Gal-9/Tim-3 signaling pathway activation suppresses the generation of Th17 cells and promotes the induction of Foxp3+regulatory T cells in renal ischemia-reperfusion injury

doi:10.21203/rs.3.rs-2244283/v1

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Gal-9/Tim-3 signaling pathway activation suppresses the generation of Th17 cells and promotes the induction of Foxp3+regulatory T cells in renal ischemia-reperfusion injury

https://doi.org/10.21203/rs.3.rs-2244283/v1

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CD4⁺T cells mediate the pathogenesis of renal ischemia-reperfusion injury (IRI). Emerging research suggests that a Th17/regulatory T cell (Treg) imbalance plays a pivotal role in the development of renal IRI. The recently identified negative checkpoint T cell immunoglobulin domain and mucin domain family 3 (Tim-3) inhibits the immune response by binding to its ligand, galectin-9 (Gal-9). However, the role of the Gal-9/Tim-3 signaling pathway in the regulation of CD4⁺T cell subsets in renal IRI remains unclear. In this study, the effect of the Gal-9/Tim-3 signaling pathway on Th17/Treg subsets in renal IRI was investigated using a mouse model. Renal IRI induced the expression of Gal-9 in renal tubular epithelial cells and increased the percentages of Tim-3⁺Th17 cells and Tim-3⁺Foxp3⁺Treg cells in the IR kidneys. The administration of rAAV9-Gal-9 suppressed kidney inflammation, reduced the mortality of mice with renal IRI, increased Foxp3⁺Treg cells, and reduced Th17 cells. In contrast, the blockade of Tim-3 in vivo with an anti-Tim-3 mAb aggravated renal inflammation, decreased Foxp3⁺Treg cells, and promoted Th17 cells. Thus, Gal-9/Tim-3 signaling pathway activation may protect against renal IRI by inhibiting Th17 cell production and inducing Foxp3⁺Treg cell expansion. Our study suggests that the Gal-9/Tim-3 signaling pathway might become a target of immunotherapy in renal IRI.

Galectin-9

T cell immunoglobulin domain and mucin domain protein 3

Renal ischemia-reperfusion injury

Th17 cell

Regulatory T cell

Ischemia-reperfusion injury (IRI) is the leading cause of ischemic acute kidney injury and an important factor leading to delayed recovery after kidney transplantation [1]. However, effective methods to alleviate renal injury and promote repair are lacking.

Renal IRI is an inflammatory disease mediated by adaptive and innate immune[2]. Intensive studies have shown that CD4⁺T cells exert a pivotal role in the renal IRI-induced inflammation[3]. Th17 cells and regulatory T (Treg) cells are two CD4⁺T cell subsets that are interrelated in development and have antagonistic effects on immune function, with some degree of plasticity[4, 5]. In the acute phase of renal IRI, Th17 cells secrete proinflammatory cytokines, such as IL-17 and IL-22[6], thereby aggravating renal injury[7, 8]. However, Foxp3⁺Treg cells release TGF-β and IL-10, reduce CD4⁺T cell proliferation, regulate other effector T cells, and down-regulate the immune response[9]. Treg cells not only prevent acute injury in the early stage of renal IRI [10–12] but also reduce renal fibrosis and promote repair[11, 13, 14]. Thus, the imbalance of Th17 and Treg subsets plays a vital role in renal IRI [15, 16]. Regulating the Th17/Treg balance toward Treg cells has become a promising approach to protect against renal IRI[16–18]. However, the exact mechanism still deserves further investigation.

In the immune microenvironment of renal IRI, the abnormal expression of immune checkpoints leads to CD4⁺T cell changes[19]. Similar to programmed death-1(PD-1), T immunoglobulin and mucin molecule 3 (Tim-3) is a negative regulatory molecule that is important for T cell tolerance[20]. Tim-3 is expressed on the surface of several immune cells, such as Th1, Th17, Treg cells, and macrophages[21].Among several identified ligands of Tim-3, Galectin-9 (Gal-9) is the unique natural ligand of Tim-3 [22]. The Gal-9/Tim-3 signaling pathway plays an important role in modulating immune responses and diseases, such as autoimmune encephalomyelitis, diabetes, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, and bronchial asthma[20]. However, whether or not the Gal-9/Tim-3 signaling pathway plays a role in the differentiation and function of Th17/Treg subsets in renal IRI is currently unknown.

