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Overexpression of Regulatory T Cell-Related Markers (FOXP3, CTLA-4 and GITR) by Peripheral Blood Mononuclear Cells from Patients with Breast Cancer

Background: Regulatory T (Treg) cells are immunosuppressor lymphocytes that play a critical role in the establishment and progression of cancers. A number of markers, especially FOXP3, CTLA-4 and GITR influence the function of Treg cells. This investigation aimed to evaluate the expression of a number of important Treg cell-related markers by peripheral blood mononuclear cells (PBMCs) from newly-diagnosed women with breast cancer. Methods: The fresh PBMCs were obtained from 20 women with breast cancer and 20 healthy individuals. The PBMCs from both groups were cultured for 32 hours in the presence or absence of PHA (10 μg/ml). After total RNA extraction from cultured PBMCs, the expression of the FOXP3, CTLA-4 and GITR transcripts was assessed using real time-PCR. Results: The mRNA expression of FOXP3, CTLA-4 and GITR in unstimulated PBMCs from patients with breast cancer were significantly higher than healthy control group (P<0.05, P<0.03 and P<0.04, respectively). Similarly, the expression of FOXP3, CTLA-4 and GITR transcripts in PHA-stimulated PBMCs from patients with breast cancer were significantly increased in comparison with healthy individuals (P<0.01, P<0.005 and P<0.01, respectively). Conclusion: The increased expression of FOXP3, CTLA-4 and GITR represent higher activity of Treg cells in patients with breast cancer that may play an important role in the tumor establishment and development.

Introduction
Breast cancer is expected to account for 29% of all newly diagnosed cancers and 15% of the all cancer-related deaths among women, worldwide (Siegel et al., 2016). The immune system plays an essential role in defense against tumor cells so that the patients with suppressed or compromised immune function have a very increased incidence of malignancy (Casey et al., 2014). Based on the immune surveillance concept, one of the major duty of the immune system is to identify and kill cancerous cells as they appear (Monzavi-Karbassi et al., 2013). The elements of the both innate and adaptive immunity, such as natural killer (NK) cells, NKT cells, macrophages, neutrophils, eosinophils, specific cytotoxic T lymphocytes (CTLs), antibodies and some cytokines exhibit antitumor activity (Casey et al., 2014;Monzavi-Karbassi et al., recognition/killing by several mechanisms, especially down-regulating of immune responses (Spranger, 2016). Some abnormalities in immune-related parameters were observed in patients with breast cancer (Jafarzadeh et al., 2015a;Jafarzadeh et al., 2015b;Jafarzadeh et al., 2016). The purpose of cancer immunotherapy is to robust the immune system to recognize and kill tumor cells by overwhelming the pathways by which tumor cells evade and suppress the immune responses (Sheikhi et al., 2016).
CD4 + T helper (Th) cells play multiple functions in the induction of immune responses against tumor cells. The effector CD4 + T cells are classified into diverse subsets including Th1, Th2, Th17 and regulatory T (Treg) lymphocytes based on the synthesis of a specific cytokine profile (Golubovskaya and Wu, 2016). Th1 cells produce cytokines such as IFN-γ, IL-2, IL-12 and tumor necrosis factor-beta (TNF-β) that exhibit strong anti-tumor activities by activating CD8 + CTLs and NK-mediated cytotoxicity, as well as improving the expression of major histocompatibility complex (MHC) and costimulatory molecules on the surface of antigen presenting cells (APCs) (Golubovskaya and Wu, 2016;Kursunel and Esendagli, 2016). Conversely, Th2 cell-related cytokines (including IL-4, IL-5, IL-6 and IL-13) inhibit anti-tumor immune responses by down-regulation of Th1 cells (Golubovskaya and Wu, 2016;Jafarzadeh et al., 2015b). A large number of pro-inflammatory cytokines release by Th17 cells, particularly IL-17 (also known as IL-17A), IL-17F, IL-21, IL-22 and GM-CSF (Etesam et al., 2016;Guery and Hugues, 2015). There are some controversies and inconsistencies regarding the role of Th17 cells in tumor immunology. The proor anti-tumor effects of Th17 cells may be exert in a tumor type-dependent manner. Therefore, Th17 cell-associated immune responses were related with both good or bad prognoses in cancer investigations (Guery and Hugues, 2015).
Regulatory T (Treg) cells play a prominent role in the regulation of the immune activities and maintain tolerance to self-antigens through a number of mechanisms such as suppression of antigen-presenting cells via CTLA-4, secretion of immunomodulatory cytokines (IL-10, TGF-β and IL-35), expression of granzyme/perforin, consumption of IL-2, and degradation of ATP (Jafarzadeh et al., 2015c;Takeuchi and Nishikawa, 2016). Treg cells make up 5-15% of the CD4 + T cells and there are two subsets of Treg cells, including natural Treg (nTreg) cells that develop in the thymus and inducible Treg (iTreg) cells that arise from naïve CD4 + T cells in the peripheral tissues after antigenic stimulation, in the presence of TGF-β and IL-2, respectively (Jafarzadeh et al., 2015c;Noack and Miossec, 2014). The differentiation, maintenance and suppressor function of Treg cells is regulated by a master transcription factor Forkhead Box P3 (FOXP3) which is encoded by a related gene that mapped on the X chromosome (Etesam et al., 2016;Jafarzadeh et al., 2015c;Szylberg et al., 2016).
Treg cells suppress a wide range of immune cells such as CD8 + T, NK, B and APCs (Jafarzadeh et al., 2015a;Muenst et al., 2016). Treg cells are aggregated into tumor tissues by chemokine CCL22 that is secreted by tumor cells and macrophages (Jafarzadeh et al., 2015a;Muenst et al., 2016). There are some investigations regarding the Treg cell-dependent immunosuppression in cancer patients (Hatanaka et al., 2014;Tohyama et al., 2013). The suppression of cell-mediated immunity and the enhancing of the number and/or function of Treg cells may lead to the tumor progression (Hatanaka et al., 2014). The immunosuppressive functions of Treg cells are influenced by some markers, particularly FOXP3, Cytotoxic T lymphocyte antigen-4 (CTLA4) and glucocorticoid induced TNFR family-related gene (GITR) (Nasser et al., 2014;Rathod et al., 2014).
The CD4 + T cells recognizing MHC-antigenic peptide which is presented on the surface of APCs (Beyersdorf et al., 2015). The recognition of MHC-antigenic peptide by T cell receptor (TCR) is necessary for T cell activation. In addition, the binding of costimulatory molecules, especially CD28, on the T cell to counterpart molecules B7-1 and B7-2 on the APCs providing a second costimulatory signal which is required for full activation of T cell (Beyersdorf et al., 2015;Buchbinder and Hodi, 2015). Since B7-1 and B7-2 molecules bind to both CTLA-4 and CD28 molecules, therefore, CTLA-4 may negatively influences T cell activation by competing with CD28 for attachment to B7 molecules. CTLA-4 is also constitutively expressed on Treg cells and its ligation positively reinforcements the immunosuppressive functions of Treg cells (Buchbinder and Hodi, 2015).
The GITR is also constitutively expressed at high amounts on the surface of Treg cells, but at low quantities on naïve and memory T lymphocytes (Knee et al., 2016). Following TCR engagement, the GITR ligation on T cells with its ligand (GITRL) or agonist antibodies, reinforces T cell activation by inducing IL-2 and IFN-γ expression, enhancing CD25 expression and promoting cell expansion (Knee et al., 2016). Although, high expression of GITR is a feature for Treg cells, its function on these cells is more complex. There are some reports showing that GITR ligation inhibits immunosuppressive activities of Treg cells (Knee et al., 2016). In a murine model it has been indicted that GITR acts as a costimulatory molecule for activation of Treg cells (Bianchini et al., 2011).
Although, there are a number of reports regarding the determination of the FOXP3 and CTLA-4 expression in the peripheral blood from patients with breast cancer (Jaberipour et al., 2010;Kawaguchi et al., 2017), however, there is no investigation regarding the measurement of FOXP3, CTLA-4 and GITR in PBMCs from malignant patients after in vitro stimulation. This study aimed to evaluate the expression of some Treg cell related markers (including FOXP3, CTLA4 and GITR) by PBMCs from patients with newly diagnosed breast cancer following the activation with phytohemagglutinin (PHA) mitogen.

Subjects
From October 2015 up to May 2016, 20 women with breast cancer (mean age: 41.25 ± 9.07 years) were selected among patients who referred to the affiliated hospitals of Jundishapur University of Medical Sciences. The patients were newly diagnosed, and enrolled to study before they receive chemotherapy, radiotherapy or immunotherapy. The presence of breast cancer and its staging was confirmed by expert oncologists based on the defined surgical and pathological observations and also according to the criteria of the Sixth Edition of the American Joint Committee on Cancer (AJCC) (Singletary et al., 2003). Moreover, 20 healthy women (mean age: 39.45 ± 7.89 years) were enrolled into the study as a control group. The healthy individuals were randomly recruited among blood donors and were in good health, without any acute or chronic illnesses. The procedure was evaluated and approved by the Ethical Committees of Rafsanjan University of Medical Sciences and the written informed consent was also obtained from participants before blood sampling.

Isolation of peripheral blood mononuclear cells (PBMCs)
Five to ten mL of fresh peripheral blood was collected relative to the amount of GAPDH expression that was calculated by the 2 -δδCt formula. The PCR products were also electrophoresed and observed on a 1% agarose gel comprising 0.5 mg/ml ethidium bromide.

