Apoptosis inhibitor of macrophage ameliorates fungus-induced peritoneal injury model in mice

Fungal peritonitis in a patient on peritoneal dialysis (PD) is a refractory injury accompanied by severe inflammation, predisposing patients to a poor prognosis. Defective clearance of necrotic tissue interferes with amelioration of tissue injury and induces abnormal tissue remodeling. In the recent reports, apoptosis inhibitor of macrophage (AIM, also called CD5L) prevents obesity, hepatocellular carcinoma and acute kidney injury. Here, we investigated potential roles of AIM in prevention of progression of fungal peritonitis models. AIM −/− mice subjected to zymosan-induced peritonitis exhibited progressive inflammation and sustained peritoneal necrosis tissue on day 28 after the disease induction, whereas there was an improvement in AIM +/+ mice. This appeared to be caused by deposition of AIM at the necrotic peritoneum in AIM +/+ mice. In vitro, AIM enhanced the engulfment of necrotic debris by macrophages derived from zymosan-induced peritonitis, M1- and M2a-like bone marrow derived macrophages, as well as by mesothelial cells. In addition, administration of recombinant AIM dramatically ameliorated severe inflammation associated with necrosis in zymosan-induced peritonitis of AIM −/− mice. Our observations suggest that AIM appears to be involved in the repair process of zymosan-induced peritonitis, and thus, could be the basis of development of new therapeutic strategies for PD-related fungal peritonitis.

a: Zymosan-induced peritonitis models were created in AIM +/+ and AIM −/− mice. They were sacrificed on days 0, 7, 14, 21 (n=6 in each group) and 28 (n=14). b: Zymosan model AIM −/− mice were treated with rAIM or PBS. Two hundred mg of rAIM was administered intravenously three times per week from day 7 to 28, following which the animals were sacrificed and assessed (n=11 in each group).     Day28

Patient profiles and demographic data
The All patients were free from peritonitis for more than 1 month before inclusion in the study. Peritoneal transport was assessed at the time of PET as the ratio of Dialysate/Plasma creatinine (D/P Cr), and its average value was 0.66±0.10. CAPD peritonitis was defined by the presence of a leukocyte count of 100 cells/mL or more in PD effluent, of which 50% or more were polymorphonuclear neutrophils (S-1). All cases of peritonitis were bacterial in origin, and 21% were culture negative. There were no cases of fungal peritonitis and of development of EPS.

Animal model and experimental design
Animal experiments were performed in accordance with the Animal Experimentation Guidelines of Nagoya University Graduate School of Medicine (Nagoya, Japan), and were approved by the Animal Experimentation Committee of Nagoya University (Approval # 25378 and DNA#14-50). AIM −/− mice (S-2) had been backcrossed to C57BL/6 (B6) for 13 generations before they were used in these experiments. Eight to ten-week-old male AIM +/+ and AIM −/− mice weighing 20-25 g were used throughout the study. Animals were maintained under specific-pathogen-free conditions and had free access to food and water. In the present study, we used a zymosan-

Sample processing and analysis
One part of each tissue sample was fixed with 10% buffered formalin overnight, following which the samples were routinely processed and embedded in paraffin, and the cut sections were stained with hematoxylin and eosin (HE) and Masson's trichrome for light microscopy and were used for immunohistochemistry (IHC). A second fragment was snap-frozen in liquid nitrogen, and the tissues were cut with a cryostat and used for IHC. A third fragment of each tissue sample was immersed in RNAlater (Ambion, Austin, TX) to isolate RNA. For preservation of the integrity and stability of total RNA, all the procedures were conducted at 4°C under sterile conditions.

RNA isolation from tissues and cultured cells, and quantitative PCR analysis
RNA isolation and synthesis of first-strand cDNA were conducted as described previously (S-10, 12). One μg of total RNA from each peritoneum sample was then reverse transcribed. Real-time polymerase chain reaction analysis was performed using an Applied Biosystems Prism 7500HT sequence detection system with TaqMan gene expression assays, as described previously (S-9, 10). A list of the TaqMan Gene Expression Assays (Applied Biosystems Inc., Foster City, CA) used in this experiment is shown in Supplementary Table 4. We used 18S ribosomal RNA as an endogenous control.

