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. 2014 Aug 15;193(4):1717-27.
doi: 10.4049/jimmunol.1302167. Epub 2014 Jul 11.

Macrophage PTEN regulates expression and secretion of arginase I modulating innate and adaptive immune responses

Emine Sahin  1 , Stefan Haubenwallner  1 , Mario Kuttke  1 , Isabella Kollmann  1 , Angela Halfmann  2 , Alexander M Dohnal  2 , Li Chen  3 , Paul Cheng  3 , Bastian Hoesel  4 , Elisa Einwallner  5 , Julia Brunner  1 , Julia B Kral  1 , Waltraud C Schrottmaier  1 , Kathrin Thell  1 , Victoria Saferding  6 , Stephan Blüml  6 , Gernot Schabbauer  7
Affiliations

Macrophage PTEN regulates expression and secretion of arginase I modulating innate and adaptive immune responses

Emine Sahin et al. J Immunol. .

Erratum in

  • J Immunol. 2014 Nov 15;193(10):5350. Dohnal, Alexander B [Corrected to Dohnal, Alexander M]

Abstract

The activation of innate immune cells triggers numerous intracellular signaling pathways, which require tight control to mount an adequate immune response. The PI3K signaling pathway is intricately involved in innate immunity, and its activation dampens the expression and release of proinflammatory cytokines in myeloid cells. These signaling processes are strictly regulated by the PI3K antagonist, the lipid phosphatase, PTEN, a known tumor suppressor. Importantly, PTEN is responsible for the elevated production of cytokines such as IL-6 in response to TLR agonists, and deletion of PTEN results in diminished inflammatory responses. However, the mechanisms by which PI3K negatively regulates TLR signaling are only partially resolved. We observed that Arginase I expression and secretion were markedly induced by PTEN deletion, suggesting PTEN(-/-) macrophages were alternatively activated. This was mediated by increased expression and activation of the transcription factors C/EBPβ and STAT3. Genetic and pharmacologic experimental approaches in vitro, as well as in vivo autoimmunity models, provide convincing evidence that PI3K/PTEN-regulated extracellular Arginase I acts as a paracrine regulator of inflammation and immunity.