Statistical analysis
The data were presented as mean ± SEM. Statistical comparison was analyzed by a computer program (SPSS version 18, Chicago, IL, USA) by using ANOVA and Student's t test, as appropriate. The P values less than 0.05 were considered to be statistically significant.

The expression of FOXP3 by PBMCs from breast cancer patients and healthy group
The fold change of the gene expression of FOXP3 in the unstimulated and PHA-stimulated PBMC from women with breast cancer and healthy subjects have been demonstrated in Table 2 and Figure 1. In healthy women, the expression of FOXP3 mRNA in PHA-stimulated cultures was significantly lower compared with unstimulated cultures (P<0.02). In patients with breast cancer, the amounts of the FOXP3 expression in the PHA-stimulated PBMCs was also lower than unstimulated cultures, but the difference did not reach to a significant level. The amounts of the FOXP3 expression were significantly increased in the unstimulated and PHA-stimulated PBMCs from patients with breast cancer compared with equal cultures from healthy women (P<0.05 and P<0.01, respectively).

The expression of CTLA4 by PBMCs from breast cancer patients and healthy group
In both healthy normal and breast cancer groups, the amounts of the CTLA4 mRNA expression in cultures stimulated with PHA was significantly higher than unstimulated PBMCs (P<0.001 and P<0.01, respectively) ( Figure 2 and Table 2). The amounts of the CTLA4 expression by unstimulated and PHA-induced PBMCs from patients with breast cancer were significantly higher than counterpart cultures from healthy women (P<0.03 and P<0.005, respectively) ( Figure 2 and Table 2).
into heparinized tubes from healthy women and patients with breast cancer. The PBMCs were separated by a standard procedure using Ficoll-Paque (Sigma, USA). Briefly, a 1:2 dilution was made by adding an equal volume of phosphate buffered saline (PBS) to a blood sample. The diluted blood was then carefully layered over the equal volume of Ficoll-Paque solution. The sample was centrifuged for 30 minutes at 20 °C, with a rate of 3000 rpm. Immediately after centrifugation, the upper layer was removed and discarded. The mononuclear cell layer was carefully transferred to a new 15 mL tube and washed three times by mixing with 10 mL of Roswell Park Memorial Institute (RPMI)-1640 media. Then, the final pellet of PBMCs were resuspended in a complete culture media of RPMI-1640 containing 10% heat inactivated fetal bovine serum (Gibco Life Techologies Ltd, Paisley, UK) and antibiotics, including penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were then seeded at 1 × 10 6 cells per milliliter in 24-well culture plates (Nunc, Thermo Fisher Scientific Inc, Denmark) in the presence or absence of 10 μg/ml of a T cell stimulator, phytohemagglutinin [(PHA) (Gibco Life Techologies Ltd, Paisley, UK)]. The PBMCs were incubated for 32 hours at 37 °C in a 5% CO 2 incubator. After 32 hours, the cells were harvested for total RNA extraction and further experiments.

RNA extraction, reverse transcription and quantitative real-time PCR
A Trizol reagent (Bionner, Korea) was used to extract the total RNA from the PBMCs, according to the manufacturer's guideline. The quality of the extracted RNA was determine by electrophoresis on the agarose gel pre-treated with ethidium bromide. A spectrophotometer system used to measure the purity and quantity of RNA, basis on the measuring its absorption at 260 nm and 280 nm. A complementary DNA (cDNA) synthesis kit (Applied Biosystems, USA) was used to generate cDNA from extracted RNA. The reverse transcription protocol was included: 70°C for 10 minutes (in the absence of reverse transcription enzyme), 20°C for 1 minute (cooling stage), addition of reverse transcription enzyme, 42°C for 60 minutes, and eventually the protocol was completed by a step at 95°C for 10 minutes to halt the function of the reverse transcription enzyme.
The analysis of the gene expression of FOXP3, CTLA4 and GITR was performed using a real-time PCR instrument (Applied Biosystems, USA) using a SYBR green master mix (Applied Biosystems, USA), mixed with 200 ng of template cDNA with 2 µL suitable primers (10 pmol stock). The primers synthesized by Bioneer Company (Korea). The sequences of the used primers were indicated in Table 1. The program of thermal cycler was entailed: 50°C for 2 minutes, 95°C for 2 minutes; 40 cycles of 95°C for 15 seconds, 60°C for 1 minutes, 95°C for 15 seconds.
For normalization of the amplified target genes, the GAPDH gene was used as an internal control or housekeeping gene. The amount of the FOXP3, CTLA4 and GITR expression in the PBMCs expressed as units Genes Primers

The expression of GITR by PBMCs from breast cancer patients and healthy group
In both normal control and breast cancer groups, the PHA-induced expression of GITR mRNA was lower than unstimulated PBMCs, but the differences were not significant ( Figure 3 and Table 2). The fold change expression of GITR by unstimulated and PHA-stimulated PBMCs from patients with breast cancer were significantly increased as compared with equal cultures from healthy subjects (P<0.04 and P<0.01, respectively) ( Figure 3 and Table 2).

Discussion
Results from our study showed that the mRNA expression of FOXP3 in PBMCs stimulated with PHA was significantly decreased as compared with unstimulated cultures. In both healthy control and breast cancer groups, the PHA-induced CTLA4 expression was significantly higher than unstimulated PBMCs. However, the amounts of the GITR mRNA expression did not significantly differ between unstimulated and stimulated PBMCs neither in the healthy women nor in patients with breast cancer. These findings represent that PHA may differentially influence the expression of   the FOXP3, CTLA-4 and GITR. In agreement with our results, it has been reported that PHA-induced expression of FOXP3 was reduced in healthy subjects and patients with atherosclerosis (Mottaghi et al., 2012). The influences exerted by PHA may be contributed to the polyclonal activation of T cells, which lead to the differentiation of naïve T CD4 + into various effector T cells, including Th1-, Th2-and Th17 cells, and these aforementioned cells may have inhibitory effects on the FOXP3 expression and Treg cell differentiation. In patients with breast cancer, the amounts of the FOXP3 expression in PBMCs stimulated with PHA was lower than unstimulated cells, but the difference was not statistically significant. This difference may be due to the higher frequency of Treg cells in patients with breast cancer in comparison with healthy individuals. The results of the present investigation indicated that the gene expression of a number of Treg cell-related markers including FOXP3, CTLA4 and GITR were significantly increased in both unstimulated-and PHA-treated PBMCs from newly-diagnosed patients with breast cancer in comparison with healthy control women. The increased expression of FOXP3 lead to an increase in the number of Treg cells and enhanced immunosuppressive function in patients with breast cancer. Moreover, the elevated amounts of the FOXP3 expression by PHA-treated PBMCs from patients with breast cancer representing an increased potential for differentiation of Treg cells that lead to the suppression of anti-tumor immunity.
The results of this study also indicated that the gene expression of CTLA4 was significantly increased in both unstimulated-and stimulated PBMCs from patients with breast cancer as compared with healthy control group. CTLA-4 palys an important role in damping of anti-tumor immunity and therefore in cancer development and progression (Buchbinder and Hodi, 2015). Increased number of Treg cells and higher expression of CTLA4 have been observed in patients with various types of cancer compared to control individuals (Kordi-Tamandani et al., 2014). Binding of CTLA-4 on T cells to B7 molecules on APCs down-regulates the activation of T-cells through various mechanisms (Beyersdorf et al., 2015). Since B7 molecules bind to both CTLA-4 and CD28 molecules, CTLA-4 may competitively block CD28-B7 interactions. Moreover, CTLA-4 can directly suppress the TCR-mediated signaling, decrease the expression of IL-2 and its receptor, and modulate the cell proliferation (Buchbinder and Hodi, 2015). It has been also reported that CTLA-4 may contribute in the generation of peripheral tolerance through induction of apoptosis or anergy in antigen specific T cells (Buchbinder and Hodi, 2015). It has been demonstrated that the CTLA-4-blocking antibodies increase antitumor immune responses through promotion of the CD8 + CTL activation and depletion of Treg cells (Buchbinder and Hodi, 2015). As mentioned the CTLA-4 is constitutively expressed on Treg cells and antibodies against CTLA-4 lead to the depletion of intratumoral Treg cells, perhaps via induction of antibody-dependent cell-mediated cytotoxicity (ADCC) (Buchbinder and Hodi, 2015).
Here, we also showed for the first time that the expression of GITR by both unstimulated and stimulated PBMCs from patients with breast cancer was higher than healthy control group. There are some reports regarding the damping of anti-tumor immunity by GITR. It has been demonstrated in patients with acute myeloid leukemia (ALL) that the GITR ligation on NK cells impair NK cytotoxicity, reduce IFN-γ production and enhance IL-10 secretion (Baessler et al., 2009). IFN-γ plays an important role in the limiting of malignant cells as it has anti-proliferative, anti-angiogenic and pro-apoptotic effects (Kursunel and Esendagli, 2016). IFN-γ also enhances antigen presentation to T cells, stimulates the synthesis of the tumoricidal intermediates such as reactive oxygen species (ROS) and nitric oxide (NO), increases the expression of IL-12, IL-18, and CD86 by APCs, promotes the differentiation of Th1 cells, potentiates the CTL function, and inhibits Th2 and Treg cell differentiation (Kursunel and Esendagli, 2016). On the other hand, IL-10 may help tumor cell to escape from immune system through damping the anti-tumor immune response (Mannino et al., 2015). It has been indicated that the blocking of GITR on NK cells enhances their cytotoxicity Figure 3. The mRNA Expression of GITR by Unstimulated and PHA-stimulated PBMCs from the Healthy Women and Breast Cancer Patients. The GITR expression by unstimulated and PHA-stimulated PBMCs from patients with breast cancer was significantly higher than equal cultures from healthy women. and IFN-γ secretion (Baessler et al., 2009).
GITR marker was also expressed at high amounts on the surface of NK cells from patients with Chronic lymphocytic leukemia (CLL) (Buechele et al., 2012). Upon interaction of GITR with GITRL on leukemia cells, the GITRL-mediated signaling lead to the secretion of IL-6, IL-8 and TNF-α, which act as proliferative and survival factors for cancer cells (Buechele et al., 2012). In addition, GITR ligation impair the NK activity and reduce the IFN-γ production by NK cells, which could be improve by GITR blocking antibodies. Thus, GITR-GITRL interaction may involve in the pathogenesis of CLL and inhibit the NK cell-mediated tumor cell killing (Buechele et al., 2012).
On the other hand, the results of a number of investigations in murine model were indicated the participation of GITR in the induction of antitumor immune responses. For example, the administration of agonistic antibodies against GITR enhances survival rates in tumor-bearing animals (Naidoo et al., 2014). In addition, the combined programmed Death 1 (PD-1) blockade and GITR stimulation induce a powerful antitumor immunity in a murine ovarian cancer model (Lu et al., 2014). In the murine models, the anti-tumor activity of GIRT has been mainly attributed to the stimulation of CTLs and Th1 cells, induction of M1 macrophages and inhibition of Treg cells (Kawano et al., 2015;Patel et al., 2016). Therefore, it seems that GITR may plays different roles in mice and humans (Nocentini et al., 2012). Furthermore, species-related differences concerning the effects of GITR on the T cells have also been indicated (Nocentini et al., 2012). In contrast to the murine system, GITR does not inhibit human Treg cells-related immunosuppression (Levings et al., 2002). It should be also noted that GITR role in the anti-tumor immunity may be exert in a tumor type-dependent manner. Further investigations are required to clearly elucidate whether GITR mediates various influences in different tumors.
In conclusion, the higher expression of FOXP3, CTLA4 and GITR represent higher activity of Treg cells in patients with breast cancer that may play a role in the tumor development. These findings emphasize that the targeting of Treg cells with aforementioned markers may be a suitable strategy for breast cancer immunotherapy.