Western blotting of human serum AIM
Western blotting was performed as described previously (S-13). Briefly, 1 μL of mouse or human serum was dissolved in NuPAGE 12% Bis-Tris Gel (Life Technologies, Carlsbad, CA) under reducing conditions, and proteins were transferred to Immobilon-P Membrane (Merck Millipore, Billerica, MA). The membranes were blocked with 5% skim milk in PBS-Tween-20 (Sigma-Aldrich) for 1 h at room temperature, then incubated overnight at 4°C with rabbit anti-AIM antibody diluted in blocking buffer and probed with goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA) diluted with blocking buffer for 120 min at room temperature. Three times of 10 min washes between steps were performed with PBS-T. Bound antibody was detected with ImmunoStar LD (Wako).
Briefly, the recombinant plasmids encoding musculus Cd5l were transfected to Chinese hamster ovary (CHO)-3E7 cells. Culture supernatants were collected and purified by rat anti-murine AIM monoclonal antibody-conjugated Protein G Sepharose (GE Healthcare, Uppsala, Sweden). Bound protein was eluted with 0.1 M Glycine-HCl (pH 3.0) and neutralized with 1 M Tris-HCl (pH 8.5). The protein was concentrated by Amicon Ultra filter concentrators (Merck Millipore). Endotoxin concentrations were measured by the chromogenic LAL endotoxin detection system (Genscript, Piscataway, NJ). The purified protein was analyzed by Western blotting and was detected as 36 KD.

Collection of cells derived from the peritoneum of Zymosan model mice on day 7
On day 7, the peritoneums of each zymosan model mice were resected and cut into small pieces 1-2 mm in size. The pieces were transferred into gentleMACS TM C tubes

Culture of mesothelial cells from AIM +/+ and AIM −/− mice
Mouse parietal peritoneal mesothelial cells were obtained by digestion of parietal peritoneum from AIM +/+ and AIM −/− mice as described previously (S-15). Briefly, the peritoneal membrane was digested with a 0.125% trypsin solution for 15 min and occasionally agitated at 37°C. Cells were cultured in DMEM/F-12 media supplemented with 20% fetal calf serum, 50 U/ml penicillin, 50 g/ml streptomycin, and 1% Biogro-2 (Biological Industries Israel Beit Haemek Ltd., Israel). In order to purify the mesothelial cells during assay of phagocytosis, CD11b positive cells were depleted using CD11b MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. The purity of mesothelial cells was more than 98%, as verified by flow cytometric analysis and immuno-histochemistry for cytokeratin, CD11b and -smooth muscle antigen.

Bone marrow derived macrophage isolation and polarization
Preparation of M1-and M2a-like macrophages was performed according to previously

Preparation of dead cell debris using mesothelial cells (Met-5A) coated with AIM
Human mesothelial cell-line (Met-5A) cells, which were purchased from American Type Culture Collection (Manassas, VA), were cultured as described previously (S-10,17). Met-5A cells were heat-killed by incubation at 65 °C for 20 min in PBS, and labeled with FVD520, -660 or -780 (eBioscience) for 30 min at 4 °C. For coating of the surface of the cell debris with AIM, labeled dead cell debris was divided into two groups, and was incubated with serum-free culture medium with or without rAIM at a concentration of 50 μg/ml at 37 °C for 1 h, as described previously (S-13).

Phagocytosis assay using the peritoneal cells derived from the peritoneum of zymosan model mice
Peritoneal cells (10 6 cells/sample) derived from zymosan model mice on day 7 were mixed with dead cell debris labeled with FVD780 with or without AIM coating, in serum free DMEM/F-12 medium supplemented with 5 g/ml insulin, 5 g/ml transferrin and 5 ng/ml selenous acid at 37°C for 10 and 30 min. Then, the cells were

Confocal microscopy studies
In addition, we observed the above phenomena using confocal microscopy (

Statistical Analyses
Variables with a normal distribution are described as mean values ± SD, and asymmetrical distributions are presented as median and interquartile range. Categorical variables are given as numbers and percentages. To assess the differences between two groups, Student's t test or the Mann-Whitney U test was used. Fisher's exact test was employed when variables were categorical. Comparisons among groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett's or Kruskal-Wallis multiple comparison tests. Differences were considered to be statistically significant if P value was <0.05. All analyses were conducted by SPSS software (SPSS, Chicago, IL).