Fractalkine aggravates LPS‐induced macrophage activation and acute kidney injury via Wnt/β‐catenin signalling pathway

Abstract Fractalkine (CX3CL1, FKN), a CX3C gene sequence inflammatory chemokine, has been found to have pro‐inflammatory and pro‐adhesion effects. Macrophages are immune cells with a critical role in regulating the inflammatory response. The imbalance of M1/M2 macrophage polarization can lead to aggravated inflammation. This study attempts to investigate the mechanisms through which FKN regulates macrophage activation and the acute kidney injury (AKI) involved in inflammatory response induced by lipopolysaccharide (LPS) by using FKN knockout (FKN‐KO) mice and cultured macrophages. It was found that FKN and Wnt/β‐catenin signalling have a positive interaction in macrophages. FKN overexpression inhibited LPS‐induced macrophage apoptosis. However, it enhanced their cell viability and transformed them into the M2 type. The effects of FKN overexpression were accelerated by activation of Wnt/β‐catenin signalling. In the in vivo experiments, FKN deficiency suppressed macrophage activation and reduced AKI induced by LPS. Inhibition of Wnt/β‐catenin signalling and FKN deficiency further mitigated the pathologic process of AKI. In summary, we provide a novel mechanism underlying activation of macrophages in LPS‐induced AKI. Although LPS‐induced murine AKI was unable to completely recapitulate human AKI, the positive interactions between FKN and Wnt/β‐catenin signalling pathway may be a therapeutic target in the treatment of kidney injury.

FKN is a CX3C gene sequence chemokine that functions as immune response and adhesion towards its unique receptor on CX3CR1-expressing immune cells. FKN occurs in two possible forms, a membrane-bound and a soluble form, which can be converted into each other in vivo. 4,5 When inflammation occurs, the membranebound FKN is broken down into a soluble form. FKN mobilizes immune cells to accumulate at the site of action. FKN act as adhesion molecules and chemical primers which regulate the development of inflammatory diseases when tissue injury occurs. 6 Studies suggested that FKN plays critical roles in the pathological process involved in atherosclerosis, 7 osteoarthritis 8 and muscle injury. 9 However, the molecular mechanism by which macrophages interfere with FKN in regulating the development of inflammatory diseases is rarely reported. Moreover, the specific mechanism has been unclear.
Wnt/β-catenin is a highly conservative signalling pathway that activates the transcriptional activity through β-catenin nuclear translocation, 10 regulates synaptic transmission, cells growth, proliferation, differentiation, adhesion and genetic stability. 11,12 All of these factors play an important role in the progression of inflammatory diseases such as acute lung injury, 13 fibrotic disease 14 and systemic lupus erythematosus. 15 However, the potential mechanisms through which Wnt/β-catenin interacts with FKN in the LPSinduced inflammatory system have not been reported. Study of the molecular mechanism may provide a new direction for clarifying the molecular mechanism of FKN which regulates the development of inflammatory diseases.
The study utilized LPS-induced macrophage and acute kidney injury (AKI) mice. The results suggest that LPS-induced macrophage activation was accelerated by FKN overexpression. However, FKN deficiency prevented LPS-induced AKI through inhibiting macrophage activation in which Wnt/β-catenin signalling contributed an essential role.

| Renal function measurement
All mice serum was collected for creatinine (Scr) and blood urea nitrogen (BUN) assays used to evaluate the renal function. Scr and BUN levels of mice were examined using the Blood Urea Nitrogen Assay kit (C013-2-1, Jiancheng Bioengineering Institute) and Creatinine Assay kit (C011-2-1, Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Urine samples were collected during a 24 hours period using metabolic cages (Nalgene, Rochester).
Urinary protein was measured using a urine protein assay kit (C035-2-1, Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

| RNA-sequencing assay
Total RNA was extracted with TRIzol Reagent (Invitrogen). Agarose gel electrophoresis was used to assess RNA integrity. Total RNA was purified with Qia Quick PCR Kit, and PCR amplification was performed. RNA-seq libraries for sequencing were constructed by F I G U R E 1 FKN positively interacted with Wnt/β-catenin signalling pathway in J774A.1 cells. A, Expression profile of FKN-regulated genes using the RNA-seq. B, Differentially expressed genes are shown in a volcano map. C, Biological processes were revealed in RNAsequencing. D, The data sets were analysed by GESA using the Hallmark collection. The correlation between FKN and Wnt/β-catenin signalling was indicated in GESA. E-G, Co-IP and IF assays detected the interaction between FKN and Wnt/β-catenin signalling and this was visualized using confocal microscopy with specific antibodies (green). Nuclei were incubated with DAPI (blue). The cytoskeletons were incubated with β-tubulin (red). Scale bars represent 10 μm Illumina HiSeq TM 2500. ABI Step OnePlus Real-Time PCR System (Life Technologies) was used for quantification and pooling. The sequence was performed according to the PE150 mode of HiSeq2500.