Conflict of interest
The authors have no any conflict of interest.


extfieldsofstudy: Chemistry Medicine

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Protective Effect of Phaleria macrocarpa Water Extract (Proliverenol) against Carbon Tetrachloride-Induced Liver Fibrosis in Rats: Role of TNF-α and TGF-β1

Phaleria macrocarpa is one of the Indonesian herbal plants which has been shown to have a hepatoprotective effect. This study was conducted to evaluate the protective effect of water extract of mahkota dewa (Phaleria macrocarpa) in liver fibrosis and to elucidate its mechanism of action. Male Sprague-Dawley rats were treated with carbon tetrachloride (CCl4) for 8 weeks to induce liver fibrosis. Rats were randomly divided into 6 groups (n=5), i.e., control group, CCl4 group, CCl4 + NAC group, CCl4 + various doses of water extract of Phaleria macrocarpa (50, 100, and 150 mg/kg body weight). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), liver histopathology, malondialdehyde (MDA), ratio GSH/GSSG, Tumor Necrosis Factor- (TNF-) α, and Transforming Growth Factor- (TGF-) β1 were analyzed. This study demonstrated that water extract of Phaleria macrocarpa and NAC significantly protected CCl4-induced liver injury as demonstrated by reduced AST, ALT, ALP, and fibrosis percentage compared with the CCl4-only group. In addition, water extract of Phaleria macrocarpa and NAC significantly reduced the levels of MDA, TNF-α, and TGF-β1 as well as increasing the ratio of GSH/GSSG. Water extract of Phaleria macrocarpa prevents CCl4-induced fibrosis in rats. The prevention of liver fibrosis was at least in part through its antioxidant and anti-inflammatory activities and through its capacity to inhibit hepatic stellate cells (HSC) activation by reducing fibrogenic cytokine TGF-β1.

Introduction
Liver fibrosis is the accumulation of extracellular matrix, or excess connective tissue, in response to chronic liver injury. The main causes of liver fibrosis were viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), autoimmune disease, or hypoxia [1][2][3][4]. The progression of liver fibrosis often develops into irreversible cirrhosis and is associated with liver cancer [4].
Carbon tetrachloride (CCl 4 ), a well-known hepatotoxin, is widely used in laboratory animals to induce liver injuries including liver fibrosis. CCl 4 requires biotransformation to produce free radicals that eventually lead to membrane lipid peroxidation and causes molecular damage in hepatocytes [5,6]. Hepatic stellate cells (HSC), whose main function is to store vitamin A and plays a pivotal role in activating the immune response via secretion of cytokines and chemokines, are activated as a result of liver injury. Once activated, HSC increase their production of extracellular matrix (ECM) protein leading to liver fibrosis. Activation of HSC is dependent on cytokines, particularly inflammation cytokines, TNF-, and fibrogenic cytokines, TGF- [2,7].
The purpose of this study was to investigate the preventive effect of water extract of mahkota dewa fruits (Proliverenol) 2 Journal of Toxicology in CCl 4 -induced liver fibrosis in rats and its mechanism of action. For comparison we used N-acetylcysteine (NAC) which has antioxidant and free radical scavenging action [6,12,13].

Materials and Methods
2.1. Animals. Thirty male Sprague-Dawley rats weighing 200-350 g were used after 1 week for acclimatization to the animal house conditions (12-hour light/dark cycle). They were obtained from the National Agency of Drug and Food Control, Indonesia. Water and food were provided ad libitum. This study was approved by the Institutional Ethics Committee, Faculty of Medicine, Universitas Indonesia.

Water Extract of Mahkota Dewa Fruits (Proliverenol).
Aqueous extracts of mahkota dewa fruits (Proliverenol) were kindly provided by Dexa Laboratories of Biomolecular Sciences, PT Dexa Medica Pharmaceutical Company (Jakarta, Indonesia). The extraction method was subcritical water extraction, using water under external pressurization above its boiling point as an extraction solvent [14]. As described by Kim et al. (2010), the extraction process was conducted at 373 K, 4.0 MPa, and 5 hours [14]. The extract was then filtrated, concentrated, dried in a conventional oven, and then stored in a well closed container. Identification of Proliverenol was done by thin layer chromatography plate using ethyl acetate: acetone: formic acid: water (8: 2: 1: 1, v/v) as the eluent [15].

Experimental Design.
The rats were randomly allocated to the six groups, namely, the control group (n = 5), the CCl 4 group (n = 5), the CCl 4 + NAC group (n = 5), the CCl 4 + Proliverenol 50 mg/kg BW group (n = 5), the CCl 4 + Proliverenol 100 mg/kg BW group (n = 5), and the CCl 4 + Proliverenol 150 mg/kg BW group (n = 5). Proliverenol and NAC were dissolved in olive oil. The model of liver fibrosis was induced by i.p. injection with CCl 4 (0.2 mL/100 g BW) mixed with olive oil as vehicle in 1:1 ratio twice weekly for 2 weeks followed by i.p. injection of mixed CCl 4 (0.1 mL/100 g BW) and olive oil (1:1 v/v) twice weekly for 6 weeks as described by Constandinou [16]. NAC and Proliverenol were given once daily by oral gavage for 8 weeks.
Seventy-two hours after the last CCl 4 injection, rats were sacrificed, and blood samples were collected in the EDTA tubes and centrifuged at 3,000 g, 4 ∘ C for 15 min, to obtain plasma. The samples were stored at -20 ∘ C until analyzed. The liver was excised from each animal, and a portion of the liver was taken for histopathological examination, while the remaining tissue was kept in freezer at -80 ∘ C until analyzed.

Biochemical
Analysis. Activities of ALT, AST, and ALP in plasma and the ratio of GSH/GSSG in the liver were determined according to the manufacturer's guideline. Hepatic lipid peroxidation was determined as thiobarbituric acting-reacting substance and is expressed as equivalent of malondialdehyde (MDA) using 1,1,3,3-tetramethoxypropane as standard. Levels of TNF-and TGF-1 in the liver were determined using ELISA kits according to the manufacturer's guideline. Protein concentrations were measured according to the manufacturer's guideline.
2.6. Liver Histopathological Analysis. Liver tissue samples were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with Masson's trichrome to identify increases in liver collagenous tissue. The degrees of liver fibrosis were evaluated by a pathologist from the Faculty of Veterinary Medicine, Bogor Agricultural University, in a blinded manner. Quantification of the degree of fibrosis was done by ImageJ software with threshold-color plug-in logarithm. Percentage of fibrosis was calculated by dividing the area of fibrosis (blue regions) by the total area.

Statistical
Analysis. SPSS 16.0 software package was used for statistical analysis. Results were expressed as means ± standard deviations (SD). Statistical analysis was performed by one-way ANOVA test followed by Tukey's postanalysis test for multiple comparisons. p <0.05 was considered as statistically significant.

Biochemical Markers.
Plasma ALT, AST, and ALP activities were measured as markers of liver injury. The effect of oral administration of Proliverenol and NAC on plasma ALT, AST, and ALP activities is presented in Figures 1(a)-1(c). Significant increases in the activities of these marker enzymes were observed in CCl 4 -treated rats. Treatment with Proliverenol and NAC significantly decreased those enzyme activities.

