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, 190 (11), 5779-87

SerpinB2 Is Critical to Th2 Immunity Against Enteric Nematode Infection

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SerpinB2 Is Critical to Th2 Immunity Against Enteric Nematode Infection

Aiping Zhao et al. J Immunol.

Abstract

SerpinB2, a member of the serine protease inhibitor family, is expressed by macrophages and is significantly upregulated by inflammation. Recent studies implicated a role for SerpinB2 in the control of Th1 and Th2 immune responses, but the mechanisms of these effects are unknown. In this study, we used mice deficient in SerpinB2 (SerpinB2(-/-)) to investigate its role in the host response to the enteric nematode, Heligmosomoides bakeri. Nematode infection induced a STAT6-dependent increase in intestinal SerpinB2 expression. The H. bakeri-induced upregulation of IL-4 and IL-13 expression was attenuated in SerpinB2(-/-) mice coincident with an impaired worm clearance. In addition, lack of SerpinB2 in mice resulted in a loss of the H. bakeri-induced smooth muscle hypercontractility and a significant delay in infection-induced increase in mucosal permeability. Th2 immunity is generally linked to a CCL2-mediated increase in the infiltration of macrophages that develop into the alternatively activated phenotype (M2). In H. bakeri-infected SerpinB2(-/-) mice, there was an impaired infiltration and alternative activation of macrophages accompanied by a decrease in the intestinal CCL2 expression. Studies in macrophages isolated from SerpinB2(-/-) mice showed a reduced CCL2 expression, but normal M2 development, in response to stimulation of Th2 cytokines. These data demonstrate that the immune regulation of SerpinB2 expression plays a critical role in the development of Th2-mediated protective immunity against nematode infection by a mechanism involving CCL2 production and macrophage infiltration.