To address this question, we evaluated the effect of the Gal-9/Tim-3 signaling pathway on renal IRI in a mouse model, including its effects on Th17 cells and Foxp3⁺Treg cells. Our results suggest that the Gal-9/Tim-3 signaling pathway may ameliorate renal IRI immunopathology through regulating the balance between Th17 and Foxp3⁺Tregs.

Animals

Male C57BL/6 WT mice (aged 8 ~ 12 weeks) with a body mass of 20 ~ 25 g were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and bred under specific pathogen-free conditions at the experimental animal center of West China School of Public Health, Sichuan University. All animal experiments conducted in this study followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines[23].

Study design

First, C57BL/6 mice were randomly divided into two groups (n = 5/group): post-IRI 3 days and 10 days. Considering the high mortality of bilateral renal IRI, a unilateral renal IRI mouse model was established by clamping the left renal pedicle for 45 min[24]. The expression of Gal-9 and Tim-3 and the percentage of Tim-3⁺Th17 cells and Tim-3⁺ Foxp3⁺Tregs were determined.

Next, the effect of recombinant adeno-associated virus-mediated Gal-9 on Th17 and Foxp3⁺Treg cells in the mouse model of renal IRI was investigated. Recombinant adeno-associated virus type 9 (rAAV9) is a serotype with kidney-targeted gene delivery [25]. Two to four weeks after rAAV9 was injected into the tail vein, the virus expression level reached a peak in the kidney[25]. Two recombinant adeno-associated virus vectors, rAAV9-Gal-9-ZsGreen (rAAV9-Gal-9) and rAAV9-ZsGreen (rAAV9 vehicle), were constructed by Biowit Biotech (Shenzhen, China), as described in the supplementary material. C57BL/6 mice were randomly divided into the following groups (n = 5/group): rAAV9-Gal-9 3 days, rAAV9-Gal-9 10 days, rAAV9 vehicle 3 days, and rAAV9 vehicle 10 days. rAAV9-Gal-9 (2×10¹¹ Vg/mice) or rAAV9 vehicle (2×10¹¹ Vg/ mice) was injected via the caudal vein. Two weeks later, the unilateral renal IRI model was established[13].The effects of rAAV9-Gal-9 on renal pathology, inflammatory factors, and the percentages of Th17 cells and Foxp3⁺Treg cells were observed. Then, a bilateral renal IRI mouse model was established by clamping the bilateral renal pedicle for 30 min[13]. The survival rates of mice in the rAAV9-Gal-9 group and rAAV9 vehicle group during the first 7 days after IRI were compared (n = 10/group).

Finally, the effect of anti-Tim-3 mAb on Th17 and Foxp3⁺Treg cells in renal IRI were evaluated in vivo. C57BL/6 mice were randomly divided into the following groups (n = 5/group): Tim-3 Ab 3 days, Tim-3 Ab 10 days, normal saline control 3 days, and normal saline control 10 days. Using the unilateral renal IRI model[13],100 µg of anti-Tim-3 mAb (mouse,14-5871-82, eBioscience, San Diego, CA, USA) or an equal volume of 0.9% normal saline was injected intraperitoneally immediately after the operation. The effects of anti-Tim-3 mAb on the percentages of Th17 cells and Foxp3⁺ Treg cells in kidneys were observed. The survival rate of mice with and without anti-Tim-3 mAb injection during the first 7 days after bilateral renal IRI was compared (n = 10/group).

Histological evaluation of kidney injury

Upon sacrifice at the indicated time points, the freshly harvested kidneys were fixed by immersion in 10% buffered formalin phosphate overnight and embedded with paraffin following routine procedures. Tissue sections (5µm) were stained with hematoxylin and eosin (H&E). Renal pathology was evaluated by a renal pathologist with a single blind method by observing the cortical and outer medullary lesions, including diffuse renal tubular dilatation, tubular formation, vacuolar degeneration, and/or abscission of renal tubular epithelial cells. In each mouse, the tubular injury score was determined by dividing the number of damaged renal tubules by the total number of renal tubules in at least 10 high-power fields [26].

Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from kidneys using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser Kit (Takara Bio, Dalian, China).