Histopathological Findings.
Histopathological examinations of the liver sections and the percentage of liver fibrosis were presented in Figures 2(a) and 2(b). Significant increases of liver fibrosis were observed in CCl 4 -treated rats. Interestingly, treatment with Proliverenol and NAC significantly decreased the percentage of liver fibrosis.

Hepatic MDA and Ratio GSH/GSSG.
Oxidative stress plays an important role in the development of liver fibrosis. MDA, the main final product of lipid peroxidation, was measured in rats' livers to determine the membrane lipid oxidative damage [17], whereas the ratio of GSH/GSSG was measured as an indicator of oxidative stress [17,18]. A significant increase of MDA levels and a significant decrease of GSH/GSSG ratio were observed in CCl 4 -treated rats, while the levels of MDA and the ratio of GSH/GSSG were decreased   and increased, respectively, in the Proliverenol and NAC groups, as compared to the CCl 4 -treated groups (Figures 3(a)  and 3(b)).

Hepatic Level of TNF-.
Inflammation is commonly associated with liver fibrosis during chronic liver injury [13,17]. To explore the mechanisms underlying the protective effect of Proliverenol on liver fibrosis, we proposed that Proliverenol might protect the liver against CCl 4 -induced injury by suppressing inflammation in the liver. The levels of proinflammatory cytokines TNF-were determined in the liver by ELISA. As shown in Figure 4(a), significantly increased levels of TNF-were observed in CCl 4 -treated rats. Treatment with Proliverenol and NAC decreased the levels of TNF-in a significant manner.

Hepatic Level of TGF-1 .
TGF-1 is the major profibrogenic cytokine [1][2][3]19]. To elucidate the underlying mechanisms of Proliverenol in the suppression of liver fibrogenesis, we measured the levels of TGF-1 in the rat model. The levels of profibrogenic cytokines TGF-1 were determined in the liver by ELISA. As shown in Figure 4(b), significantly increased levels of TGF-1 were observed in CCl 4 -treated rats. On the other hand, treatment with Proliverenol and NAC significantly decreased the levels of TGF-1 .

Discussion
This study has demonstrated that intraperitoneal injection of CCl 4 to rats for 8 weeks leads to a marked elevation in the activities of plasma ALT, AST, and ALP as compared with the control group.  myofibroblast-like cells which resulted in the secretion of several profibrogenic cytokines such as TGF-, endothelin-1, and platelet-derived growth factor [21,22]. Treatment with Proliverenol and NAC significantly reduced the activities of those marker enzymes as compared with CCl 4 -treated rats. This result implied that Proliverenol and NAC potentially prevent liver damage by suppressing the leakage of enzymes through cellular membranes and hence restore the enzymes' activities in the hepatocytes. However, there was no evidence of a dose-response relationship with regard to efficacy parameters including fibrosis percentage, MDA, GSH/GSSG ratio, TNF-, and TGF-among treatments. Our results with Proliverenol are in agreement with results obtained by Nailufar et al. in ethanol-induced liver damage using DLBS1433 (Proliverenol). They showed that, among various concentrations of DLBS1433-60, maximum antioxidant activity was showed by DLBS1433-60 at a concentration of 100 ppm with no further increase of antioxidant activity if the concentration was increased [23]. The histopathological analysis revealed that chronic administration of CCl 4 produced a marked increase in collagen deposition, as evidenced by the blue stains around the portal triad and increased the percentage of liver fibrosis. Treatment with Proliverenol and NAC significantly reduced the percentage of liver fibrosis as compared with CCl 4 -treated and control rats.
The   and Kamalakkannan et. al (2005) [12]. This increase may be associated with the generation of trichloromethyl radicals ( • CCl 3 ) and trichloromethyl peroxyl radical ( • O 2 CCl 3 ) following metabolism of CCl 4 by cytochrome P450 enzyme [24][25][26]. In this study, we demonstrated that the levels of MDA were decreased in the CCl 4 + Proliverenol groups as well as in the CCl 4 + NAC group. We believe that the antioxidant capacity to scavenge reactive oxygen species is the main mechanism of action of Proliverenol and NAC to protect the hepatic parenchyma against the trichloromethyl and trichloromethyl peroxyl radicals.
GSH is an important nonenzymatic antioxidant required to maintain the normal redox state of cells [17,27]. Oxidative stress induced by CCl 4 results in the increased utilization of GSH and subsequently decreased the ratio GSH/GSSG in tissue, thereby affecting functional and structural integrity of cell and organelle membranes [17]. Our results showed that oral administration Proliverenol and NAC with CCl 4 increased the ratio GSH/GSSG in liver tissues as compared to CCl 4 administered alone, which indicated their potential to counteract the oxidative damage induced by CCl 4 and to reinforce the antioxidant defense in normal conditions. TNF-is a key factor that triggers an inflammatory cascade involving the induction of cytokine after liver injury. The injured liver, predominantly Kupffer cells and infiltrating macrophages and neutrophils, will produce TNF-. In particular, TNF-plays a dichotomous role in the liver, where it not only induces hepatocyte proliferation, apoptosis, and inflammation but also is known to suppress collagen 1 gene expression [13,17,20,27,28]. Our results showed that oral administration of Proliverenol and NAC with CCl 4 reduced the levels of TNF-in the liver as compared to CCl 4 administered alone. This result suggested that protective effect of Proliverenol and NAC on CCl 4 -induced liver fibrosis was at least, in part, due to their capabilities to suppress TNFproduction. Activation of HSC was triggered by TGF-1 , which was released from Kupffer cells as well as oxidative stress caused by CCl 4 [16]. Besides, activation of HSC can also be provoked by a range of chronic injuries to the liver such as viral hepatitis, toxins, and autoimmune disorders [29]. TGF-1 is a major profibrogenic cytokine. Its main functions are regulating the production, degradation, and accumulation of the ECM in liver fibrosis. TGF-1 leads fibrogenesis through autocrine and paracrine effects of HSC [17,20,30]. Our study showed that oral administration of Proliverenol and NAC with CCl 4 reduced the levels of TGF-1 in liver tissue as compared to CCl 4 administered alone. This suggested that protective effect of Proliverenol and NAC on CCl 4induced liver fibrosis was associated with their abilities to inhibit HSC activation by reducing TGF-1 production.
In conclusion, the protective effect of Proliverenol in CCl 4 -induced liver fibrosis in rats appears to stem from its antioxidant capacities as indicated by protection against increased lipid peroxidation and decreased ratio GSH/GSSG and its ability to suppress proinflammatory cytokines TNFand to inhibit HSC activation by reducing profibrogenic cytokines TGF-1 . Thus, we believe that Proliverenol might be a therapeutic candidate for the prevention of liver fibrosis.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.


s2fieldsofstudy: Biology Medicine

text:
Klotho ameliorates sepsis-induced acute kidney injury but is irrelevant to autophagy

Background The role of Klotho (KL) in sepsis-induced acute kidney injury (AKI) and the potential relationship between KL and autophagy in septic AKI were investigated. Materials and methods A murine model of sepsis-induced AKI was established by cecal ligation and puncture (CLP). Mice undergoing CLP and immortalized proximal tubular epithelial human HK-2 cells that were exposed to lipopolysaccharide (LPS) were treated with recombinant KL, autophagy stimulator rapamycin (Rap), and autophagy suppressor 3-methyladenine (3-MA). Results Autophagy activation and KL reduction reached maximum levels in mice 24 hours after CLP. Recombinant KL and/or Rap significantly attenuated CLP-induced renal dysfunction (P<0.05) and partially restored endogenous renal KL expression (P<0.05). Recombinant KL had no impact on CLP-induced autophagy and apoptosis, whereas Rap significantly stimulated autophagy and reduced apoptosis in mice. 3-MA significantly exacerbated renal dysfunction, increased apoptosis, and inhibited autophagy in mice with CLP-induced AKI (all P<0.05). In LPS-treated HK-2 cells, Rap significantly enhanced autophagy and reduced apoptosis (all P<0.05), whereas recombinant KL had no impact, and 3-MA inhibited autophagy and significantly increased apoptosis (P<0.05). Conclusion Recombinant KL alleviates renal dysfunction and restores renal KL expression in mice with sepsis-induced AKI, but the underlying mechanism may not be related to autophagy induction.

with AKI, and KL expression is also decreased in mice with ischemia-reperfusion injury (IRI). [17][18][19] In addition, injection of recombinant KL increases endogenous KL expression and attenuates kidney damage in rats with AKI. 17 Therefore, KL may have a protective effect on kidney function. The cellular mechanism underlying KL-mediated renoprotection may relate to autophagy stimulation. In vitro, overexpression of KL can induce autophagy in hepatocellular carcinoma and gastric cancer cells. 20,21 Moreover, autophagy can protect the kidney from toxin-mediated AKI and ischemia-reperfusioninduced AKI. 22,23 To investigate the role of KL in sepsis-induced AKI, we established a mouse model of sepsis-induced AKI by cecal ligation and puncture (CLP) and investigated the effects of administration of recombinant KL on endogenous KL expression and kidney function in mice with CLP-induced AKI. An in vitro cell line model, human proximal tubular epithelial cell line HK-2, was also used to investigate the role of KL in autophagy. humidity under a 12-hour light/dark cycle and had free access to food and water. The mice habituated to their environment for 7 days before they were used for experiments. Every effort was made to minimize the number and suffering of the mice.

