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. 2010 Aug 17;3(135):ra62.
doi: 10.1126/scisignal.2000955.

Arginine usage in mycobacteria-infected macrophages depends on autocrine-paracrine cytokine signaling

Joseph E Qualls  1 , Geoffrey Neale, Amber M Smith, Mi-Sun Koo, Ashley A DeFreitas, Huiyuan Zhang, Gilla Kaplan, Stephanie S Watowich, Peter J Murray
Affiliations

Arginine usage in mycobacteria-infected macrophages depends on autocrine-paracrine cytokine signaling

Joseph E Qualls et al. Sci Signal. .

Abstract

Nitric oxide (NO) produced by macrophages is toxic to host tissues and invading pathogens, and its regulation is essential to suppress host cytotoxicity. Macrophage arginase 1 (Arg1) competes with NO synthases for arginine, a substrate common to both types of enzymes, to inhibit NO production. Two signal transduction pathways control the production of Arg1 in macrophages: One pathway dependent on the Toll-like receptor adaptor protein myeloid differentiation marker 88 (MyD88) induces the expression of Arg1 during intracellular infections, whereas another pathway, which depends on signal transducer and activator of transcription 6 (STAT6), is required for Arg1 expression in alternatively activated macrophages. We found that mycobacteria-infected macrophages produced soluble factors, including interleukin-6 (IL-6), IL-10, and granulocyte colony-stimulating factor (G-CSF), that induced expression of Arg1 in an autocrine-paracrine manner. Arg1 expression was controlled by the MyD88-dependent production of these cytokines rather than by cell-intrinsic MyD88 signaling to Arg1. Our study revealed that the MyD88-dependent pathway that induced the expression of Arg1 after infection by mycobacteria required STAT3 activation and that this pathway may cause the development of an immunosuppressive niche in granulomas because of the induced production of Arg1 in surrounding uninfected macrophages.

