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Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells
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Contents lists available at ScienceDirect
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Short Communication
Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells Mithun Das Gupta a,1 , Mirjam Fliesser a,1 , Jan Springer a , Tanja Breitschopf a , Hannes Schlossnagel a , Anna-Lena Schmitt a , Oliver Kurzai b , Kerstin Hünniger b , Hermann Einsele a , Jürgen Löffler a,∗ a
University Hospital Wuerzburg, Medical Clinic and Polyclinic II, Wuerzburg, Germany Septomics Research Centre, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology–Hans Knoell Institute, Jena, Germany b
a r t i c l e
i n f o
Article history: Received 3 February 2014 Received in revised form 10 April 2014 Accepted 19 April 2014 Keywords: Aspergillus fumigatus Dendritic cells Microrna Monocytes
a b s t r a c t Aspergillus fumigatus is responsible for severe and often fatal infections in immunocompromised patients. The human immune response against this pathogenic mould is still not fully understood. Recently, microRNAs (miRNAs) have been characterized as regulators of inflammation and immune response in various diseases. MiRNAs specifically bind to mRNA target sequences, thereby leading to gene silencing by target degradation and/or translational repression. To investigate the possible role of miRNAs during A. fumigatus infection, we studied the expression of two major immune relevant miRNAs, miR-132 and miR-155, in human monocytes and dendritic cells (DCs). Both cell types are crucial for the immune response against A. fumigatus. Here, we demonstrate for the first time that miR-132 and miR-155 are differentially expressed in monocytes and DCs upon stimulation with A. fumigatus or bacterial lipopolysaccharide (LPS). Interestingly, miR-132 was induced by A. fumigatus but not by LPS in both cell types. Our data suggest that miR-132 may be a relevant regulator of the immune response directed against A. fumigatus. © 2014 Elsevier GmbH. All rights reserved.
Introduction Aspergillus fumigatus is the major pathogen causing invasive aspergillosis (IA), one of the most fatal infections in patients with acute leukaemia and recipients of allogeneic hematopoietic stem-cell transplants (Segal, 2009). Diagnosis of IA is difficult and although antifungal agents are widely applied in the clinic, every second patient dies from this infection. A better characterization of the human immune response directed against A. fumigatus may help to develop novel treatment strategies as well as improve diagnosis of IA. Monocytes are important for the innate immune response against A. fumigatus, for example by damaging fungal hyphae (Diamond et al., 1983). Dendritic cells (DCs) are antigen presenting cells that bridge innate and adaptive immunity. DCs initiate a T-helper cell response against A. fumigatus, which is important
∗ Corresponding author. Tel.: +49 931 201 36412; fax: +49 931 201 36409. E-mail address: loeffler [email protected] (J. Löffler). 1 These authors contributed equally to the manuscript.
to overcome the infection (Bozza et al., 2002). A. fumigatus is recognized on the cell surface of monocytes and DCs by toll-like receptors (TLR2 and TLR4) and the c-type lectin receptor dectin-1 (Segal, 2009). Downstream of these receptors, an intricate regulatory system coordinates the inflammatory response of the immune cells. Recent reports have identified microRNAs (miRNAs) as important regulators of TLR signalling (O’Neill et al., 2011). MiRNAs are short, approximately 22 nucleotide long RNA sequences that bind to complementary sequences in the 3 -untranslated region (3 -UTR) of multiple target mRNAs, thereby leading to gene silencing by translational repression and/or mRNA target degradation (Bartel, 2009). Upon activation of TLRs, the expression of certain miRNAs is up-regulated in immune cells. These miRNAs may then modulate TLR signalling by targeting downstream molecules or TLR expression itself. Among others, miR-132 and miR-155 have been characterized as “fine-tuners of TLR signaling” (O’Neill et al., 2011). Briefly, miR-132 was shown to modulate peptidoglycan-induced TLR2 signalling by targeting IL-1R-associated kinase 4 (IRAK4) downstream of TLR2 in the human monocytic THP-1 cell line (Nahid et al., 2013). In human DCs, miR-155 has been shown to target
http://dx.doi.org/10.1016/j.ijmm.2014.04.005 1438-4221/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Das Gupta, M., et al., Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.005
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TGF-beta activated kinase 1, an adaptor in the TLR/IL1 signalling cascade, thereby controlling the intensity of the DC inflammatory response (Ceppi et al., 2009). However, gene regulation by miRNAs is highly complex and miRNA profiles may vary between different cell types and pathogenic stimuli. Very little is known so far about the possible role of miRNAs during fungal infections and about miRNA regulation in primary human immune cells responding to fungal pathogens like A. fumigatus. Here, to our knowledge for the first time, we present data on the regulation of miR-132 and miR-155 in primary human monocytes and DCs after stimulation with the pathogenic mould A. fumigatus. We stimulated both cell types with different A. fumigatus morphologies (conidia, germ tubes, hyphae) or with the bacterial TLR4 ligand ultrapure lipopolysaccharide (LPS). Our data demonstrate that miR-132 is specifically induced in human monocytes and DCs by stimulation with A. fumigatus but not by LPS, indicating a unique role of miR-132 in the response of human primary innate immune cells during Aspergillus infection. Materials and methods Cell culture and fungus Human monocytes were isolated from peripheral blood mononuclear cells of