Dimethyl sulfoxide (D8418), Hoechst 33342 (B2261), Tween 80 (P8074), and paraformaldehyde (158127) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, penicillin, streptomycin, and fetal bovine serum (FBS) were products of ThermoFisher/Gibco (Carlsbad, CA, USA). CPT-11 (#HY-16562A) was purchased from MedChem Express (Princeton, NJ, USA), dissolved in ethanol before freshly diluted in phosphate-buffered saline (PBS) containing 2% Tween 80 in each experiment, and the final concentration of ethanol, never exceeded 5%. Fluorescence-labeled monoclonal antibodies CD11b-FITC (#11-0112), F4/80-PE (#12-4801), MHCII-APC (#17-5321), CD23-PE (#12-0232), CD19-eFluor660 (#50-0193), and CD11b-AlexaFluor488 (#53-0112) were obtained from eBioscience (San Diego, CA, USA); monoclonal anti-mouse F4/80-AlexaFluor 647 (#123121) were bought from BioLegend (San Diego, CA, USA). The rabbit monoclonal antibody against GATA6 (#5851) was purchased from Cell Signaling Technology (Danvers, MA, USA). CF568 goat-anti-rabbit IgG (H+L), highly cross-adsorbed (#201031) was obtained from Biotium (Hayward, CA, USA).

Female C57BL/6 mice (6–8 weeks of age) were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China). All animal experiments were performed in accordance with the guidelines for the care and use of animals approved by the Committee on the Ethics of Animals Experiments of Jinan University.

After 1 week of acclimatization, mice were randomly divided into groups with six mice in each group, and administered intraperitoneally (i.p.) with vehicle, CPT-11 (200 mg/kg body weight, at day 0) or untreated (control), respectively. The mice were sacrificed at day 3, day 7, day 14, day 28, and day 56, respectively.

In another experiment, mice were i.p. injected with CPT-11 (200 mg/kg body weight). After 7 days, the mice were injected (i.p.) with viable Escherichia coli bacteria (1 × 10⁹ CFU/mouse), which was freshly prepared as described previously (27). Their survival was observed and recorded every 6 h for four consecutive days.

Further in a separate experiment, mice were orally administered with CPT-11 (400 mg/kg body weight) once (at day 0) or twice (at day 0 and day 1), vehicle or left untreated. The mice were sacrificed at day 3, day 7, or day 14, respectively. The PECs were collected and analyzed as described below. The intestines and colons were isolated and fixed in 4% neutral formaldehyde. Paraffin slices of the tissues were stained with hematoxylin and eosin. Images were captured under a Zeiss Axio Observer D1 microscope armed with a color CCD (ZEISS).

After indicated treatment and being sacrificed, each mouse was injected with 1.5 ml washing buffer (germ-free PBS containing 0.5 mM EDTA and 5% calf serum) into the peritoneal cavity and the peritoneal lavage fluid was collected. The PECs were washed once with PBS-F (PBS containing 0.1% NaN₃ and 3% FBS) by centrifugation at 300 × g for 5 min, and then stained with FITC labeled anti-CD11b, PE labeled anti-F4/80, and APC labeled anti-MHCII, or PE-conjugated anti-CD23 and eFluor660-conjugated anti-CD19 monoclonal antibodies at 4°C for 30 min. Red blood cells, if there were, were lysed with ACK lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA). After washing once with PBS-F, cells were fixed with 4% paraformaldehyde in PBS and then analyzed on a flow cytometer (FACSCalibur; Becton Dickinson). Data were acquired and analyzed by using the CELLQuest software (Becton Dickinson).

Immunofluorescence analysis was performed essentially as previously described (28). Briefly, PECs were collected by centrifugation at 300 × g for 5 min and re-suspended in complete DMEM medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Then the cells were seeded in glass-bottomed dishes (5 × 10⁵ cells/dish). After 2-h incubation at 37°C in a humidified incubator of 5% CO₂, unattached cells were discarded. After washed with PBS, the adherent macrophages were fixed in 4% paraformaldehyde for 15 min, and permeabilized with 2 ml cold methanol (−20°C) for 10 min. Then the cells were incubated with AlexaFluor488-CD11b (1:80), AlexaFluor647-F4/80 (1:100), and GATA6 (1:300) antibodies overnight, followed by being stained with CF568-conjugated goat-anti-rabbit IgG (1:750) for 1 h. The nuclei were revealed by Hoechst 33342 staining (5 µg/ml in PBS) for 10 min. The cells were observed by the Zeiss Axio Observer D1 microscope with a Zeiss LD Plan-Neofluar 100×/0.6 Korr M27 objective len (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Fluorescence images were captured and analyzed by the Zeiss ZEN software.

Peritoneal exudate cells were collected with 2 ml washing buffer (germ-free PBS containing 0.5 mM EDTA and 5% calf serum), centrifuged at 300 × g for 5 min at 4°C, and then washed once with 2 ml cold PBS. The cells were re-suspended in PBS at 2 × 10⁶ cells/ml, being ready for transplantation.