The SYBR Green qRT-PCR assay (Bio-Rad, Hercules, CA, USA) was performed to quantify mRNA levels of Gal-9 (NM_001159301.1), Tim-3 (NM_134250.2), Foxp3 (NM_001199348.1), and RORγt (NM_001293734.1) by CFX384 real-time PCR (Bio-Rad). Specific primers (Table 1) were purchased from Tsingke Biotechnology Co., Ltd. (Beijing, China). The PCR system was as follows: 10 µL of SYBR ® Green I Mix, 1 µL of forward primer (10 µmol/L), 1 µL of reverse primer (10 µmol/L), 2 µL of cDNA, and 6 µL of dH₂O. The reaction conditions were as follows: 98℃ for 2 min; 98℃ for 2 s, 60℃ for 5 s (40 cycles); 75℃ 10 s, 95℃ for 2 s. The expression level of each gene was normalized to the level of β-actin and the relative fold expression values were calculated using the 2^−∆∆CT method[27].

Table 1

Primer sequences for quantitative real-time PCR
Gene	Forward primer sequence	Reverse primer sequence
Gal-9	5′-AGCGAAGTCTGCTTGGGAGG − 3′	5′-CAAGTTCTTCAGGCGGTGGTAAT-3′
Tim-3	5′-ACAGTTCCCTGGTCTTATGAATGAT-3′	5′-CTCCGTGGTTAGGGTTCTTGG-3′
Foxp3	5′-TCGAGGAGCCAGAAGAGTTT-3′	5′-ACTTCTCTCTGGAGGAGGCA-3′
RORγt	5′-GCTGACCCCTAAGAAACCCC-3′	5′-GAAGCAGTTTGGGACCCCTT-3′
β-actin	5′-AGTGTGACGTTGACATCCGT-3′	5′-GCAGCTCAGTAACAGTCCGC-3′

Immunohistochemistry

Paraffin-embedded kidney tissues were sectioned at a thickness of 4 µm and mounted on slides. Kidney sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Immunostaining was performed using the Streptavidin Peroxidase Immunohistochemical Staining Kit (SP-9001; ZSGB-Bio, Beijing, China), according to the manufacturer’s recommendations. Antigen retrieval was performed for 6 min in citrate buffer (pH = 6.0) in a pressure cooker. Subsequently, samples were blocked by goat serum for 10 min and incubated with primary antibodies against Gal-9 (rabbit polyclonal to galectin 9, ab69630, Abcam, Cambridge, UK; diluted 1:600 in PBT) overnight at 4°C. Next, secondary antibodies (biotinylated anti-rabbit IgG) and Strept ABC-Complex were added at 37°C for 15 min. Afterwards, samples were incubated with DAB substrate for 2 min, counterstained with hematoxylin for 4 min, dehydrated, cleared, and mounted. Images were captured at ×20 magnification. The expression and distribution of Gal-9 were determined in at least 5 high-power fields.

Western blotting

Using the Tissue Protein Extraction Kit (CW0891; Cwbio IT Group, Beijing, China), protein was isolated from about 20 mg of kidney tissue. An aliquot of protein (∼30 µg) was separated, blotted onto the PVDF membrane, and probed with antibodies specific for Gal-9 (ab69630; Abcam) or GAPDH (sc-166574; Santa Cruz, Houston, TX, USA). Immunoblots were developed using SuperSignal™ West Dura Chemiluminescent Substrate (3707; Thermo Fishier Scientific) and visualized by the Bio-Rad Gel Doc System. Optical densities of bands of interest were determined using Image J 1.46r (National Institutes of Health, Bethesda, MD, USA) and normalized against the appropriate loading controls.

Flow cytometry

The following antibodies were used for flow cytometry (FCM): CD3e-eFluor, CD4-FITC, CD25-PE, Tim-3-APC, Foxp3-PE-Cy5.5, and IL-17A-PE (all eBioscience, San Diego, CA, USA). Anti-mouse CD16/CD32 antibody (BioLegend, San Diego, CA, USA) was used to block Fc receptors. For FCM, kidney mononuclear cells (KMNCs) were isolated by density gradient centrifugation according to an established protocol [28]. For each analysis, two IR kidneys were combined. The Trypan blue exclusion method was used for counting using a blood cell counter.