clP in mice
Mice were randomized into the following nine groups (n=5 in each group): normal (N), sham (S), CLP model (CLP), CLP model treated with KL (CLP+KL), CLP model treated with Rap (CLP+Rap), CLP model treated with KL and Rap (CLP+KL+Rap), CLP model treated with CMC solution (CLP+CMC), CLP model treated with 3-MA (CLP+3-MA), and CLP model treated with KL and 3-MA (CLP+KL+3-MA). CLP-induced sepsis in mice was established as previously described. 25,26 Briefly, the mice underwent 1 cm midline laparotomy under anesthesia (intraperitoneal injection of pentobarbital sodium, 50 mg/kg). The caecum was ligated at 1 cm from the end with a 4-0 silk suture. A 22-gauge needle was used to perforate the caecum with two holes near the ligation site. A small amount of cecal content was squeezed out of the caecum through the two holes to induce sepsis. After the ligated and punctured caecum was returned to the peritoneal cavity, the muscle layer and epidermal layer were sutured, and 1 mL of 0.9% sterile saline solution (37°C) was administered subcutaneously. Successful establishment of CLP-induced sepsis was confirmed by the following criteria: 1) survived 6 hours after the surgery; 2) developed the characteristic symptoms of sepsis, including diarrhea, piloerection, and hypoergia; and 3) exhibited a peak in mortality at 24 hours, with 40%-60% mortality at 72 hours after the surgery. Sham-operated mice underwent laparotomy and bowel manipulation without ligation or puncture. None of the mice received antibiotic treatment. The mice were allowed free access to food and water before and after the surgery. Recombinant mouse KL (0.01 mg/kg) was administered by intraperitoneal injection 1 hour after CLP surgery, as previously described. 24,27 Rap (1 mg/kg) or 3-MA (30 mg/kg) was administered by intraperitoneal injection 2 hours before CLP surgery to activate or suppress autophagy, respectively. 28 The mice were sacrificed by cervical dislocation at 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, and 5 days after CLP surgery. Blood samples were collected, and plasma was stored at -80°C. Both kidneys of each mouse were removed, and each kidney was cut into four pieces. Three pieces were instantly snap-frozen in liquid nitrogen and stored at -80°C. The remaining piece was fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in PBS and then prepared as

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Kl ameliorates sepsis-induced aKi paraffin-embedded or epon-embedded tissue blocks for light microscopy or electron microscopy, respectively.

assessment of mouse renal function
Mouse renal function was assessed by blood urea nitrogen (BUN) and serum creatinine (Scr) levels. Blood samples were collected 1 day after CLP surgery. Blood samples were centrifuged at 2,500 rpm for 10 min to separate the serum. BUN and Scr concentrations were measured using a kit (Nanjing Jiancheng Biology Engineering Institute, Nanjing, China, C013-2 and C011-2) according to the manufacturer's instructions.

light microscopy of mouse kidney specimens
Paraffin-embedded kidney specimens were sectioned (3 μm thickness). Tissue sections were stained with H&E and periodic acid-Schiff (PAS) according to the standard protocols and observed under a light microscope (BX53; Olympus Corporation, Tokyo, Japan).

electron microscopy
Approximately 1 mm 3 epon-embedded kidney specimens were fixed in 1% osmium tetroxide, stained with uranyl acetate and lead citrate, and cut into ultrathin sections. The sections were observed under a Hitachi H7500 electron microscope (Hitachi Ltd., Tokyo, Japan).

immunohistochemistry (ihc)
Paraffin-embedded kidney specimens were sectioned (3 μm thickness) and incubated with 3% H 2 O 2 for 10 min. After tissue sections were washed with PBS, they were boiled in 10 mM citrate buffer (pH 6.0) for 4 min for antigen retrieval and then blocked with 10% goat serum in PBS ( . The tissue sections were counterstained with hematoxylin, dehydrated, and mounted on glass slides. Tissue sections that were incubated with secondary antibody alone were used as a negative control for IHC staining. Images at 200× magnification were acquired using a biological imaging microscope (BX53; Olympus Corporation). The intensity of positive staining, which appeared brown, was determined using Image-Pro plus 6.0 image analysis software. The mean optical density (MOD) was calculated to represent the intensity. MOD values increased as protein expression increased.

Apoptosis analysis by flow cytometry
HK-2 cells were seeded in 6-well plates at a density of 2×10 6 cells per well. After reaching 80% confluency, the cells were treated with the eight drug combinations. The cells were then trypsinized and incubated in 500 μL of binding buffer containing 5 μL of Annexin V-FITC and 5 μL of propidium iodide (BD Biosciences, San Jose, CA, USA [556547]) in the dark for 15 min. The cells were analyzed with a flow cytometer (FACSCalibur, BD, Franklin Lakes, NJ, USA). Flow cytometry data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).

Western blot
Mouse renal tissues were homogenized, and HK-2 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium Immunofluorescence assay HK-2 cells cultured on coverslips were fixed in 4% polyoxymethylene for 15 min and then blocked in 1% bovine serum at room temperature for 60 min. After incubation with anti-LC3B (1:400, Cell Signaling [3868]) overnight at 4°C, the coverslips were incubated with donkey anti-rabbit IgG-conjugated FITC secondary antibody (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 1 hour at room temperature. After the coverslips were extensively washed with PBS, they were mounted with ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific) and viewed under a fluorescence microscope (Olympus Corporation).

statistical analysis
All in vitro experiments were repeated at least three times. The results are expressed as the mean ± SD. Student's t-test or one-way ANOVA was used to compare values among the different groups. A 2-sided P-value was used, and P,0.05 was considered statistically significant. SPSS 23.0 statistical analysis software was used.

ethics statement
The experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Wenzhou Medical University (approval no: wydw2016-0079), whose guidelines and protocols were followed for the welfare of the animals. These were also in line with international guidelines for the care and use of laboratory animals.

Results
autophagy was maximally activated 1 day after clP in mice One day after CLP, H&E and PAS staining revealed vacuolar degeneration, loss of brush borders, exposure of the basement membrane, and infiltration of the interstitium by inflammatory cells in proximal tubular epithelial cells, indicating obvious renal tissue damage ( Figure 1A). Electron microscopy showed autophagic vacuoles engulfing abnormal mitochondria and other cytoplasmic components 1 day after CLP ( Figure 1A). CLP affected the expression of autophagy-associated proteins, including LC3 and P62, in a time-dependent manner. The ratio of LC3-II/LC3-I protein levels peaked 1 day after CLP and then gradually decreased ( Figure 1B), whereas P62 protein levels changed in the opposite manner. P62 protein levels were the lowest 1 day after CLP and increased at later times ( Figure 1B). In addition, compared with the sham and normal control groups, the CLP group exhibited a gradual decrease in KL protein levels after CLP that reached the lowest levels 1 day after CLP and recovered at later times ( Figure 1C). These data clearly indicate that CLP induces maximal autophagy activation and maximal KL reduction 1 day after CLP in mice.
recombinant Kl alleviated clP-induced renal dysfunction and damage without impacting autophagy and apoptosis Compared with the no treatment control, treatment with KL alone (0.01 mg/kg) or Rap (1 mg/kg) alone significantly reduced Scr and BUN levels in mice with CLP, and KL+Rap further synergistically reduced Scr and BUN levels (all P,0.05, Figure 2A), suggesting that both Rap-mediated autophagy activation and KL may alleviate CLP-induced renal dysfunction. By contrast, Scr and BUN levels were significantly increased in the 3-MA (30 mg/kg), an autophagy suppressor, group compared with those in the no treatment control group, and KL reduced Scr and BUN levels only slightly in the presence of 3-MA (P,0.05, Figure 2A)

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Kl ameliorates sepsis-induced aKi suppression by 3-MA may decrease CLP-induced renal dysfunction and block beneficial KL-mediated effects. H&E staining ( Figure 2B) also showed that CLP-induced renal tissue damage was attenuated by KL alone, Rap alone, and KL+Rap, whereas 3-MA blocked the beneficial effects of KL.
Compared with the sham control group, the CLP+media, CLP+KL, CLP+Rap, CLP+KL+Rap, and CMC groups exhibited a significant increase in the ratio of LC3-II/LC3-I levels and a significant reduction in P62 levels (all P,0.05, Figure 3A), suggesting that CLP stimulated autophagy. Notably, compared with CLP+media or CLP+KL, Rap significantly increased LC3-II/LC3-I (all P,0.05, Figure 3A), indicating further autophagy stimulation in addition to CLPinduced autophagy. The ratios of LC3-II/LC3-I and P62 levels in the CLP+3-MA and CLP+KL+3-MA groups were similar to those in the sham control group ( Figure 3A). These data demonstrate that Rap activates autophagy and that 3-MA suppresses autophagy in mice with CLP, whereas KL does not further increase CLP-induced autophagy. Electron microscopy revealed obvious autophagy in the CLP, CLP+KL, CLP+Rap, and CLP+KL+Rap groups, with milder autophagy in the CLP+3-MA and CLP+KL+3-MA groups ( Figure 3B). Notes: (A) images of h&e staining, Pas staining, and electron microscopy of kidney tissue 24 hours after clP. Mouse kidney tissues were collected 24 hours after clP and sectioned. Tissue sections were fixed and stained with H&E and PAS, which showed vacuolar degeneration and loss of brush borders (black arrows) in proximal tubular epithelial cells. images at 400× magnification were acquired using a biological imaging microscope (BX53; Olympus Corporation, Tokyo, Japan). Tissue sections were also observed under a hitachi h7500 electron microscope (hitachi ltd., Tokyo, Japan), which revealed engulfment of degraded cytoplasmic components by autophagosomes (red arrows). In mice with CLP, compared with the no treatment control, treatment with KL alone, Rap alone, or KL+Rap significantly increased endogenous KL levels, whereas treatment with 3-MA alone or KL+3-MA did not affect endogenous KL levels (all P,0.05, Figure 4). In addition, CLP significantly increased CASP3 expression in the kidney ( Figure 5). In mice with CLP, recombinant KL had no impact on CASP3 expression, Rap significantly reduced CASP3 levels, and 3-MA significantly increased CASP3 expression (all P,0.05, Figure 5). These data indicate that recombinant KL may not affect apoptosis in the kidneys of mice with CLP, whereas Rap may reduce apoptosis, and 3-MA may induce apoptosis.
recombinant Kl did not appear to affect autophagy in lPs-treated hK-2 cells To further investigate the association between autophagy and KL-mediated renoprotection, we treated HK-2 cells with LPS to induce autophagy. LPS reduced KL expression in HK-2 cells in a dose-and time-dependent manner. The dose response

