Alternative activation of macrophages by IL-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion

Abstract

Alternatively activated macrophages play an important role in host defense in the context of a T helper type 2 (Th2) microenvironment such as parasitic infection. However, the role of these macrophages during secondary challenge with Th1 pathogens is poorly defined. In this study, thioglycollate-elicited mouse peritoneal macrophages were treated with interleukin-4 (IL-4) or IL-13 in vitro and challenged with Neisseria meningitidis. After 8 to 12 hours of IL-4 pretreatment, the nonopsonic phagocytic uptake of N meningitidis was markedly reduced, depending on the common IL-4Ralpha chain, but independent of Scavenger receptor A and macrophage receptor with collagenous structure (MARCO), 2 known receptors for N meningitidis. Inhibition of phagocytosis extended to several other microbial particles, zymosan, and other bacteria. Concomitantly, IL-4 potentiated the secretion of proinflammatory cytokines, after additional bacterial stimulation, which depended on the MyD88 signaling pathway. Similar results were obtained after intraperitoneal stimulation of IL-4 and N meningitidis in vivo. Further in vitro studies showed a striking correlation with inhibition of Akt phosphorylation and stimulation of the mitogen-activated protein kinase pathway; inhibition of phagocytosis was associated with inhibition of phagosome formation. These findings are relevant to host defense in mixed infections within a Th2 microenvironment and shed light on immunologic functions associated with alternative priming and full activation of macrophages.

Figure 1

Alternative activation of MΦs decreases N meningitidis uptake. (A) Flow cytometric analysis of ThioMΦ ingestion of Rhodamine Green labeled Neisseria (RdGnX-N.m.). ThioMΦs were stimulated with IL-4 for 48 hours and challenged with ethanol-fixed RdGnX-N.m. (100 bacteria/MΦ) for 2 hours at 37°C. The mean fluorescence for each treatment was determined by flow cytometry. The histogram shows the effect of IL-4 treatment on uptake of N meningitidis. (B) IL-4 induces a dose-dependent inhibition of N meningitidis uptake. ThioMΦs were stimulated with different concentrations of IL-4 for 48 hours and challenged as described. The mean fluorescence for each population was determined by flow cytometry. The average mean fluorescence for each condition is shown. Error bars indicate SDs. (C) IL-4–inhibited RdGnX-N.m. uptake by MΦs in a time-dependent manner. ThioMΦs were stimulated for different periods with IL-4 and challenged as above. Unchallenged cells served as a negative control. The mean fluorescence for each population was determined by flow cytometry. The average mean fluorescence for each condition is shown. Error bars indicate SDs. (D) The inhibition of N meningitidis uptake is specific for alternative activation of MΦs. (Left) ThioMΦs were stimulated with IL-13 (10 ng/mL) for 48 hours and challenged with ethanol-fixed RdGnX-N.m. The mean fluorescence for each treatment was determined by flow cytometry. The histogram shows the effect of IL-13 treatment on uptake of N meningitidis. (Right) IL-4 inhibited N meningitidis uptake by ThioMΦs via IL-4Rα. WT or IL-4Rα^−/− ThioMΦs were incubated for 48 hours with IL-4 (5 ng/mL) and challenged with RdGnX-N.m. as described. After fixation with 2% paraformaldehyde, the mean fluorescence for each population was determined by flow cytometry. The figure showed the average mean fluorescence intensity of 3 independent experiments. Error bars indicate SDs.

Figure 2

IL-4 priming of MΦs does not modify SRA and MARCO expression or decrease N meningitidis binding at MΦ surface. (A) Effect of IL-4 on N meningitidis binding at the MΦ surface. ThioMΦs were treated for 48 hours with IL-4 and incubated for 2 hours at 4°C with RdGnX-N.m. (Nm; 100 bacteria/MΦ). The average mean fluorescence intensity of 2 independent experiments is presented as a bar diagram. Error bars indicate SDs. (B) Effect of IL-4 on surface expression of SRA and MARCO. ThioMΦs were treated for 48 hours with IL-4, and SRA and MARCO cell surface expressions were determined by flow cytometry using αMARCO mAb (ED31) or αSRA mAb (2F8). One experiment representative of 2 is shown. (C) Effect of alternative activation on RdGnX-N.m. uptake by WT, SRA^−/−, MARCO^−/−, and SRA^−/−/MARCO^−/− MΦs. ThioMΦs were treated for 48 hours with IL-4 and incubated for 2 hours at 37°C with RdGnX-N.m. (100 bacteria/MΦ). The average mean fluorescence intensity for 4 independent experiments is presented as a bar diagram. Error bars indicate SDs.