According to previous studies[16, 29], similar methods was applied to identify Tim-3 expression on Th17 subsets. KMNCs were stimulated for 4 h with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma, St. Louis, MO, USA) and ionomycin (1 µg/mL; Calbiochem-Merck, Darmstadt, Germany) in the presence of 1×Brefeldin A (BioLegend) in X-VIVO10 medium (Biozym, Hessisch Oldendorf, Germany). After blocking Fc receptors, KMNCs were stained for 30 min at 4°C with fluorochrome-labeled antibodies against CD3, CD4 and Tim-3. After fixation with 4% paraformaldehyde, KMNCs were permeabilized with 0.1% Nonidet P-40 for 20 min and intracellular staining was performed with anti-IL-17A.

According to previous studies[13, 14], similar strategies was adopted to identify Tim-3 expression on Foxp3⁺Tregs. KMNCs were incubated with an anti-CD16/CD32 antibody for 10 min. Cells were surface-stained for 30 min at 4°C with fluorochrome-labeled antibodies against CD3, CD4, CD25 and Tim-3. For the evaluation of Foxp3, a commercially available Foxp3 kit (00-5523-00, eBioscience) was used. All data were collected using a FACS AriaII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo version 10.0 (Tree Star, Ashland, OR, USA).

Kidney bio-plex protein array

To examine inflammatory cytokines generated by renal IRI, protein levels of IFN-γ, TNF-α, IL-17, IL-6, IL-4, and IL-10 were measured in mouse IR kidneys using the Bio-Plex Multiple Protein Array Kit (Merck Millipore, Billerica, MA, USA), as described previously[30]. The total protein concentration of each sample was determined using a Bicinchoninic Acid Protein Assay Kit (CW0014S; Cwbio IT Group) and was used to normalize the measured cytokine levels.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics Version 21.0 (IBM Corp., Armonk, NY, USA). Quantitative data are expressed as the means ± standard deviations. The unpaired, two-tailed Student’s t-test was used for pairwise comparisons. Cumulative survival was analyzed by the log rank test (Kaplan–Meier). P-values less than 0.05 were considered statistically significant.

Gal-9 expression in the kidneys from rats with renal IRI

To explore the regulatory effect of the Gal-9/Tim-3 signaling pathway on the immune balance of Th17/Treg cells, we used immunohistochemistry, qRT-PCR, and western blotting to observe the dynamic changes in Gal-9 expression in a mouse model of renal IRI. Immunohistochemistry revealed little Gal-9 expression in contralateral kidneys, with increased expression of Gal-9 in IR kidneys (Fig. 1A). Gal-9 expression was mainly located in tubular epithelial cells, and almost no expression was detected in the glomerulus. Compared with levels in the contralateral kidneys, Gal-9 mRNA and protein expression levels in the IR kidneys were increased in the 3 day and 10 day groups (Fig. 1B-D). These results indicated that Gal-9 expression was significantly elevated in IR kidneys of mice with IRI.

Tim-3 expression on the surface of Th17 and Foxp3 ⁺ Treg cells in the kidneys from rats with renal IRI

The expression of Tim-3 mRNA in the IR kidneys was higher in the 3 day and 10 day groups than in the contralateral kidneys (Fig. 2E). To investigate whether the Gal-9/Tim-3 signaling pathway regulates the Th17/Treg balance, we evaluated Tim3 expression on Th17 and Foxp3⁺Treg cell subsets. Compared with those of the contralateral kidneys, the percentages of Tim-3⁺Th17 and Tim-3⁺Foxp3⁺Treg cells were significantly higher in the 3 day and 10 day groups (Fig. 2C, D). These results indicated that the Gal-9/Tim-3 signaling pathway may be activated in renal IRI.

Effects of rAAV9-Gal-9 on renal histopathology, inflammation and survival after renal IRI in mice

First, we investigated effect of Gal-9 on the histopathology of renal IRI. Mice were divided to four groups, as described in the Methods section. HE-stained kidney sections from rAAV9-vehicle treated mice and rAAV9-Gal-9-treated mice were assessed for renal tissue injury (Fig. 3A). As shown in Fig. 3B and C, tubular injury score in rAAV9-Gal-9-treated mice was significantly lower than that in rAAV9 vehicle controls (P < 0.05). These results suggest that rAAV9-Gal-9 treatment may reduce renal tissue damage in renal IRI.