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Kl ameliorates sepsis-induced aKi of HK-2 cells to LPS showed that 10 μg/mL LPS maximally reduced KL expression, increased the LC3-II/LC3-I ratio, and decreased P62 levels ( Figure 6A). A time-course assay revealed that 24-hour treatment with 10 μg/mL LPS resulted in maximal effects ( Figure 6B). Therefore, 24-hour treatment with 10 μg/mL LPS appears to maximally reduce KL expression while inducing autophagy in HK-2 cells.
Recombinant human KL (0.4 μg/mL) was added after HK-2 cells were incubated with LPS at 10 μg/mL for 1 hour, and the cells were then incubated for another 23 hours. Similar to the results from the animal model, recombinant KL did not further increase LPS-induced autophagy, whereas Rap significantly increased the LC3-II/LC3-I ratio and decreased P62 levels ( Figure 7A). In addition, recombinant KL and Rap increased endogenous KL levels only slightly in LPS-treated HK-2 cells ( Figure 7B). These findings indicate that exogenous recombinant KL may not further enhance LPS-induced autophagy and may not substantially affect endogenous KL expression in LPS-treated HK-2 cells. Furthermore, an apoptosis assay revealed that Rap significantly reduced apoptosis, whereas 3-MA increased apoptosis of LPS-treated HK-2 cells (all P,0.05, Figure 8). By contrast, recombinant KL did not affect apoptosis in LPS-treated HK-2 cells (Figure 8).

autophagy-related signal in hK-2 cells following different treatments
Following treatment with recombinant KL, Rap and 3-MA, the LC3-II fluorescence signal varied among the different groups. There were no marked changes in the HK-2 group. However, after the cells were exposed to LPS, the fluorescent signal exhibited the largest increase in the HK-2+LPS+Rap Mice received recombinant KL (0.01 mg/kg) 1 hour after CLP. Rap (1 mg/kg) or 3-MA (30 mg/kg) was administered by intraperitoneal injection 2 hours before CLP surgery. Mouse renal tissues were collected 24 hours after clP. sections were stained with anti-Kl (1:100, abcam, cambirdge, UK) at 4°c overnight. images at 200× magnification were acquired using a biological imaging microscope (BX53; Olympus Corporation, Tokyo, Japan). Abbreviations: rap, rapamycin; 3-Ma, 3-methyladenine; clP, cecal ligation and puncture; aKi, acute kidney injury; cMc, carboxymethyl cellulose; ihc, immunohistochemistry.

Discussion
The current study established a mouse model of CLPinduced AKI. In the mouse model, autophagy activation and KL protein reduction in the kidney reached maximal levels 24 hours after CLP. These CLP-associated adverse effects appeared to be acute and spontaneously partially resolved 5 days after CLP. A similar acute response in the kidney was observed in a rat model of IRI. 17 17 Therefore, 24 hours after CLP may be an optimal time to investigate the molecular mechanisms underlying sepsis-induced AKI and test the effects of agents on sepsis-induced AKI. Here, we tested recombinant KL, Rap, and 3-MA.

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Kl ameliorates sepsis-induced aKi erythropoietin-mediated attenuation of kidney damage was associated with increased KL expression in the kidney. 19 KL-deficient transgenic rats show more severe kidney dysfunction and renal histological damage than wild-type mice after IRI, whereas transgenic rats overexpressing KL develop milder kidney damage after IRI. 17 In addition, administration of recombinant KL to rats with IRI alleviates IRI-induced AKI. 17

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Kl ameliorates sepsis-induced aKi The molecular and cellular mechanisms underlying KL-mediated renoprotection remain unclear. Our results showed that exogenous recombinant KL and Rap significantly restored endogenous KL expression in mouse renal tissues. KL is a tumor suppressor in gastric cancer, 29 and restoration of KL expression induces autophagy in gastric cancer and hepatocellular carcinoma cells. 20,21 Therefore, in the current study, we hypothesized that KL might alleviate CLP-induced AKI by enhancing autophagy.
We observed maximal autophagy activation 24 hours after CLP. In a rat model of CLP-induced AKI, Hsiao et al also demonstrated that autophagy was activated 3 hours  30 Autophagy induction is also detected in kidney tissues during ischemia-reperfusion in mice. 28,31 The role of autophagy in renal protection and recovery from AKI has been shown in previous studies. Kimura et al established proximal tubular epithelial cell-specific autophagy-deficient transgenic mice; compared with wild-type mice, transgenic mice exhibited obvious cellular hypertrophy and degeneration, developed more severe kidney dysfunction, and showed increased proximal tubule cell apoptosis during ischemia-reperfusion. 22 Inhibition of autophagy by 3-MA enhances renal tubular cell apoptosis and deteriorates renal tissue damage, whereas stimulation of autophagy by Rap reduces apoptosis and tissue damage in mice during ischemiareperfusion. 28,31 Similarly, the current study also revealed that activation of autophagy by Rap improved kidney function, reduced apoptosis, and attenuated renal tissue injury, whereas suppression of autophagy by 3-MA exacerbated kidney dysfunction, increased apoptosis, and deteriorated renal tissue damage in mice with CLP-induced AKI. Our in vitro results also showed that activation of autophagy by Rap significantly reduced apoptosis and inhibition of autophagy by 3-MA-enhanced apoptosis in LPS-treated HK-2 cells. These findings clearly demonstrate that autophagy plays a protective role in AKI induced by CLP or ischemia-reperfusion.
Notably, our results did not show that KL attenuates CLP-induced AKI by enhancing autophagy. Administration of recombinant KL did not affect the expression levels of LC3-II, LC3-I, or P62 in the renal tissue of mice with CLPinduced AKI and LPS-treated HK-2 cells, indicating that recombinant KL may not enhance autophagy in our in vivo and in vitro AKI models. Therefore, the beneficial effects of KL on renal function in mice with CLP-induced AKI may not be associated with autophagy. Moreover, in both a mouse model in vivo and HK-2 cells in vitro, recombinant KL did not affect apoptosis.

Conclusion
In summary, the current study established a mouse model of CLP-induced AKI. The acute kidney responses, including renal dysfunction, renal KL expression reduction, and autophagy activation, were maximally affected 24 hours after CLP. Rap-mediated autophagy stimulation and 3-MA-mediated autophagy suppression reduced and exacerbated AKI in mice, respectively. Although recombinant KL attenuated renal dysfunction and increased endogenous KL expression in mice with CLP-induced AKI, it did not affect autophagy or apoptosis in the kidney. Therefore, KL-mediated renoprotection may be associated with mechanisms other than autophagy in sepsis-induced AKI.


s2fieldsofstudy: Biology Chemistry

text:
Bio-Orthogonal Chemistry and Reloadable Biomaterial Enable Local Activation of Antibiotic Prodrugs and Enhance Treatments against Staphylococcus aureus Infections

Systemic administration of antibiotics can cause severe side-effects such as liver and kidney toxicity, destruction of healthy gut bacteria, as well as multidrug resistance. Here, we present a bio-orthogonal chemistry-based strategy toward local prodrug concentration and activation. The strategy is based on the inverse electron-demand Diels–Alder chemistry between trans-cyclooctene and tetrazine and involves a biomaterial that can concentrate and activate multiple doses of systemic antibiotic therapy prodrugs at a local site. We demonstrate that a biomaterial, consisting of alginate hydrogel modified with tetrazine, is efficient at activating multiple doses of prodrugs of vancomycin and daptomycin in vitro as well as in vivo. These results support a drug delivery process that is independent of endogenous environmental markers. This approach is expected to improve therapeutic efficacy with decreased side-effects of antibiotics against bacterial infections. The platform has a wide scope of possible applications such as wound healing, and cancer and immunotherapy.

