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Macrophage control of phagocytosed mycobacteria is increased by factors secreted by alveolar epithelial cells through nitric oxide independent mechanisms.

Petursdottir DH, Chuquimia OD, Freidl R, Fernández C - PLoS ONE (2014)

Bottom Line: Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo.We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC) can influence the functionality of resident pulmonary macrophages (PuM).We show that; a) in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b) the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS)1; c) AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS), secretion of nitric oxide (NO), or IL-12, d) in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
Tissue-resident macrophages are heterogeneous with tissue-specific and niche-specific functions. Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo. We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC) can influence the functionality of resident pulmonary macrophages (PuM). We have previously reported that factors secreted by AEC increase control of intracellular growth of BCG in macrophages. In the current study, we also aimed to investigate possible mechanisms by which AEC-derived factors increase intracellular control of BCG in both primary murine interstitial macrophages, and bone marrow-derived macrophages and characterize further the effect of these factors on macrophage differentiation. We show that; a) in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b) the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS)1; c) AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS), secretion of nitric oxide (NO), or IL-12, d) in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production. Collectively, our data show that PuM were restricted in inflammatory responses mediated by IFN-γ through SOCS1 and that factors secreted by AEC- enhanced the microbicidal capacities of macrophages by iNOS independent mechanisms.

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AECsup has opposite effects to IFN-γ on iNOS, and Arg-1 expression in BMM and PuM and mediates killing through an iNOS independent mechanism.BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup or 1 mM of NG-monomethyl-L-arginine (NMMLA) together with either IFN-γ or AECsup or left untreated. Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of a) iNOS, b) Arg-1 ± SD from 3-4 experiments. In c) the effect of the iNOS inhibitor NMMLA on intracellular growth of BCG is shown. Bacterial growth was evaluated by determining RLU. Data are expressed as mean ± SD. The differences between groups of BMM and PuM were analyzed using non-parametric, one-way ANOVA (Kruskal-Wallis) with Dunn‘s post-test. * significantly different from medium control, P<0.05
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pone-0103411-g005: AECsup has opposite effects to IFN-γ on iNOS, and Arg-1 expression in BMM and PuM and mediates killing through an iNOS independent mechanism.BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup or 1 mM of NG-monomethyl-L-arginine (NMMLA) together with either IFN-γ or AECsup or left untreated. Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of a) iNOS, b) Arg-1 ± SD from 3-4 experiments. In c) the effect of the iNOS inhibitor NMMLA on intracellular growth of BCG is shown. Bacterial growth was evaluated by determining RLU. Data are expressed as mean ± SD. The differences between groups of BMM and PuM were analyzed using non-parametric, one-way ANOVA (Kruskal-Wallis) with Dunn‘s post-test. * significantly different from medium control, P<0.05

Mentions: Up-regulation of iNOS and the subsequent synthesis of reactive nitrogen species is currently believed to be one of the primary mechanisms downstream of IFN-γ-induced killing of mycobacteria [22]. On the other hand, Arg-1 can compete with iNOS for their common substrate L-arginine, and consequently, the expression of iNOS and Arg-1 is often reciprocally regulated in macrophages [26]. To reveal possible mechanisms for the observed intracellular bacterial control, we followed the expression of iNOS and Arg-1 induced in PuM and BMM upon treatment with AECsup or IFN-γ. We found that IFN-γ but not AECsup induced iNOS in both BMM and PuM (Figure 5a). In contrast, in both PuM and BMM, Arg-1 was induced only by AECsup and not by IFN-γ (Figure 5b). Notably, the levels of Arg-1 induced in BMM were higher than the levels induced in PuM. Addition of NMMLA, an inhibitor of iNOS, to cultures of BMM infected with BCG and treated with AECsup or IFN-γ, inhibited the bacterial load reduction mediated by IFN-γ but had no effect on AECsup activity (Figure 5c) confirming that AECsup increased intracellular killing using an iNOS independent pathway.


