Th2 cytokine-induced alterations in intestinal smooth muscle function depend on alternatively activated macrophages - PubMed (original) (raw)

Th2 cytokine-induced alterations in intestinal smooth muscle function depend on alternatively activated macrophages

Aiping Zhao et al. Gastroenterology. 2008 Jul.

Abstract

Background & aims: Enteric nematode infection induces a strong type 2 T helper cell (Th2) cytokine response characterized by increased infiltration of various immune cells, including macrophages. The role of these immune cells in host defense against nematode infection remains poorly defined. The present study investigated the role of macrophages and the arginase pathway in nematode-induced changes in intestinal smooth muscle function and worm expulsion.

Methods: Mice were infected with Nippostrongylus brasiliensis and treated with clodronate-containing liposome to deplete macrophages or given S-(2-boronoethyl)-I-cysteine in drinking water to inhibit arginase activity. Segments of intestinal smooth muscle were suspended in organ baths to determine responses to acetylcholine, 5-hydroxytryptamine, or nerve stimulation. The phenotype of macrophages was monitored by measuring mRNA expression of the specific molecular markers by real-time polymerase chain reaction or viewed by immunofluorescence staining.

Results: Infection increased the infiltration of macrophages and up-regulation alternatively activated macrophage markers by a mechanism dependent on interleukin-4 (IL-4) or interleukin-13 (IL-13) activation of signal transducer and activator of transcription 6. Elimination of alternatively activated macrophages blocked smooth muscle hypercontractility and the increased smooth muscle thickness, and impaired worm expulsion. In addition, specific inhibition of arginase activity interfered with smooth muscle contractility, but only partially affected the protective immunity of the host.

Conclusions: These data show that the phenotype of macrophages is determined by the local immune environment and that alternatively activated macrophages play a major role in the effects of Th2 cytokines, IL-4 and IL-13, on intestinal smooth muscle function.

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Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1

Figure 1

N. brasiliensis infection induced changes in the mRNA expressions of macrophage molecular markers. Mice were inoculated subcutaneously with 500 N. brasiliensis (Nb) infective third stage larvae or treated with vehicle (VEH), and studied 9 days later. Intestinal strips were taken from the mice for total RNA extraction. Real-time quantitative PCR was performed to measure the mRNA expression. The fold increases were relative to the individual vehicle groups (VEH) after normalization to 18s rRNA. * p<0.05, **p<0.01 compared with the respective WT-VEH (n≥5 for each group).

Figure 2

Figure 2

Increased infiltration and alternative activation of macrophages in the whole section (A–F) or smooth muscle layer (G, H) of small intestine from mice infected with N. brasiliensis. Mice were inoculated subcutaneously with 500 N. brasiliensis infective third stage larvae (B, D, F, H) or treated with vehicle (A, C, E, G), and studied 9 days later. Frozen tissue blocks of mid-jejunum were prepared and the sections were cut for immunofluoresence staining for anti-F4/80-Alexa647 (A, B) or anti-CD206-FITC (C, D). Fluorescent channels were photographed separately and then merged together to locate the alternatively activated macrophages in the whole section of the small intestine (E, F). For smooth muscle layer, only the merged picture is shown (G, H). All the pictures are the representatives from each group of at least 5 mice. Original magnification, x200; lm: longitudinal smooth muscle layer; cm: circular smooth muscle layer.

Figure 3

Figure 3

Dependence of infection-induced macrophage recruitment and activation on IL-4/IL-13 activating Stat6 and innate verse adaptive immune response. WT, SCID, IL-4−/−, IL-13−/−, or Stat6−/− mice were inoculated subcutaneously with 500 N. brasiliensis (Nb) infective third stage larvae or treated with vehicle (VEH), and studied 9 days later. One group of SCID mice was given (i.v.) exogenous IL-13 for 7 days. Intestinal strips were taken for total RNA extraction. Real-time quantitative PCR was performed to measure the mRNA expression. The fold increases were relative to the individual vehicle groups (VEH) after normalization to 18s rRNA. * p<0.05, **p<0.01 compared with the respective WT-VEH (n≥5 for each group).