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1
Nematode infection induced up-regulation of SerpinB2 expression in the small intestine dependent on STAT6. C57BL/6 mice (WT) mice were infected with H. bakeri (Hb) or N. brasiliensis (Nb) third stage larvae. (A) The kinetics of SerpinB2 mRNA expression in the small intestine of mice at days 4, 7, 10, and 14 post Hb infection (DPI) was analyzed by qPCR. The fold changes were relative to the vehicle (VEH) group after normalization to 18S rRNA. (B) The intestinal lysates (60µg) were analyzed on 4–12% Bis-Tris NuPage gels and probed with anti-mouse SerpinB2. The 46-kDa SerpinB2 protein and a cleavage product of SerpinB2 with lower molecular weight (~37 kDa) were detected (arrows). Negative controls: identically prepared intestinal lysates from SerpinB2−/− mice. Positive controls: LPS-treated RAW 264.7 murine macrophages (12µg) and bone marrow-derived macrophages (BMDM, 40 µg). Immunoblot was reprobed with anti-GAPDH as a loading control. n.s. indicates nonspecific immunoreactive bands as evidenced by their presence in intestinal lysates from SerpinB2−/− mice. (C) qPCR analysis of the SerpinB2 expression in intestines from WT and STAT6−/− mice at day 10 post Nb infection. *p<0.05 versus VEH (n≥5 for each group).
Figure 2
Figure 2
Impaired Th2 protective immunity against H. bakeri infection in mice deficient in SerpinB2. WT or SerpinB2−/− mice were infected with H. bakeri (Hb). (A) Numbers of adult worms in the lumen of small intestine of C57BL/6 mice (WT) mice at day 14 post infection (DPI). qPCR was performed to measure the mRNA expression of (B) IL-13, (C) IL-4, (E) IL-13Rα2, (F) IL-25, and (G) IFNγ in the small intestine of mice at day 4, 7, 10, and 14 DPI. The fold changes were relative to the WT-vehicle (WT-VEH) groups after normalization to 18S rRNA. (D) ELISA analysis of the in situ IL-13 production of the intestines. **p<0.01 versus WT-Hb; *p<0.05 versus the respective VEH; ϕp<0.05 versus the respective WT (n≥5 for each group).
Figure 3
Figure 3
Abolished intestinal smooth muscle hyper-contractility/hypertrophy and delayed increase in mucosal permeability in mice deficient in SerpinB2 in response to H. bakeri infection. C57BL/6 mice (WT) or mice with SerpinB2 deficiency (SerpinB2−/−) were infected with H. bakeri (Hb) and studied at day 14 post infection. Intestinal strips were suspended longitudinally in organ baths for in vitro contractility studies in response to (A) acetylcholine (ACH, 10nM-0.1mM), (B) EFS (20Hz, 100V), or (C) the amplitude of spontaneous contraction. (D) Thickness of the smooth muscle layer was measured by microscopic examination of tissue sections of intestine cut from frozen blocks and Giemsa stained. *p<0.05 versus the respective vehicle (VEH). (E) Muscle-free mucosa from mice at days 7, 10, 14 post infection (DPI) was mounted in micro snapwell for the measurement of trans-epithelial electrical resistance (TEER). *p<0.05, **p<0.01 versus WT-VEH; ϕϕp<0.01 versus SerpinB2−/−-VEH (n≥5 for each group).
Figure 4
Figure 4
Impaired monocyte recruitment, CCL2 production, and alternative activation of macrophages in the intestines of mice deficient in SerpinB2. C57BL/6 mice (WT) or mice with SerpinB2 deficiency (SerpinB2−/−) were infected with H. bakeri (Hb). Segments of small intestine were collected at days 4, 7, 10, 14 post infection (DPI) and analyzed for mRNA expression of (A) F4/80, (C) arginase I, or (D) CD206 by qPCR. The fold changes are relative to the WT-vehicle (WT-VEH) groups after normalization to 18s rRNA. (B) Tissue sections of intestine were cut from frozen blocks collected at 4 DPI, and stained with anti-F4/80 (blue) and anti-CCL2 (red) for visualizing macrophages and CCL2 production, respectively. The images are representative from 5 mice per group. *p<0.05 versus the respective vehicle (VEH); ϕp<0.05 versus the respective WT (n≥5 for each group).
Figure 5
Figure 5
Attenuated up-regulation of CCL2 expression in the intestines in response to H. bakeri infection and a defect of monocyte recruitment in response to CCL2 stimulation in mice with SerpinB2 deficiency. (A) Segments of small intestine were collected at days 4, 7, 10, 14 post H. bakeri infection (DPI) for analyzing mRNA expression of CCL2 by qPCR. The fold changes were relative to the WT-vehicle (WT-VEH) groups after normalization to 18s rRNA. *p<0.05 versus the respective VEH; ϕp<0.05 versus the respective WT (n≥5 for each group). (B) Bone marrow-derived macrophages (BMDM) or enriched peritoneal exudate cells (PECs) were prepared from WT or SerpinB2−/− mice, and analyzed for CCR2 expression by qPCR. (C) WT or SerpinB2−/− mice were injected with CCL2 and peritoneal lavages were collected at 18 hours post injection for macrophage counting. (n=4 per group; *p<0.05)
Figure 6
Figure 6
In vitro macrophage production of CCL2, but not the development of M1 or M2 phenotype, was impaired by SerpinB2 deficiency. Macrophages were generated from bone marrow mononuclear (BMDM) cells of WT or SerpinB2−/− mice. Cells were treated with IL-4, IL-13, or LPS for 24 hours, and analyzed for mRNA expression of (A) arginase I, (B) nitric oxide synthase 2, or (C) CCL2 by qPCR. The fold changes are calculated relative to the WT-BMDM-VEH after normalization to 18S rRNA. *p<0.05 versus the respective VEH; ϕp<0.05 versus the respective WT-BMDM. Data shown in bar graphs are the means ± SEM and are representative of two independent experiments performed in triplicate.

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