Figure 3

Alternative activation potentiates N meningitidis–induced proinflammatory cytokine secretion via MyD88, independent of SRA and MARCO. (A) WT ThioMΦs were cultivated 48 hours in the presence or absence of IL-4 and incubated for 24 hours with or without N meningitidis (Nm; 100 bacteria/cell). The culture supernatant was analyzed for production of TNF-α and IL-6 by ELISA and by FlowCytomix for IL-12 p70 production. Data represent the mean ± SEM of replicates from 1 experiment, representative of 3 experiments. (B) Increased proinflammatory secretion depended on the IL-4 receptor pathway but was independent of pathogen recognition receptor expression. (Left) WT (■) and IL-4Rα^−/− (□) ThioMΦs were cultivated 48 hours in the presence or absence of IL-4 and incubated for 24 hours with or without N meningitidis (100 bacteria/cell). The culture supernatant was analyzed for production of TNF-α and IL-6 by ELISA. Data represent the mean ± SEM of replicates from 1 experiment, representative of 3 experiments. (Right) WT, SRA^−/−, MARCO^−/−, and SRA^−/−/MARCO^−/− ThioMΦs were cultivated for 48 hours in the presence or absence of IL-4 and challenged with or without N meningitidis (100 bacteria/cells) for 24 hours. The culture supernatant was harvested and analyzed for IL-6 secretion by ELISA. (C) MyD88^−/− ThioMΦs were cultivated for 48 hours in the presence or absence of IL-4 and challenged with or without N meningitidis for 24 hours. Cell supernatants were assayed for TNF-α by ELISA. (D) Flow cytometry of ingestion of RdGnX-N meningitidis by WT and MyD88^−/− ThioMΦs. ThioMΦs were treated for 48 hours with different concentrations of IL-4 and incubated for 2 hours at 37°C with RdGnX-N meningitidis (100 bacteria/MΦ). The average mean fluorescence intensity of 3 independent experiments is shown as a bar diagram. Error bars indicate SDs.

Figure 4

IL-4 impairs uptake of RdGnX-N meningitidis and increases antibacterial proinflammatory response in vivo. (A left) Flow cytometry profile of uptake of RdGnX-N.m. (Nm) by peritoneal cells. RdGnX-N meningitidis cells (10⁸) were injected in the peritoneum of C57/BL6J mice. Animals were preinjected with IL-4, and the inflammatory infiltrate was collected by lavage. Mice preinjected with PBS were used as control. (Right) Scatter plot showing effect of IL-4 on RdGnX-N meningitidis uptake by MΦs. (B) Cytokine ELISA for TNF-α and IL-6 production in peritoneal lavage fluid. Ethanol-killed N meningitidis cells (2 × 10⁶) were injected into the peritoneum of C57/BL6J mice, preinjected with IL-4. The inflammatory infiltrate was collected by lavage, and the supernatant was assayed for TNF-α and IL-6. Mice preinjected with PBS were used as controls. Each symbol represents 1 animal.

Figure 5

IL-4 pretreatment and N meningitidis challenge inhibit Akt phosphorylation, but stimulate p42/p44 and p38 pathways. (A) Phagocytosis of N meningitidis by ThioMΦs is dependent on the p38 and PI3K pathway. ThioMΦs were treated with a kinase inhibitor (PD98059, 50μM; wortmannin, 100nM; SB202190, 50μM) for 1 hour before and during challenge (2 hours at 37°C) with RdGnX-N.m. (Nm; 100 bacteria/cell), and phagocytosis was determined by flow cytometry. (Right) FACS profile of RdGnX-N.m. uptake after treatments. The mean fluorescence of 1 experiment representative of 3 is shown as a bar diagram (left). (B) Proinflammatory cytokine secretion is dependent on the p38 pathway. ThioMΦs were treated with a kinase inhibitor (PD98059, 50μM; wortmannin, 100nM; or SB202190, 50μM) for 1 hour before and during challenge (24 hours at 37°C) with N meningitidis (100 bacteria/cell), and cytokine production was determined by ELISA. Results from 1 experiment of 2 are presented. (C) ThioMΦs were stimulated for 48 hours in the presence or absence of IL-4 and challenged with N meningitidis (100 bacteria/cell) for different periods. Expression of total protein levels and of the phosphorylated forms of p38, p42/p44, and Akt was determined by Western blotting. The results of 1 experiment, representative of 2, are presented. A vertical line has been inserted to indicate a repositioned gel lane.

Figure 6

Microscopy analysis of zymosan phagocytosis by untreated and IL-4–treated ThioMΦs. ThioMΦs were plated on bacteriologic plastic for TEM or on coverslips for scanning electron microscopy in the same well. Cells were treated for 48 hours with IL-4 and challenged with zymosan (20 particles/MΦ) for 30 minutes at 37°C. Scanning electron microscopy showed that zymosan particles were no longer seen on the cell surface of untreated ThioMΦs, because all had been internalized by the cells (TEM data). In IL-4–treated ThioMΦs, many incomplete phagocytic cups were observed on the surface of MΦs, showing the arrest of the phagocytic cup closure (white arrows) induced by IL-4 treatment. Moreover, TEM showed that few zymosan particles were internalized in IL-4–treated ThioMΦs compared with untreated MΦs.

Figure 7

Schematic summary of effect of IL-4 priming on phagocytic and secretory capacities of MΦs. IL-4 treatment (first step) inhibited the PI3K pathway, required for N meningitidis (Nm) phagocytosis and increased proinflammatory cytokine secretion after N meningitidis challenge (second step), consistent with enhanced p38 phosphorylation.