■ INTRODUCTION
Bacterial infections are a critical health threat of our time. 1,2 The standard approach to treat bacterial infections is through systemic administration of antibiotics. The choice of the antibiotic for a given infection is based on the minimum inhibitory concentration (MIC) profile of the targeted bacteria. 3 The MIC is defined as the minimum concentration of an antibiotic that will inhibit bacterial growth in vitro during an overnight incubation. 4 Historically, the MIC correlates with a specific dosing regimen (magnitude and frequency). Ideally, a supratherapeutic dose would be used to ensure complete elimination of the infection and to prevent emergence of antibiotic-resistant bacterial strains. However, high doses of systemic antibiotic therapy cause adverse side-effects including musculoskeletal, hepatic, and renal toxicity. 5−7 There is a growing need for better antibiotics. We believe that improved drug delivery methods that supply high doses of antibiotics specifically to the area of infection would fulfill this need.
An example that underscores the aforementioned challenges is the eradication of implant-associated infections (IAIs). 8 IAIs due to bacterial biofilms formed on the surface of soft tissues and medical devices are more resistant to conventional antibiotics than planktonic bacteria. Staphylococcus aureus is one of the most frequent germs causing biofilm-associated infections on the medical indwelling devices such as contact lenses, catheters, endotracheal tubes, mechanical heart valves, pacemakers, and prosthetic implants. 9−12 Among S. aureus isolates the methicillin-resistant (MRSA) strains remain the most frequent multidrug-resistant hospital-associated infection.
Microorganisms like S. aureus adhere to an extracellular polymeric matrix and develop into organized, complex communities with structural and functional heterogeneity, known as biofilms. Depletion of metabolic substances and accumulation of waste products causes biofilm bacteria to enter into a slow-growing state, rendering them more resistant to antibiotic drugs than their planktonic counterparts. 13 Moreover, the spread of antibiotic-resistant strains, including MRSA isolates, further reduces therapeutic options. A drug delivery method capable of supplying multiple high doses of antibiotics directly to the location of the biofilm infected implant would greatly improve the efficacy of IAI treatment.
It has recently been shown that biocompatible polymers, strategically implanted in the vicinity of the infected part of the body, can serve as local depots of large doses of medication. 14 Currently, the TYRX envelope 15 is the only clinically approved implantable mesh that is impregnated with rifampin and minocyclin. Biocompatible hydrogels have also been explored to achieve local release of antibiotics. 16−18 Hydrogels are a particularly important class of modern drug delivery materials due to their physical similarity to the physiological environment. 19 They can be engineered to resemble the native interstitial matrix. The biocompatible material has a high water content (70−99%), and the physical properties, such as stiffness, porosity, and shape, can be tuned for different medical applications. 20−22 The reported hydrogel systems release drugs either by spontaneous diffusion or through controlled or uncontrolled hydrogel degradation. These systems have a key limitationthey cannot be reloaded. The course of treatment is predetermined at the time of hydrogel implantation. Additional invasive procedures are necessary to alter the course of treatment or to provide additional medication. Moreover, the systems are usually extremely specific to the therapeutic being delivered.
A number of approaches have emerged in recent years to address this problem. In 2014, we presented our first step toward the construction of an implantable biomaterial based on click chemistry for targeting small molecules and showed that we could enhance the delivery of a suitable radiolabeled molecules by an order of magnitude to an implanted polysaccharide. 23 Mooney and co-workers designed a hydrogel drug-reloading system based on nucleic acid sequence complementarity. 24 In the described construct, small molecule drugs were modified with DNA strands that were captured by the complementary strands attached to the implanted hydrogel. Chen et al. reported self-healing hydrogel enabling reloading via diffusive chemical transport across the gel−gel interface. 25 Hsieh and co-workers described a reloadable system involving hydrogel suspended anti-PEG immunoglobulin M (IgM) antibodies that were capable of catching pegylated proteins. 26 None of these systems, however, have been successfully applied in vivo to tackle bacterial infections.
Herein, we report a reloadable hydrogel system that is capable of activating antibiotic prodrugs at the site of bacterial infection. It is based on the previously reported local drug activation approach that combines the temporal flexibility of systemic drug delivery and the spatial control of injectable biomaterials and could enhance the localization of small molecule therapeutics. 27 Our previous report demonstrated that the approach is capable of local activation of therapeutically meaningful quantities of a chemotherapeutic agent. The approach has been applied to treat soft-tissue sarcoma xenografts in mice and showed superior efficacy and significantly lower side-effects than conventional doxorubicin standard of care treatment. In this report, we demonstrate that the local drug activation approach is indeed reloadable, and its versatility can be utilized to tackle planktonic and biofilm infections.
To illustrate our approach, we chose two FDA-approved antibiotics, vancomycin and daptomycin that have widespread clinical significance in the treatment of S. aureus infections, including MRSA strains, and whose versatility is limited by harmful side-effects. Vancomycin is a tricyclic glycopeptide antibiotic that is indicated to fight severe infections caused by Gram-positive bacteria. It has a well-understood mechanism of action involving inhibition of bacterial cell wall biosynthesis. Vancomycin is often used as a drug of "last resort" against microorganisms that proved resistant against other antimicrobial agents. 28 Vancomycin is also used in the treatment of planktonic bacteria in the surrounding of the device-related infections. 29,30 Wider indication of vancomycin is limited due to its adverse effects like hypotension and tachycardia, phlebitis, nephrotoxicity, and ototoxicity. 31 Meanwhile, daptomycin is a cyclic lipopeptide antibiotic. Its activity is dependent on the presence of Ca 2+ that facilitates oligomerization and insertion into bacterial membranes. Oligomers of daptomycin are thought to form toxic pores inside of bacterial membranes causing membrane depolarization. Daptomycin has a narrow therapeutic window. It is approved at a dose of 4 mg/kg for the treatment of complicated skin and soft-tissue infection and at a dose of 6 mg/kg for S. aureus bloodstream infection. 32 Daptomycin has recently been reported to be effective against biofilms and therefore is a promising option for IAI treatment. 30 However, during phase 1 clinical trials, higher doses of daptomycin such as 8 mg/kg led to unacceptable adverse effects involving the musculoskeletal system with accompanying increases in creatine phosphokinase levels. 33 Moreover, its applications to joint infections have been limited due to limited biodistribution and the aforementioned side-effect profile. 34 In this work we will show that systemically administered prodrugs of vancomycin and daptomycin can be converted into active antibiotics at the site of bacterial infection using bio-orthogonal chemistry.

■ RESULTS AND DISCUSSION
Our local prodrug activation approach is based on the inverse electron-demand Diels−Alder (IEDDA) chemistry between trans-cyclooctene (TCO) and tetrazine (Tz). During the past decade, this chemistry has become increasingly popular for in vivo bioconjugation due to fast reaction kinetics and inertness of the two bio-orthogonal groups to endogenous biomolecules and bioprocesses. 35,36 IEDDA chemistry has been shown to be virtually nontoxic and highly effective under physiological conditions. 37 As illustrated in Figure 1, the strategy starts with the injection of a biocompatible tetrazine-modified alginate gel (TAG) at the site of an infection. A prodrug, synthesized by covalent modification of a clinically tested antibiotic with a releasable TCO moiety, is injected intravenously ( Figure 1B). When the prodrug reaches the site of the hydrogel, the bioorthogonal agents (TCO and Tz) react with each other through the IEDDA chemistry, concentrating the therapeutic payload at the site of an infection ( Figure 1C). 27,38 Finally, the resulting intermediate isomerizes spontaneously releasing the active antibiotic from the hydrogel to perform its therapeutic function locally ( Figure 1D). The multivalency of Tz on the hydrogel's surface allows catching of multiple doses of systemically administered TCO-modified compounds.
To evaluate the strategy, TAG was prepared by modifying an ultrapure medium viscosity (>200 mPa s) sodium alginate, containing a minimum of 60% guluronate monomer units, with the Tz ( Figure S1). Based on 1 H NMR analysis, TAG contained about 400 nmol of Tz per mg of the material (∼8% of alginate monomers modified). The extent of Tz loading reflects the maximum amount of prodrugs that can theoretically be activated. To determine if Tz incorporation altered the flow properties of alginate, the viscosities of 2.5% w/v aqueous solutions of TAG and unmodified gel (UG) were measured. In general, aqueous polysaccharide solutions are known to behave as non-Newtonian fluids, such that their dynamic viscosities decrease in response to an increase in the applied shear rate. Consistently, both TAG and UG displayed such a shear-thinning or pseudoplastic behavior ( Figure S2). Furthermore, zeta (ζ) potential, a surrogate measurement for surface charge, at 1 mg/mL dilution in distilled water at pH 7.0 was found to be −78.6 ± 5.2 mV for TAG and −75.6 ± 5.7 mV for UG. These values suggest that the low extent of modification with Tz did not affect the inherent surface charge of the alginate gel. Additionally, the high magnitude of ζ (> ±30 mV) of these gels indicates that they are stable in water and do not aggregate, as consistent with the literature.
The prodrugs of vancomycin and daptomycin, shown in Scheme 1, were synthesized by covalent modification of the parent antibiotics with the releasable TCO moiety. The detailed syntheses are illustrated in Figures S9 and S16.