In preparing BMSs, the bone marrow from hind femora was flushed out with 10 ml of sterile cold PBS and the cells were collected by centrifugation at 300 × g for 5 min at 4°C. Red blood cells were lysed in ACK lysis buffer for 10 min at 37°C, stopped with cold DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were washed once with 2–3 ml cold PBS, and then re-suspended in PBS to 2 × 10⁷ cells/ml. All procedures were performed in an aseptic surrounding.

Mice were i.p. treated with CPT-11 for 7 days, and then 0.5 ml of peritoneal exuded cells (1 × 10⁶ cells) or BMCs (1 × 10⁷ cells) were injected into the peritoneal cavity. After additional 21 days, the mice were sacrificed and their PECs were collected and analyzed by flow cytometry and immunofluorescence microscopy, respectively.

All experiments were performed three times independently, with one representative experiment shown. Data were expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance followed by Tukey post hoc test and unpaired Student’s t-test were used to analyze the statistical significance among multiple groups and between two groups, respectively. Kaplan–Meier survival curves were adopted for analysis of mice survival, and the statistical difference between two groups was determined using the log-rank (Mantel–Cox) test. P-values < 0.05 were considered statistically significant.

The dynamic process of CPT-11-induced damage to the peritoneal macrophages was assessed in mice administered with CPT-11 (i.p.). After CPT-11 treatment, the phenotypes of the PECs from the mice of each group were analyzed every week by flow cytometry. The LPMs (F4/80^hiMHC-II^low) and SPMs (F4/80^lowMHC-II^hi) were gated and their ratios and numbers were determined, respectively (Figure 1A). The results showed that neither the ratios nor the numbers of LPMs and SPMs were significantly changed by vehicle when compared with untreated control. In contrast, the ratios and numbers of LPMs were greatly decreased by CPT-11 treatment. Notably, LPMs in CPT-11-treated group were hardly detectable at day 7. However, the numbers of SPMs were not significantly changed by CPT-11, despite their increased ratios within the CD11b⁺ population (Figures 1B,C). Together with previous studies (13), these results indicated that the resident peritoneal macrophages were rapidly depleted by i.p. CPT-11 treatment.

Next, we sought to explore the recovery dynamic process of resident peritoneal macrophages after CPT-11 treatment. Flow cytometry revealed that LPMs were detected again at day 14 and from then on their numbers were gradually increased (Figure 2A). However, although the ratios of LPMs in CPT-11 groups were time-dependently increased, their cell numbers were still much lower than those of vehicle-treated control group. The cell numbers of LPMs in CPT-11-treated group were ~30% of that in vehicle-treated control group even after 56 days. Again, the numbers of SPMs (F4/80^lowMHC-II^hi) were barely influenced by CPT-11 treatment (Figures 2A,B). These results indicated that CPT-11 administration had prolonged deleterious effects on the resident peritoneal macrophages.

To verify the flow cytometric data showing that LPMs can be recovered from CPT-11 treatment, we next used immunofluorescent staining to detect the expression of GATA6, a functional marker of LPMs (9), in PECs. The PECs were cultured ex vivo. The macrophages among them were attached to the culture dish thus being selected by discarding the non-adherent cells, and were further observed by immunofluorescence microscopy using antibodies against CD11b, F4/80, and GATA6. As shown in Figure 3A, the expression of GATA6 was not significantly influenced by vehicle treatment; but after CPT-11 treatment, the cells expressing GATA6 were drastically decreased at day 3 and were barely detectable at day 7, indicative of almost complete loss of functional LPMs during these early stages. Supporting this, the expression of CD11b (a marker of myeloid cells) and F4/80 (a marker for mature macrophages) was also suppressed by CPT-11 treatment (Figure 3A). At day 14, however, GATA6 expression was detected in a minor population of the peritoneal macrophages and since then the GATA6-expressing cells were gradually increased. Concomitant with the re-expression of GATA6, both CD11b and F4/80 expressions were gradually reappeared, indicative of the recovery of mature macrophages.

Quantification of immunofluorescent observation showed that the percentages of GATA6⁺ macrophages were rapidly decreased within 1 week by CPT-11 treatment but slowly recovered after day 14 (Figure 3B), which was consistent with the flow cytometric analysis of LPMs (Figures 1B and 2B). Notably, although the percentage of GATA6⁺ LPMs in CPT-11-treated group (day 56) was comparable with the counterpart of corresponding vehicle-treated control group, their absolute cell counts (~7.5 × 10⁵ PECs) were much fewer than the latter group (~1.5 × 10⁶ PECs). These results also showed that CPT-11 administration induced a rapid loss but a very slow recovery of the functional LPMs with GATA6 expression, indicating a prolonged deterioration of these innate immune cells in the peritoneal cavity of mice.