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Figures

Fig. 1
Fig. 1
Mycobacteria-infected cells secrete factor(s) that induce the expression of Arg1. (A) Wild-type mice (n = 4) were infected intranasally with BCG. Four weeks after infection, mice were sacrificed and lungs were prepared for histology by staining for acid-fast bacilli (pink, top) and for Arg1 (brown, bottom). Scale bars represent 500 µm (left panels) and 50 µm (right panels). (B) BMDMs from wild-type and Myd88−/− mice (n = 2 mice) were infected with BCG (at MOIs of 100 and 10) for 12, 24, and 48 hours. Supernatants (a 1:2 dilution of the combined supernatants from both sets of infections) were used to stimulate BMDMs from wild-type or Myd88−/− mice for either 4 or 24 hours. RNA was analyzed by qRT-PCR. Data shown are the mean fold-increase in Arg1 mRNA ± the standard deviation (SD), and are representative of two experiments (A and B). (C) BMDMs from wild-type mice were infected with Mtb strains CDC1551 and HN878 (n = 3). Supernatants from these infections were used to stimulate wild-type and Myd88−/− BMDMs (n = 3). RNA was analyzed by qRT-PCR. Data shown are the mean fold-increase in Arg1 mRNA ± the standard deviation (SD), and are from one experiment. (D and E) BMDMs from wild-type and Myd88−/− mice were infected with BCG over the indicated times. RNA from these infected cells was subjected to Affymetrix expression analysis. (D) Profiles of the fold-increase in gene expression of wild-type (dark blue traces, with Arg1 as the yellow trace) and Myd88−/− (gray traces, with Arg1 as the red trace) over time in log2 scale after infection with BCG. (E) Gene expression heat map clustered by secreted or extracellular GO terms.
Fig. 2
Fig. 2
The activation of STAT3, but not STAT6, is critical for the induction of Arg1 expression after infection with BCG. (A) BMDMs from wild-type mice were left untreated, or were infected with BCG (at MOIs of 100 or 10). Whole-cell lysates were analyzed by Western blotting for the indicated proteins. Data shown are from one experiment with pooled BMDMs from six mice. Culture supernatants from wild-type BMDMs infected with BCG for 12, 24, and 48 hours were used to stimulate BMDMs from wild-type and Stat6−/− mice (B), and BMDMs from wild-type and Il4ra−/− mice (C) for 4 and 24 hours. Whole-cell lysates were analyzed by Western blotting for the indicated proteins. Data shown are from one experiment with three mice (B) and from one experiment with one mouse (C). (D) IL-6, IL-10, and G-CSF were detected by Luminex in supernatants from BMDMs from wild-type mice infected with BCG at MOIs of 100, 10, and 1. (E) BMDMs from Stat3+/flox mice or Stat3Δ/flox;Tie2cre mice were stimulated with supernatants from BMDMs from wild-type mice infected with BCG at an MOI of 100. Whole-cell lysates were analyzed by Western blotting (n=1 based on deletion efficiency of STAT3). Data shown in D and E are from two experiments, except for the G-CSF data in (D) which are from one experiment.
Fig. 3
Fig. 3
The expression of Arg1 is increased in BCG-infected macrophages from Il10−/− mice compared to that in cells from wild-type mice. (A to C) BMDMs from wild-type, Stat6−/−, Myd88−/−, and Il10−/− mice were left untreated or were infected with BCG (at MOIs of 100, 10, and 1). RNA was analyzed by gel electrophoresis followed by Northern blotting (A, MOIs of 100 and 10 are shown) or by qRT-PCR (C). (B) Culture supernatants from the above BCG-infected cells were collected and analyzed by ELISA for the presence of IL-6 (data from cells infected at MOIs of 100 and 10 are shown). Data are presented as the mean concentration of IL-6 (ng/ml) ± SD. (D) Culture supernatants from wild-type and Il10−/− BMDMs infected with BCG (combined supernatants from infections at MOIs of 100 and 10) were used to stimulate BMDMs from wild-type mice for 4 and 24 hours. Whole-cell lysates were analyzed by Western blotting for the indicated proteins. (E) Supernatants were collected from wild-type BMDMs infected with BCG (at an MOI of 10) for 24 hours. Supernatants were diluted 1:2 and used to stimulate BMDMs from wild-type;Tie2cre mice (n = 6 mice) or Socs3flox/flox;Tie2cre mice (n = 10 mice). Twenty-four hours after stimulation, RNA was analyzed by qRT-PCR. Data are from one experiment with at least three mice per group (A to D) or from two experiments combined and presented as the mean expression ± SD (E).
Fig. 4
Fig. 4
IL-6, IL-10, and G-CSF are required for STAT3-dependent production of Arg1 after infection with BCG. (A) Supernatant transfer model: solid arrows indicate the induced production of Arg1 and dashed gray arrows indicate the loss of Arg1 expression in supernatant "recipients" from BCG-infected "donors". (B and C) BMDMs from wild-type and Myd88−/− mice were stimulated with supernatants from BCG-infected (MOI = 100) wild-type and Il6−/− BMDMs. (D and E) Myd88−/− BMDMs were stimulated with supernatants from BCG-infected wild-type BMDMs (at an MOI of 100) for 48 hours in the presence of cytokine-neutralizing antibodies. (F) BMDMs from wild-type mice were infected with BCG (at an MOI of 100) for 24 hours in the presence of cytokine-neutralizing antibodies. (B and E) RNA was analyzed by qRT-PCR. (C, D, and F) Whole-cell lysates were analyzed by Western blotting. Data are presented as mean values ± the standard error of the mean (SEM) for B and E and are representative of two experiments (B to E) or one experiment (F). (G) Arg1 and other “alternatively activated” genes are expressed in macrophages after activation of STAT6 by stimulation of cells with IL-4 or IL-13. (H) IL-10 enhances this established model of alternative activation of macrophages by increasing the cell-surface abundance of IL-4Rα, the common receptor subunit for both IL-4 and IL-13. (I) BCG-infected macrophages produce IL-6, IL-10, and G-CSF after the activation of MyD88, thus inducing the STAT3-dependent expression of Arg1 without triggering alternative activation of macrophages.
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