Scheme 1. Prodrugs of Vancomycin and Daptomycin Modified with the Releasable TCO Group
Vancomycin was modified at the N-terminus which is involved in binding the C-terminal portion of bacterial cell wall p r e c u r s o r p e p t i d e s e n d i n g i n t h e s e q u e n c e −Lys−D-Ala−D-Ala. This binding sequesters substrates necessary for construction of the bacterial cell wall, eventually causing cell death. Extensive studies by Williams have shown that acylation of the N-terminus of vancomycin causes a 17fold decrease in antibiotic activity. 39 Daptomycin was converted into a prodrug by modifying the most reactive, ornithine residue (Orn 6 ). The precise role of this residue in daptomycin's mechanism of action is not fully understood. However, Marahiel reported that a prodrug formed by acylation of the ornithine residue is 4 times less active. 40 Due to its well-known reactivity and documented significance, Orn 6 was chosen as the site of attachment of the releasable TCO group.
In Vitro Prodrug Activation. TAG can efficiently activate multiple doses of prodrugs of vancomycin and daptomycin, TCO−Vanco and TCO−Dapto, under simulated physiological conditions. When 25 mg of TAG and 5 nmol of either prodrug were mixed in phosphate buffered saline for 2 h at room temperature, over 99% of the compounds detected in the supernatant were activated vancomycin and daptomycin as shown by LC-MS and HPLC analyses (Figure 2A). Subsequent measurements over a 24 h period detected only the release of the corresponding antibiotic. Calibration of the acquired spectra revealed that TCO−Vanco and TCO−Dapto are rapidly captured by TAG and that over 80% of the activated antibiotics are released within 24 h ( Figure 2B). To test the ability of TAG to be reloaded, the hydrogel was treated with second and third doses of 5 nmol of TCO−Vanco and TCO−Dapto 24 and 48 h after the first dose. As illustrated in Figure 2, TAG is capable of efficiently "catching" multiple doses of prodrugs and subsequently releasing their corresponding activated antibiotics. The kinetics of drug release after the second and the third doses followed essentially the same trend as those seen after the first dose.
Microcalorimetric Analysis of Inhibition of Growth of Planktonic Bacteria. Isothermal microcalorimetry experiments using two laboratory strains of S. aureus (MSSA and MRSA) confirmed efficient in vitro activation of the antibiotic prodrugs by TAG. The experiments were based on the known principle that metabolically active (live and reproducing) bacteria produce heat that can be measured using an isothermal calorimeter. 41 Heat generated by MSSA and MRSA bacteria when treated with different concentrations of TCO−Vanco or TCO−Dapto in the presence of either TAG or UG was plotted as a function of time ( Figures S4−S7). Analogous experiments using the parent antibiotics were carried out as a positive control ( Figure S9). An in-depth data analysis is described in Tables S1 and S2. A threshold of 4 μW for total heat production was selected as a marker of bacterial growth, and the time required for bacteria heat to reach that threshold was measured as "time to detection" (TTD). For example, bacteria without any antibiotic treatment were the fastest to reach the heat threshold and hence had a low TTD (Figure 3). Conversely, higher TTD values indicate a delay in heat production due to bacterial susceptibility to antibiotics. Inability to reach the 4 μW threshold within 24 h corresponds to 99% inhibition of bacterial growth. The TTD was assigned to be nondetectable (ND), and the minimum antibiotic dosage required to achieve 99% inhibition of bacterial growth was considered as the minimum heat inhibitory concentration (MHIC), the term which is analogous to MIC. Figure 3 shows TTD values corresponding to MRSA and MSSA bacteria treated with different concentrations of TCO− Vanco and TCO−Dapto in the presence of TAG or UG (negative control). In all cases, the untreated bacteria was able to reach the heat production threshold within 5 h. Increasing prodrug dosage consistently resulted in a delay in heat production and higher TTDs. Figure 3 illustrates that bacterial treatment in the presence of TAG resulted in significantly higher TTD values, relative to UG. For example, MRSA treated with 2 μg/mL TCO−Vanco and TAG showed TTD of 15 h, while the same treatment with UG had a TTD of 9 h ( Figure 3B). The difference was even more dramatic when MRSA was treated with 0.5 μg/mL TCO−Dapto. TTD of 23 h was observed with TAG, while the same treatment with UG had a TTD of 4 h ( Figure 3D). Analogous trends were observed with MSSA treated with TCO−Vanco and TCO−Dapto. The MHIC values derived from these data are listed in Table 1. The MHIC values indicate that the antibiotic prodrugs have higher antibiotic activity when activated in the presence of TAG.
Microcalorimetric Analysis of Inhibition of Growth of Biofilm Bacteria. Among S. aureus strains, MRSA remains the most prevalent microorganism associated with hospital infections. 42 We tested the ability of our prodrug activation system to eradicate MRSA biofilms grown on porous glass beads. First the beads were immersed in either TAG or UG and then incubated with different concentrations of daptomycin or TCO−Dapto. TCO−Vanco and vancomycin were not tested as the antibiotic has limited efficacy against S. aureus biofilms. 43 After antibiotic treatment, the beads were sonicated, and the sonication fluid was plated on agar plates. The colony-forming units (CFUs) were counted after 24 h. The results are shown in Figure 4 as CFU/mL. The lowest antibiotic concentration that inhibited colony formation on the agar plate was defined as the minimum biofilm eradication concentration (MBEC). 44 TCO−Dapto is significantly more effective at eliminating MRSA biofilms in the presence of TAG. MBEC was achieved at 64 μg/mL for TCO−Dapto with the tetrazine-modified gel, which is comparable to the standard daptomycin treatment. Meanwhile in the presence of UG, the TCO−Dapto was unable to eradicate biofilms even at concentrations of 256 μg/mL.
Elimination of S. aureus Infection in Mice. The hypothesis that TAG is reloadable and capable of concentrating and activating antibiotic prodrugs at a location of choice was first tested using a fluorescent model system. TAG or UG (negative control) were implanted at the dorsum of mice, while TCO−TAMRA was administered systemically. Figure S8 shows that TAG was able to enhance the local delivery of the fluorescent payload over days, after multiple doses. The areas injected with UG or without a gel injection (negative controls) did not have an equivalent enhancement of fluorescence, confirming that the bio-orthogonal reaction is the reason for the enhancement. Moreover, the fact that the fluorescence decreased to background levels in all cases confirmed that the cargo was not trapped indefinitely but rather was concentrated and activated (released from the material).
To study the in vivo behavior of TAG, we synthesized a gel to contain Cyanine 5.5 dye (0.732 nmol/mg) in addition to Tz. We found that TAG injected in the thigh muscle remains at the site of the injection for at least 3 months ( Figure 5). Fluorescence was observed throughout the study, and representative images of mice immediately postinjection, on days 7 and 90, are shown ( Figure 5A). The fluorescence signal observed on day 7 showed a gradual decrease over time up to 58% on day 90 ( Figure 5B). Postnecropsy relative fluorescence biodistribution showed Cy5.5 signal in the lymph nodes and liver, indicating that TAG is likely cleared from the body, with a preference for lymphatic and hepatic routes over renal clearance ( Figure 5C). Histopathological analysis of the gel site showed the presence of infiltrating macrophages without any associated fibrosis or cell death, indicating a mild inflammatory response only ( Figure 5D). Together, these results suggest that TAG undergoes gradual biodegradation, persists at the injection site for at least 90 days, and does not induce a fibrotic inflammatory response. These findings suggest that TAG is suitable for extended treatment durations.
The total therapeutic benefit of the platform comes from two components. On one hand we have the activation component. This can be determined by the difference of activity (MHIC) of the prodrug with TAG or UG as shown in Table 1. On the other hand, it is challenging to determine the benefits of the concentration step in vitro as it is hard to model the amount of prodrug that would be concentrated at the area labeled with TAG. In this study we used an in vivo efficacy study using a bioluminescent MRSA strain 45 and TCO− Vanco, a prodrug that has a limited benefit from the activation factor as determined by the difference between the MHIC against MRSA when used with TAG and UG of only 16% (Table 1). Fluorescently labeled TAG and UG were synthesized to contain ∼0.5% Cy5 loading (w/w), in addition to tetrazine in the case of TAG. Bioluminescent MRSA was injected along with the Cy5-modified hydrogels into the thighs of neutropenic mice. Mice were subsequently administered a systemic dose of TCO−Vanco. Figure 6A shows that the bioluminescent bacteria was completely eradicated within 24 h of receiving TCO−Vanco. Analogous treatments involving UG or saline in combination with TCO−Vanco were unsuccessful at clearing the infection. Upon acquiring the in vivo images, mice were sacrificed, and bacterial load of the harvested thigh  tissue was assessed in terms of colony-forming units (CFU) obtained per gram of the harvested tissue. As illustrated in Figure 6B, treatment with TAG and TCO−Vanco resulted in a 800-fold reduction of bacterial load relative to UG and TCO− Vanco. The difference of almost 3 orders of magnitude in eliminating bacteria surpasses the 16% improvement by the activation that was observed in vitro (Table 1). These results suggest that TAG is very efficient at concentrating prodrugs from the plasma circulation at the desired location. This is consistent with the unexpected results seen in our previous work with chemotherapeutic prodrugs, where Tzmediated activation of 10 daily prodrug doses led to a sustained remission of tumors. 27 Future studies will evaluate directly the pharmacokinetics of these antibiotic prodrugs as well as other prodrugs to quantify the effect directly.

■ CONCLUSION
Herein we have described a hydrogel-based reloadable platform that utilizes bio-orthogonal IEDDA chemistry to achieve local activation of systemically administered antibiotic prodrugs. The local drug activation platform was shown to be capable of activating multiple doses of prodrugs of vancomycin and daptomycin in vitro. Isothermal microcalorimetry experiments showed that the prodrugs activated by TAG were significantly more potent at inhibiting bacterial growth than the corresponding negative controls using unmodified hydrogel. In vivo experiments showed that treatment of TAG and TCO−Vanco is more effective than UG and TCO−Vanco, in eradicating luminescent MRSA infections.
A number of elements of the local drug activation platform require further optimization. First, the prodrug of daptomycin requires structural modification, as TCO−Dapto was found to have poor aqueous solubility, which limited its in vivo testing. We plan to report our efforts in this area shortly. In addition, we envision that other prodrug modifications would additionally attenuate the antibacterial activity of the resulting prodrug,  Statistical analysis was done with unpaired Welch's t test, and error bars indicate standard deviation of means of n = 3. ** p < 0.005 compared to saline, # p < 0.005 compared to unmodified alginate-Cy5 control. as daptomycin's mode of action heavily relies on its hydrophobicity for incorporation into bacterial membranes. 46,47 This will be advantageous, as it will allow administration of higher systemic dosages of prodrugs toward local activation in the area of interest. These strategies will undoubtedly be pursued in subsequent studies. ■ ACKNOWLEDGMENTS