Macrophage control of phagocytosed mycobacteria is increased by factors secreted by alveolar epithelial cells through nitric oxide independent mechanisms.

Petursdottir DH, Chuquimia OD, Freidl R, Fernández C - PLoS ONE (2014)

AECsup has opposite effects to IFN-γ on iNOS, and Arg-1 expression in BMM and PuM and mediates killing through an iNOS independent mechanism.BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup or 1 mM of NG-monomethyl-L-arginine (NMMLA) together with either IFN-γ or AECsup or left untreated. Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of a) iNOS, b) Arg-1 ± SD from 3-4 experiments. In c) the effect of the iNOS inhibitor NMMLA on intracellular growth of BCG is shown. Bacterial growth was evaluated by determining RLU. Data are expressed as mean ± SD. The differences between groups of BMM and PuM were analyzed using non-parametric, one-way ANOVA (Kruskal-Wallis) with Dunn‘s post-test. * significantly different from medium control, P<0.05
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4121081&req=5

pone-0103411-g005: AECsup has opposite effects to IFN-γ on iNOS, and Arg-1 expression in BMM and PuM and mediates killing through an iNOS independent mechanism.BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup or 1 mM of NG-monomethyl-L-arginine (NMMLA) together with either IFN-γ or AECsup or left untreated. Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of a) iNOS, b) Arg-1 ± SD from 3-4 experiments. In c) the effect of the iNOS inhibitor NMMLA on intracellular growth of BCG is shown. Bacterial growth was evaluated by determining RLU. Data are expressed as mean ± SD. The differences between groups of BMM and PuM were analyzed using non-parametric, one-way ANOVA (Kruskal-Wallis) with Dunn‘s post-test. * significantly different from medium control, P<0.05
Mentions: Up-regulation of iNOS and the subsequent synthesis of reactive nitrogen species is currently believed to be one of the primary mechanisms downstream of IFN-γ-induced killing of mycobacteria [22]. On the other hand, Arg-1 can compete with iNOS for their common substrate L-arginine, and consequently, the expression of iNOS and Arg-1 is often reciprocally regulated in macrophages [26]. To reveal possible mechanisms for the observed intracellular bacterial control, we followed the expression of iNOS and Arg-1 induced in PuM and BMM upon treatment with AECsup or IFN-γ. We found that IFN-γ but not AECsup induced iNOS in both BMM and PuM (Figure 5a). In contrast, in both PuM and BMM, Arg-1 was induced only by AECsup and not by IFN-γ (Figure 5b). Notably, the levels of Arg-1 induced in BMM were higher than the levels induced in PuM. Addition of NMMLA, an inhibitor of iNOS, to cultures of BMM infected with BCG and treated with AECsup or IFN-γ, inhibited the bacterial load reduction mediated by IFN-γ but had no effect on AECsup activity (Figure 5c) confirming that AECsup increased intracellular killing using an iNOS independent pathway.

Bottom Line: Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo.We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC) can influence the functionality of resident pulmonary macrophages (PuM).We show that; a) in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b) the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS)1; c) AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS), secretion of nitric oxide (NO), or IL-12, d) in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
Tissue-resident macrophages are heterogeneous with tissue-specific and niche-specific functions. Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo. We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC) can influence the functionality of resident pulmonary macrophages (PuM). We have previously reported that factors secreted by AEC increase control of intracellular growth of BCG in macrophages. In the current study, we also aimed to investigate possible mechanisms by which AEC-derived factors increase intracellular control of BCG in both primary murine interstitial macrophages, and bone marrow-derived macrophages and characterize further the effect of these factors on macrophage differentiation. We show that; a) in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG) by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b) the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS)1; c) AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS), secretion of nitric oxide (NO), or IL-12, d) in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production. Collectively, our data show that PuM were restricted in inflammatory responses mediated by IFN-γ through SOCS1 and that factors secreted by AEC- enhanced the microbicidal capacities of macrophages by iNOS independent mechanisms.

Show MeSH
Related in: MedlinePlus