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Figures

FIGURE 1.
FIGURE 1.
Loss of PTEN leads to a significant increase in Arginase I expression. (A–F) Quantitative real-time analysis of unstimulated PTEN−/− and WT control macrophages. RNA was isolated from tPMs or resident macrophages, and quantitative PCR was performed with (A) PTEN, (B and C) Arginase I, (D) Stabilin1, (E) YM1, and (F) Fizz-specific primers. Target genes were normalized to GAPDH or HPRT as indicated. (G) Western blot analysis of unstimulated PTEN−/− and PTEN+/+ macrophages with Abs specific for Arginase I and β-actin. (H) Quantitative real-time analysis of Arginase I levels in PTEN−/− and PTEN+/+ macrophages 8 h after stimulation with LPS (100 ng/ml). (I) Western blot analysis of PTEN−/− and PTEN+/+ macrophages 24 h poststimulation with LPS. Blots were probed with Abs specific for Arginase I, iNOS, and β-actin. (J and K) tPMs of WT or PTEN−/− mice were stimulated with LPS and treated with the PI3K inhibitor wortmannin (100 nM) or the Arginase inhibitor N-hydroxy-nor-l-arginine (l-nor-Arg) at the indicated concentrations overnight. IL-6 levels were measured by means of ELISA. (L) Naive tPMs of PTEN−/− or WT control mice were subjected to immunoblotting. Levels of PTEN, STAT6, phosphorylated STAT6, and β-actin, as loading control, were determined by Western blot analysis. *p < 0.05, **p < 0.01. Data are representative of three individual experiments (error bars represent SD).
FIGURE 2.
FIGURE 2.
C/EBPβ is upregulated and shows higher Arginase I enhancer binding activity in PTEN-deficient macrophages. (A) tPMs of PTEN−/− or WT control mice were stimulated with LPS (100 ng/ml) for 8 h. Levels of C/EBPβ and Actin were determined by Western blot analysis. (B) Quantification of immunoblots of unstimulated PTEN−/− or WT tPMs is presented and normalized to β-actin. (C) Lysates of naive tPMs used for the ABCD were tested for C/EBPβ expression. GAPDH was used to confirm equal input. (D) ABCD pull-down (PD) of C/EBPβ with biotinylated Arginase I enhancer oligos from lysates of tPMs obtained from PTEN−/− and WT littermate control mice: PD, unconjugated competitor oligos (comp) and negative controls (nc). The prestained marker (35–55 kDa) is presented on the left. The first lane of the immunoblot indicates the marker and the cross-reactivity to the 55-kDa marker protein. (E and F) Naive or LPS-stimulated (24 h) tPMs of PTEN−/− or WT control mice were subjected to immunoblotting. Levels of STAT3, phosphorylated STAT3, and Actin, as loading control, were determined by Western blot analysis. *p < 0.05. Data are representative of experiments with three mice per group (A and B) (error bars represent SD).
FIGURE 3.
FIGURE 3.
Constitutively active PI3K promotes Arginase I expression and release into the extracellular space. (A) SNs of unstimulated tPMs of WT and PTEN-deficient mice immunoblotted against Arginase I. Equal loading is presented by Coomassie blue staining and cross-reactive albumin. (B, top panel, and C) Western blot analysis and quantification of Arginase I in SNs of tPMs of WT and PTEN-deficient mice (n = 5). (B, bottom panel) Arginase I immunoblot analysis of tPMs activated with LPS (100 ng/ml) or IL-4/IL-13 (5 ng/ml) for 24 h. (D) Bone marrow DCs incubated with conditioned medium of WT or PTEN−/− tPMs and subsequently activated with LPS. Cytokine secretion was assessed after stimulation for 24 h. (E) Cytokine production of BMDCs preincubated with recArgI (30 μg/ml) and subsequently activated with LPS for 24 h. *p < 0.05, **p < 0.01 (error bars represent SD).
FIGURE 4.
FIGURE 4.
Arginase I potently inhibits T cell polarization. (A and D) CD4+-specific cytokine staining for expression of IFN-γ and IL-17 in OT-II T cells stimulated for 3 d with OVA-loaded (50 μg/ml), LPS-pulsed (100 ng/ml), and recArgI (30 μg/ml) pretreated or untreated (control) bone marrow DCs, followed by PMA/Ionomycin T cell stimulation. (B and E) Relative abundance of IFN-γ+ and IL17+ cell populations among CD4+ T cells, expressed as percentage. (G and H) Representative FACS analysis and quantified proliferation of CFSE-stained CD4+ T cells 3 d after coculture with control or recArgI (30 μg/ml) pretreated DCs. (C and F) SNs of cocultures were assessed by ELISA for IFN-γ and IL-17A production by responder T cells. (I) [3H]Thymidine incorporation in proliferating CD4+ T cells was measured. *p < 0.05, **p < 0.01. ns, not significant. Data are representative of experiments with at least three mice per group (error bars represent SD).
FIGURE 5.
FIGURE 5.
Treatment with recombinant Arginase I at early stages of disease development protects mice from development of EAE. (A–C) Disease development as indicated by clinical score in recArgI-treated versus control WT mice. (D–G) Cytokine secretion after ex vivo restimulation with MOG35–55 peptide of spleen cells and draining inguinal lymph node (dLN) cells from MOG35–55–immunized mice belonging to groups 1 and 2 (see Table I). (H) Clinical score of 2D2 TCR tg mice after transfer of recArgI pretreated or untreated BMDCs, which were pulsed with MOG35–55 peptide (30 μg/ml) and LPS (100 ng/ml). (I) Disease progression of 2D2 TCR tg mice receiving recArgI pretreated or untreated DCs pulsed with MOG35–55 peptide (50 μg/ml) and primed with LPS (100 ng/ml) and additional administration of CFA and pertussis toxin. *p < 0.05 in the EAE, (two-way ANOVA between the groups as indicated) *p < 0.05, **p < 0.01 for the evaluation of cytokine release. ns, not significant. Data are representative of experiments with four to eight mice per group (error bars represent SD).
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