Figure 4

Figure 4

Clodronate-liposome treatment depleted both resident and recruited macrophages in the intestine, indicated by the decreased mRNA expressions of macrophage markers in uninfected mice, or abolishing of the upregulation of the markers in _N. brasiliensis_–infected mice. Mice were infected with N. brasiliensis (Nb) or treated with vehicle (VEH). Clodronate- (Cl2MDP) or control PBS- (PBS) containing liposomes were administrated (i.v., 0.2 ml at days 0, 1, 3, 5, 7, and 9 after inoculation) to deplete macrophages. Intestinal strips were taken for total RNA extraction. Real-time quantitative PCR was performed to measure the mRNA expression. The fold increases were relative to the individual vehicle groups (VEH) after normalization to 18s rRNA. *p<0.05 vs the respective WT-VEH; ϕp<0.05 vs the respective PBS (n≥5 for each group).

Figure 5

Figure 5

Depletion of macrophages by clodronate-liposome treatment attenuated nematode infection-induced intestinal smooth muscle hypercontractility. Mice were infected with N. brasiliensis (Nb) or treated with vehicle (VEH). Clodronate- (Cl2MDP) or control PBS- (PBS) containing liposomes were administrated (i.v., 0.2 ml at days 0, 1, 3, 5, 7, and 9 after inoculation) to deplete macrophages. Intestinal strips were taken from the mice and suspended longitudinally in organ baths for in vitro contractility studies in response to (A) acetylcholine (ACH), (C) serotonin (5-HT), or (B) for spontaneous contraction. *p<0.05 vs the respective WT-VEH; ϕp<0.05 vs the respective PBS (n≥5 for each group).

Figure 6

Figure 6

Depletion of macrophages abolished nematode infection-induced increase in intestinal smooth muscle thickness (A) and was associated with a reduced mRNA expression of IGF-1 (B). Mice were infected with N. brasiliensis (Nb) or treated with vehicle (VEH). Clodronate-(Cl2MDP) or control PBS- (PBS) containing liposomes were administrated (i.v., 0.2 ml at days 0, 1, 3, 5, 7, and 9 after inoculation) to deplete macrophages. Changes in smooth muscle thickness were assessed in Giemsa-stained sections (A); or whole tissue was processed for the measurement of mRNA expression of IGF-1 by real-time quantitative PCR (B). *p<0.05 vs the respective WT-VEH; ϕp<0.05 vs the respective PBS (n≥5 for each group).

Figure 7

Figure 7

Inhibition of arginase abolished nematode infection-induced intestinal smooth muscle hypercontractility. Mice were infected with N. brasiliensis (Nb) or treated with vehicle (VEH), and were given 0.2% S-(2-boronoethyl)-I-cysteine (BEC) via drinking water at day 2–9 post infection for arginase inhibition in vivo. Intestinal strips were taken from the mice and suspended longitudinally in organ baths for in vitro contractility studies in response to (A) acetylcholine (ACH, 10nM-0.1mM), (C) serotonin (5-HT, 100µM), or (B) for spontaneous contraction. *p<0.05 vs the respective WT-VEH H2O; ϕp<0.05 vs the respective WT-Nb H2O (n≥5 for each group).

Figure 8

Figure 8

Depletion of macrophages resulted in impaired expulsion of N. brasiliensis, but did not affect N. brasiliensis infection-induced upregulation of IL-4 or IL-13. Mice were infected with N. brasiliensis (Nb) or treated with vehicle (VEH). Clodronate- (Cl2MDP) or control PBS-(PBS) containing liposomes were administrated (i.v., 0.2 ml at days 0, 1, 3, 5, 7, and 9 after inoculation) to deplete macrophages. Intestinal strips were taken for the measurement of the mRNA expression of IL-4 and IL-13 by real-time quantitative PCR (A). Separate groups of mice were infected with N. brasiliensis and treated with Cl2MDP. At the day 9 post infection, the intestine was collected for worm counting (B) and feces was collected for egg counting (C). *p<0.05 vs the respective WT-VEH; ϕp<0.05 vs the respective PBS (n≥5 for each group).

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