Then, we assessed IFN-γ, IL-4, IL-6, IL-10, IL-17, and TNF-α protein levels in IR kidneys (Fig. 4). Levels of IFN-γ, IL-17, and TNF-α were lower and IL-10 and IL-4 levels were higher after rAAV9-Gal-9 treatment than in the control group. However, IL-6 levels remained unchanged after rAAV9-Gal-9 treatment.

Next, we observed the effect of rAAV9-Gal-9 treatment on the survival rate of mice with bilateral renal IRI. As shown in Fig. 5, there was no significant difference in mortality between the rAAV9 vehicle group and rAAV9-Gal-9 group at 3 days after IRI. However, the mortality of rAAV9-Gal-9 mice with bilateral kidney IRI was significantly lower than that of rAAV9 vehicle mice 7 days after IRI. These results indicate that rAAV9-Gal-9 treatment attenuates the progression of renal IRI in mice.

Effects of rAAV9-Gal-9 on Th17 and Foxp3 ⁺ Treg subsets in IR kidneys in vivo

Our results have showed that rAAV9-Gal-9 treatment inhibits inflammation in IR kidneys. Additionally, previous studies also have revealed that Gal-9 regulates the development of Th17 and Foxp3⁺Treg cells in vitro and in vivo [31]. Therefore, we investigated the effect of rAAV9-Gal-9 treatment on the percentages of Th17 (CD4⁺IL-17⁺) and Foxp3⁺Treg (CD4⁺CD25⁺Foxp3⁺) cells in renal IRI. The rAAV9 vehicle 3 day group and rAAV9-Gal-9 3 day group did not exhibit significant differences in the percentage of Th17 cells and the mRNA expression of RORγt in IR kidneys (Fig. 6B, C). However, the percentage of Th17 cells and RORγt mRNA expression levels in IR kidneys of the rAAV9-Gal-9 10 day group were lower than those in the rAAV9 vehicle 10 day group (Fig. 6B, C). Of note, the percentage of Foxp3⁺Treg cells and the mRNA expression levels of Foxp3 in IR kidneys in the rAAV9-Gal-9 10 days group were significantly higher than those in the rAAV9 vehicle 10 day group (P < 0.01) (Fig. 7B, C).

Effects of Tim-3 blockage on renal histopathology, inflammation and survival after renal IRI in mice

We also investigated whether in vivo anti-Tim-3 mAb blockade altered the kidney histology in a unilateral renal IRI mouse model. Hematoxylin-stained kidney sections from control mice and Tim-3 mAb-treated mice were assessed for kidney injury (Fig. 8A). As shown in Fig. 8C, after anti-Tim-3 mAb blockade treatment, mice in the Tim-3 Ab 10 day group had a significantly higher tubular injury score than that of the control group (P < 0.05). These results suggest that blocking Tim-3 may exacerbate renal tissue damage caused by renal IRI.

Next, we examined the production of inflammatory cytokines in IR kidneys. As shown in Fig. 9, the expression of pro-inflammatory cytokines (IFN-γ, IL-6, IL-17, and TNF-α) were significantly higher in anti-Tim-3 mAb-treated mice than in the control group. Compared with levels in the control group, mice treated with Tim-3 Ab showed no significant differences in IL-10 and IL-4 levels.

Then, we observed the effect of anti-Tim-3 mAb treatment on the survival rate of mice with bilateral renal IRI. As shown in Fig. 10, there was no significant difference in mortality between the control group and Tim-3 Ab treatment group at 7 days after IRI, but the mortality of anti-Tim-3 mAb-treated mice with bilateral kidney IRI was significantly higher than that of control mice 4 days and 6 days after IRI.

Effects of Tim-3 blockage on Th17 and Foxp3 ⁺ Treg subsets in IR kidneys in vivo

Our results showed that anti-Tim-3 mAb treatment aggravated inflammation in IR kidneys. Then we investigated the effect of anti-Tim-3 mAb treatment on the percentages of Th17 and Foxp3⁺Treg cells in renal IRI. As shown in Fig. 11B and C, although there were no significant differences between the Tim-3 Ab 10 day group and control 10 day group in the percentage of Th17 cells and the mRNA expression of RORγt in IR kidneys, the percentage of Th17 cells and RORγt levels in IR kidneys of the Tim-3 Ab 3 day group were higher than those in the control 3 day group. Although there were no significant differences in the percentage of Foxp3⁺Treg cells in IR kidneys between the Tim-3 Ab 3 day group and control 3 day group, the percentage of Foxp3⁺Treg cells in IR kidneys in the Tim-3 Ab 10 day group were lower than those in the control 10 day group (Fig. 12B). Nevertheless, there were no significant differences in the mRNA expression of Foxp3 in IR kidneys between the Tim-3 Ab group and control group (Fig. 12C).

