Cellular renewal and improvement of local cell effector activity in peritoneal cavity in response to infectious stimuli

Abstract

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (MØ) subsets, namely small peritoneal MØ (SPM) and large peritoneal MØ (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for β-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-γ stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal MØ subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

Figure 1. Identification of resident peritoneal MØ subsets.

PC from C57BL/6 were harvested and stained with fluorochrome-labeled antibodies directed against F4/80, CD19, CD11c and IA^b for flow cytometry analysis. (A) Doublet cells were excluded according to forward scatter profiles (FSC-A and FSC-H). Subsequently, (B) CD19^high cells and (C) CD11c^high cells were also excluded, and (D) F4/80⁺ cells were selected. (E) F4/80 and IA^b expression defined three populations: LPM (F4/80^highIA^b-neg), SPM (F4/80^lowIA^b-high) and granulocytes (F4/80^lowIA^b-neg). These three subpopulations were purified by cell sorting on a FACS Vantage, and their morphology was evaluated ex vivo from cytospin slides (F), or after in vitro culture in chamber slides for 12 h (G). Slides were stained with hematoxylin and eosin (H&E) and analyzed by optical microscopy (40×).

Figure 2. Zymosan and T. cruzi injection alters the MØ compartment of PerC.

C57BL/6 mice were injected i.p. with zymosan (1 mg/mouse) or T. cruzi (10⁶ parasites/mouse) and at 30 min (A) or 48 h (B) after stimulation, PC from naive and injected mice were harvested and stained as described in M&M. Sequential gates were made as shown in Fig. S1. Plots show the frequencies of each subpopulation. (C) F4/80^lowMHCII^int cells present within PerC 48 h after injections were evaluated according the expression of Ly6C. Gray lines represent FMO , , the black lines show F4/80^lowMHCII^int cells from zymosan- (hairline) or T. cruzi-(bold line)-exposed PerC. (D) Total numbers of SPM, LPM and monocytes 48 h after zymosan or T. cruzi exposure (within MØ gate) are shown in panel. Data are representative of more than 3 independent experiments.

Figure 3. Zymosan and T. cruzi inoculation leads to PerC cellular renewal and improves local cell effector activities.

C57BL/6 mice were injected i.p. with zymosan (1 mg/mouse) or T. cruzi (10⁶ parasites/mouse) and 48 h later PC from naive and injected mice were harvested and cultured for 4 h. Non-adherent cells were removed by sequential washes. (A) β-Gal staining was performed as described in M&M. Slides were analyzed by optical microscopy (40×) and pictures were captured by Sony Camera (zoon 3.0). (B and C) Adherent PCs cells from 48 h-exposed mice and controls were stimulated in vitro with media or LPS (1 µg/mL). (B) Nitrite concentrations in culture supernatants were determined by the Griess reaction after 48 h of in vitro culture. Numbers represent the mean ± SD of triplicate samples. ***p<0.001 in relation to non-stimulated group and &&& p<0.001 when compared to the control group. (C) Frequencies of IL-12-producing F4/80⁺ cells were evaluated by intracellular staining as described in M&M. Bars indicate the standard deviation (SD). *** p<0.001 when compared to the control group. Data are representative of more than 3 independent experiments.

Figure 4. SPM are more responsive than LPM and monocytes to infectious stimuli.

C57BL/6 mice were injected i.p. with zymosan (1 mg/mouse) or T. cruzi (10⁶ parasites/mouse) and PCs were harvested 30 min or 48 h after stimulation. (A) SPM and LPM from C57BL/6 zymosan-exposed mice were FACS-sorted 30 min after injection and the presence of internalized zymosan particles was observed by optical microscopy. Slides were made with 10⁵ cells from purified MØ subsets, stained with H&E and analyzed by optical microscopy (40×). (B) Numbers represent the mean ±SD of internalized zymosan particles per cell in each MØ subset. *** p<0.001 when compared to the LPM group. (C) PC from control or 48 h-exposed mice were cultured for 6 h in the presence of brefeldin A with or without LPS (1 µg/ml) plus rIFN-γ (5 ng/ml). Titles above plots indicate in vivo - in vitro stimulations. Values inside gates represent the frequencies of IL-12-producing cells in each subpopulation. Data are representative of more than 3 independent experiments.