated mitophagy, and we are also required to focus on other aspects of mitochondrial dynamics that affect immunity and inflammation. In addition, the depletion of DC immune function is related to apoptosis and degree of differentiation and maturation and ability to activate T cells effectively. We should further investigate the mechanism to regulate immune cells better to treat sepsis patients.
Nevertheless, the data in the present study could demonstrate the protective effect of Pink1/Parkin-mediated mitophagy in preventing DC apoptosis. Future experiments should focus on the signaling pathways and regulatory mechanisms of mitophagy-mediated apoptosis in septic DC and other immune cells.