Multiple signals, factors, epigenetic modifications, metabolic pathways, and microbiota can regulate the Th17/Treg balance[32]. Different from previous studies, our study mainly focused on Gal-9/Tim-3 signaling pathway in renal IRI. Gal-9 is expressed in multiple organ systems and is localized in the nucleus, cell surface, cytoplasm, and extracellular matrix[33], with extremely low expression in normal renal tissues[26, 34]. Serum Gal-9 levels are increased in patients or mice with acute rejection of kidney transplantation, chronic kidney disease, and anti-glomerular basement membrane glomerulonephritis[35–37]. The relation between Gal-9 and IRI has been reported sporadically in hepatic IRI. Liver IRI or liver transplantation in mice may induce the increased expression of Gal-9 in liver cells [38–40]. Although Gal-9 levels are elevated in IRI is not a new finding, what our study shows for the first time is that renal IRI can induce the expression of Gal-9 in IR kidneys, particularly in renal tubular epithelial cells, with almost no expression in the glomerulus. Therefore, IRI can induce the expression of Gal-9 not only in liver cells but also in renal parenchymal cells. In addition, renal tubular epithelial cells may be the main source of Gal-9 in IR kidneys.

This present study ties Gal-9 and its receptor, Tim-3, to the pathogenesis of renal IRI. Tim-3 signaling pathway is associated with acute and chronic kidney inflammation[41, 42]. Abnormal Tim-3 expression can be detected on the surface of renal parenchymal cells or immune cells in IgA nephropathy[43], lupus nephritis[44], diabetic nephropathy [45], renal tumor[46], and renal transplantation rejection[47]. Tim-3 also participates in the pathogenesis of liver IRI [26, 38, 40], brain IRI [48, 49], and lung IRI [21]. The activation of the Gal-9/Tim-3 signaling pathway has a protective effect on liver IRI via negative regulation[38]. An anti-Tim-3 antibody increases the infiltration of inflammatory cells and aggravates IRI injury. In the brain of an ischemic stroke model [48], the expression of Tim-3 on the surface of CD4⁺T cells is increased and is correlated with serum IL-17 and TNF-α. Our results showed that renal IRI may induce increased Tim-3 mRNA expression in IR kidneys. More importantly, levels of Th17 cells and Foxp3⁺Treg cells that express the Gal-9 receptor Tim-3 are also markedly increased in mice with renal IRI. Therefore, Tim-3 is not only expressed on the surface of macrophages in the acute phase of renal IRI mice[50, 51], and the expression of Tim-3 on the surface of Th17 cells and Foxp3⁺ Treg cells in renal IRI was up-regulated. Our further results revealed that rAAV9-Gal-9 administration can inhibit renal inflammation and decrease the 7-day mortality of mice with bilateral renal IRI, however Tim-3 blockage may promote renal inflammation. Thus, our findings support the notion that renal IRI can activate the Gal-9/Tim-3 signaling pathway, which have a protective effect in renal IRI. Further research is needed to clarify the exact molecular mechanisms.

Previous studies have shown that Gal-9 can induce Naïve CD4⁺T cell differentiation into Foxp3⁺ Treg cells, inhibit the differentiation of Th17 cells, promote the secretion of anti-inflammatory cytokines and attenuate inflammation-related organ injury[52, 53]. Tim-3 may affect acute kidney injury by regulating Treg cells and further participate in the progression of chronic kidney disease[51]. In renal IRI, Zhan et al. [54] found that the protective effect of delayed ischemic preconditioning combined with Gal-9 is better than that of ischemic preconditioning alone, and its effect is mediated by the expansion of Treg cells. Mesenchymal stem cells attenuate sepsis-associated acute kidney injury by the induction of Tregs and inhibition of Th17 cells via the Gal-9/Tim-3 pathway[55]. Wei et al.[56]found that quercetin could restore the Th17/Treg balance via Tim-3 and protect against liver injury induced by triptolide in mice. Thus, we investigated whether the renal protective effect of Gal-9/Tim-3 signaling pathway activation was related to its regulation of Th17/Treg imbalance. In our study, the frequency of Th17 cells and the mRNA expression of RORγt in IR kidneys were decreased in the rAAV9-Gal-9 3 days group and increased in the Tim-3 Ab 10 days group. Additionally, the percentage of Foxp3⁺Treg cells and the mRNA expression of Foxp3 in IR kidneys were increased in the rAAV9-Gal-9 10 days group. These results indicated that the protective role of Gal-9/Tim-3 signaling pathway activation may be related to regulating Th17/Treg imbalance.