Apart from resident macrophages, B cells, particularly B1 cells constitute another major population of the PECs. Thus, we next assayed the dynamic process of these cells after CPT-11 treatment. The PECs were stained with CD19 and CD23 antibodies, and analyzed by flow cytometry. Among them, B1 cells were identified as CD19⁺CD23⁻ cells while B2 cells as CD19⁺CD23⁺ ones. The results showed that the numbers of B cells (CD19⁺) were not significantly changed by vehicle treatment when compared with untreated control (data not shown), but were greatly reduced by CPT-11. Three days after CPT-11 treatment, only ~3% of the PECs were B cells (Figures 4A,B). Consequently, the numbers of both B1 and B2 cells were reduced by CPT-11 administration (Figures 4C,D). Interestingly, the numbers of total B cells as well as B1 and B2 cells were restored at day 56, comparable with that of vehicle-treated group (Figure 4). These results demonstrated that the peritoneal B cells were quickly impaired by CPT-11 treatment but were recoverable within 2 months.

As CPT-11 treatment rapidly reduced the numbers of both LPMs and B cells within 1 week but the recovery processes were slow, we next sought to explore whether adoptive cell transfer accelerated the recovery processes. Mice were received syngeneic PECs and BMCs at day 7 after CPT-11 treatment. The phenotypes of the PECs were analyzed 21 days after the adoptive transfer. The results showed that the adoptive transfer with PECs significantly increased the ratios and numbers of LPMs (F4/80^hiMHC-II^low) in CPT-11-treated mice when compared with those of CPT-11-treated control group without adoptive transfer, although these values were still lower than those of vehicle-treated control group. The adoptive transfer with BMCs also slightly increased the numbers of LPMs when compared with those of CPT-11-treated group without adoptive transfer, but the differences were not statistically significant (Figures 5A,B). Besides, the ratios and numbers of SPMs were not significantly changed by the adoptive PECs or BMCs (Figures 5A,C).

Consistent with the flow cytometry analysis, immunofluorescence microscopy also revealed that GATA6 expression in the peritoneal macrophages was greatly suppressed by CPT-11 treatment, but transfer of PECs into the peritoneal cavity significantly increased the percentage of GATA6⁺ macrophages when compared with CPT-11-treated control group (Figures 6A,B). The adoptive transfer of BMCs also increased GATA6 expression, although this was not statistically significant (Figure 6B).

Similarly, the adoptive transfer of PECs also rescued the B cell population (including B1 and B2 subpopulations) in the peritoneal cavity of the CPT-11-treated mice. However, transfer of BMCs was unlikely to help the recovery of B cell counts that had been decreased by CPT-11 treatment (Figure 7). Together, the adoptive transfer of PECs, instead of BMCs, promoted the recovery of resident peritoneal macrophages and B cells in CPT-11-treated mice.

We next explored the functional consequence of the impairment of resident peritoneal macrophages in CPT-11-administered mice. The mice were first i.p. injected with CPT-11 and 7 days later their peritoneal cavity were infected with viable E. coli. As shown in Figure 8, CPT-11 treatment significantly reduced the mouse survival rate when compared with that of vehicle-treated or untreated control groups. This suggested that the reduction of peritoneal macrophages and B cells upon CPT-11 treatment had significantly weakened the innate immunity against bacterial infection.

Oral administration of CPT-11 has been shown to be active against tumors in nude mice (29), but most of the animal experiments using CPT-11 were performed by i.p. injection according to the protocol of Battelle Columbus laboratories, which has displayed similar pharmacokinetics to those by intravenous (i.v.) administration (29, 30). In this study, we investigated whether CPT-11 by oral intake also changed the components of the PECs and induced intestinal inflammation in mice as it does by i.p. injection (20, 31). Different from i.p. route, oral administration of CPT-11 twice (at day 0 and day 1) at the dose of 400 mg/kg in C57BL/6 mice did not significantly reduced the percentages of peritoneal macrophages (LPMs and SPMs) (Figure S1 in Supplementary Material) and of B cells (Figure S2 in Supplementary Material) at day 3. Nor did it changed the expression of GATA6 in the peritoneal macrophages (Figure S3 in Supplementary Material). However, the total peritoneal exude cells reduced about one-third (from ~1.5 × 10⁶ to ~9.5 × 10⁵). Moreover, oral CPT-11 administration induced severe epithelial vacuolation in the full-length of the intestines (Figure S4 in Supplementary Material) and the animals began to die from day 4 and on (data not shown).

In another experiment, the drug was administered orally at the same dose only once (at day 0). The total number of the PECs in the CPT-11 group were decreased to ~9 × 10⁵ (~1/3 reduction when compared with vehicle-treated or untreated control) at both day 7 and day 14. Again, oral CPT-11 treatment did not change the percentages of peritoneal macrophages at day 7 (data not shown), but those of LPMs were decreased by CPT-11 at day 14 (Figure S5 in Supplementary Material). The ratios and numbers of peritoneal B cells were unchanged until day 14, except that the percentages of B2 cells seemed increased by CPT-11 at day 14 (Figure S6 in Supplementary Material). However, the expression of GATA6 in the peritoneal macrophages was unchanged (Figure S7 in Supplementary Material). Therefore, in addition to induction of exaggerated intestinal inflammation, oral CPT-11 administration showed a delayed cytotoxicity to the peritoneal macrophages when compared with i.p. CPT-11 treatment.