| Co-Immunoprecipitation (Co-IP) Assay
J774A.1 cells were lysed with a pre-cooled IP lysis buffer for 30 minutes. Cell lysates were pre-processed with magnetic bead and then incubated with FKN antibody or control IgG at 4°C overnight. The antibody was captured on magnetic bead and inspected using Western blots probed with anti-Wnt-4 and antiβcatenin antibodies.

| Cell apoptosis assay
The fluorescein isothiocyanate-Annexin V/propidium iodide (FITC-Annexin V/PI) apoptosis kit (556 547, BD Biosciences) was used to detected cell apoptosis. Cells were seeded into 6-well plates and cultured for 48 hours. The cells of each group were collected, rinsed three times with 1 × PBS and then centrifuged for 3 minutes.
The cells were then adjusted to 1 × 10 6 cells/ml and incubated for 15 minutes in a binding buffer containing Annexin V-FITC and PI.
The resulting apoptosis was detected through flow cytometry using a FACS Canto II (BD Biosciences) within 1 hours.

| Western blotting
All samples which had 40 μg total protein were loaded on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes.

| HE staining and PAS staining
Kidney tissue samples were fixed in 10% formalin and then embedded in paraffin for histopathology. A total of 3-4 µm serial sections were stained followed by routine de-waxing and hydration. These sections were then stained with haematoxylin-imidine red (HE) or periodic acid-Schiff (PAS) stains according to standard procedures. These sections were visualized using a light microscope (H550S, NIKON). Mice kidney sections were prepared using standard procedures.

| Statistical analysis
All data are expressed in the form of means ± standard deviations. SPSS 23.0 and GraphPad Prism 8.0 were used to analyse statistical data. P < .05 was considered as statistically significant. Each experiment was repeated three times.

| FKN positively interacted with Wnt/β-catenin signalling pathway in macrophage
To identify the biological process of FKN in J774A.1 cells, RNAsequencing was performed on cells with or without FKN overexpression ( Figure 1A,B). The biological process showed that synaptic transmission increased with FKN interference ( Figure 1C). GSEA analysis showed that FKN overexpression was positively correlated with the activation of the Wnt/β-catenin signalling pathway ( Figure 1D). J774A.1 cells lysates were collected for co-IP analysis confirming the interaction between FKN and Wnt/β-catenin signalling ( Figure 1E). FKN and β-catenin protein distribution were found throughout the cytoplasm and nuclei after IF staining. FKN overexpression enhanced the localization of FKN and β-catenin protein.
Combination treatment with Wnt3a also enhanced the protein localization in J774A.1 cells (Figure 1F,G). A positive interaction between FKN and Wnt/β-catenin signalling pathway in J774A.1 cells was also confirmed.

| FKN inhibited LPS-induced macrophage apoptosis via Wnt/β-catenin signalling pathway
The effects of FKN on the apoptosis of LPS-induced J774A.

| FKN transformed LPS-induced macrophage into M2 phenotype via Wnt/β-catenin signalling pathway
Western blotting, ELISA and IF analysis were used to detect the ex-

| FKN deficiency attenuated LPS-induced AKI via inhibition of Wnt/β-catenin signalling pathway
The above data from J774A.1 cells demonstrated that overexpres-

| FKN deficiency prevented macrophage proliferation and polarization in LPS-induced AKI via inhibition of Wnt/β-catenin signalling pathway
Whether

| D ISCUSS I ON
The mechanism of FKN-regulated macrophage activation during LPS-induced inflammatory response is reported in this study. The molecular mechanism involved in the anti-inflammatory effects of FKN was further explored based on the study results.
The molecular mechanism involved in Wnt/β-catenin signalling was studied through RNA-sequencing. This process was altered by FKN.

| CON CLUS IONS
In summary, FKN deficiency exhibits anti-inflammatory activity in LPS-stimulated inflammatory mice model through suppression of macrophage proliferation and polarization. Wnt/β-catenin signalling has a potent biological function through which it exerts its anti-inflammatory activity. These findings strongly suggest that FKN could be a potential molecular target for the treatment of LPSinduced inflammatory diseases in the future.

E TH I C S S TATEM ENT
All animal experiments strictly followed the guidelines of the National Institute of Health and were approved by the Ethics Committee of Youjiang Medical University for Nationalities.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest associated with this manuscript.