However, our study indicated that the Foxp3 mRNA expression were similar between the Tim-3 Ab group and control group. When treated with rAAV9-Gal-9, the Foxp3 mRNA expression and the percentage of Foxp3⁺Tregs were up-regulated at 10 days. Why Tim-3 blockage has little effect on Foxp3⁺Treg subsets in IR kidneys in vivo? Our study does and cannot answer this question and was not meant to. Depending upon the cell types and cell status in different physiological conditions, Gal-9 may exerts its biological functions via multiple receptors, including CD44 and Tim-3[33].Although previous studies have indicated that Gal-9 selectively induces apoptosis in Th17 cells through its interaction with Tim-3[57], recent study also has indicated that Gal-9 inhibited the development of Th17 cells in a Tim-3-independent manner[58]. Recent studies have found that CD44 expression is positively correlated with Foxp3 expression and suppressive function of CD4⁺Treg cells[59, 60]. Therefore, except for Tim-3, Gal-9 may regulate Th17/Foxp3⁺Tregs balance through other receptor in renal IRI, which is worthy of further study.

Collectively, our data suggest that the Gal-9/Tim-3 signaling pathway may play an important role in the pathogenesis of renal IRI. Activation of the Gal-9/Tim-3 signaling pathway may regulate the Th17/Treg balance via reducing Th17 cells and increasing Foxp3⁺Treg cells, alleviate the renal inflammatory response, and protect against renal IRI. Targeting Gal-9/Tim-3 signaling pathway is a potential new therapeutic option to promote the repair of renal IRI. Further research is needed to clarify the molecular mechanisms that drive increased Gal-9/Tim-3 expression of Th17/Treg cells in renal IRI. In addition, future studies should aim to characterize the expression of Gal-9/Tim-3 on other immune cells such as macrophage in renal IRI.

Data availability

All data associated with this study are present in the paper and/or the supplementary materials. Further reasonable inquiries can be directed to the corresponding author.

Acknowledgement

We would like to thank Editage (www.editage.cn) for English language editing.

Funding

This work was supported by Sichuan Science and Technology Support Program (no. 2017JY0076).

Authors and affiliations

Department of Pediatrics, West China Second University Hospital, Sichuan University, China.

Ya-Mei Wang, Ying Xiong & Yu-Hong Tao.

Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, China

Ya-Mei Wang, Ting Feng, Hong Li, Ying Xiong & Yu-Hong Tao.

Department of Pediatrics, Cangzhou Center hospital, Cangzhou, China

Ya-Mei Wang

Corresponding author

Correspondence to Yuhong Tao

Author contribution

Y-HT developed the concept, designed experiments, interpreted the data, and prepared the manuscript. Y-MW conducted experiments, collected and analyzed data, and wrote the manuscript. TF, HL and YX guided experiment. All authors revised the manuscript and approved the submitted version.

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

This study was conducted in accordance with the Scientific Procedures Act 1986 and all animal procedures were approved by the Animal Experimentation Ethics Committee of Sichuan University.

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Gal-9/Tim-3 signaling pathway activation suppresses the generation of Th17 cells and promotes the induction of Foxp3+regulatory T cells in renal ischemia-reperfusion injury

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Abstract

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Introduction

Methods

Animals

Study design

Histological evaluation of kidney injury

Quantitative real-time PCR (qRT-PCR)

Immunohistochemistry

Western blotting

Flow cytometry

Kidney bio-plex protein array

Statistical analysis

Results

Gal-9 expression in the kidneys from rats with renal IRI

Effects of rAAV9-Gal-9 on renal histopathology, inflammation and survival after renal IRI in mice

Effects of Tim-3 blockage on renal histopathology, inflammation and survival after renal IRI in mice

Discussion

Conclusions

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References

Supplementary Files

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