healthy volunteers by magnetic-activated cell sorting using microbeads conjugated to anti-human CD14 monoclonal antibodies according to manufacturer’s instructions (Miltenyi Biotec). Monocytes were either used immediately or differentiated into DCs by five days incubation with GM-CSF and IL-4 as described before (Mezger et al., 2009). Experiments were performed with 5 × 106 monocytes or 1 × 106 DCs per condition in 1 ml RPMI 1640 medium (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich) and 120 g/ml gentamicin (Merck). Conidia of the A. fumigatus isolate ATCC 46645 (American Type Culture Collection) were prepared as previously described (Morton et al., 2011). Germ tubes and hyphae were grown by incubating conidia in RPMI 1640 medium without FCS at 200 rpm and room temperature overnight followed by incubation at 37 ◦ C until the respective fungal morphology was microscopically visible. Hyphae had an approximate length of 50–100 M. In order to maintain stable conditions over time, experiments with different A. fumigatus morphologies or different multiplicities of infection (MOIs) were performed with fungus inactivated by incubation in 70% ethanol for 30 min. Immune cells were stimulated with A. fumigatus conidia, germ tubes or hyphae with a multiplicity of infection (MOI) of 1 (if not indicated otherwise) or with 1 g/ml ultrapure LPS (Invivogen) or left untreated (ctrl). Co-cultures with monocytes (5 × 106 /ml) were performed for 2, 4 and 6 h and co-cultures with DCs (1 × 106 /ml) for 4, 6 and 9 h, respectively. Cytokine quantification To confirm activation of immune cells, the pro-inflammatory cytokines interleukin 6 (IL-6) and tumour necrosis factor ␣ (TNF-␣) were quantified in culture supernatants of immune cells cultivated for 4 and 6 h (monocytes) or for 6 and 9 h (DCs) with ultrapure LPS (1 g/ml) and A. fumigatus germ tubes (MOI = 1), respectively. Enzyme-linked immunosorbent assays (ELISA) were performed using Quantikine Colorimetric Sandwich ELISA assays (R&D Systems) according to manufacturer’s instructions. MiRNA quantification RNA was extracted using a total RNA isolation kit according to manufacturer’s instructions (mirVana miRNA Isolation Kit,
Ambion). 10 ng of total RNA was constantly used for the miRNA specific reverse transcription (RT). Subsequent quantification of these specific miRNAs was performed by quantitative real-time PCR (qPCR) with TaqMan probes as described by the manufacturer (MicroRNA RT Kit and miRNA-specific TaqMan MicroRNA Assays, both Applied Biosystems). For qPCR, three technical replicates were amplified. Data analysis was performed by the 2Ct method (Livak and Schmittgen, 2001), calculating expression levels as fold changes relative to the expression at 0 h. Statistical analysis was performed by paired t-test using GraphPad Prism.
Results and discussion In this study, we investigated the expression of two major immune relevant miRNAs, miR-132 and miR-155 in human monocytes and DCs after stimulation with A. fumigatus or the bacterial TLR 4 ligand ultrapure LPS. Initially, to confirm activation of immune cells by these two stimuli, we quantified the cytokines IL-6 and TNF-␣ in cell culture supernatants. In both cell types, IL-6 and TNF-␣ were up-regulated compared to untreated control cells after co-incubation for 4 and 6 h (monocytes) and 6 and 9 h (DCs) with LPS or A. fumigatus (Fig. 1A–D), thus confirming immune cell activation in our experimental setup. Expression of miR-155 and miR-132 was measured in monocytes and DCs co-cultivated for 2, 4 and 6 h (monocytes) or for 4, 6 and 9 h (DCs) with A. fumigatus germ tubes or LPS, respectively. To further characterize specificity of miR-132 regulation, monocytes and DCs were additionally co-cultivated with different fungal morphologies (conidia, germ tubes or hyphae) and different multiplicities of infection (MOI 1, 2.5 or 5). In monocytes, A. fumigatus germ tubes induced the expression of miR-132 (Fig. 2A) but not of miR-155 (Fig. 2B). In contrast, stimulation with LPS only enhanced the expression of miR-155, but not of miR-132 (Fig. 2A and B). In DCs, miR-132 expression was again increased by A. fumigatus germ tubes, whereas LPS did not induce miR-132 expression (Fig. 2C). MiR-155 was enhanced in DCs by LPS and, in contrast to monocytes, also by A. fumigatus germ tubes (Fig. 2D). Taken together, expression of miR-132 was specifically induced by A. fumigatus but not by LPS in monocytes as well as in DCs. Therefore, we focused our subsequent experiments on miR-132 expression kinetics. A. fumigatus conidia possess a layer of surface hydrophobins that prevent the recognition by immune cells. Upon germination, this rodlet layer is lost and the fungus is recognized by immune cells (Aimanianda et al., 2009). Therefore, the immune response against A. fumigatus is dependent on the morphology of the fungus. Our study supports these data for miRNA expression by stimulating monocytes and DCs with different A. fumigatus morphologies. In both cell types, conidia were unable to induce miR-132 expression, whereas both germ tubes and hyphae increased miR-132 levels (Fig. 3A and B). In DCs, expression of miR-132 was comparable between germ tubes and hyphae (Fig. 3B). Interestingly, monocytes stimulated with hyphae showed significantly higher miR-132 levels compared to germ tubes (Fig. 3A). Furthermore, in monocytes, miR-132 levels increased over time whereas in DCs, expression was already at a maximum level after 4 h (Fig. 3A and B). To evaluate the dependency of miR-132 up-regulation on the immune stimulatory capacity of the fungus, we tested the relevance of the multiplicity of infection (MOI) for miR-132 induction. Higher MOIs (= higher numbers of fungal cells per immune cell) possess a stronger immune-stimulatory capacity. We observed markedly increased miR-132 expression levels in monocytes stimulated with higher MOIs (Fig. 3C), whereas in DCs, MOIs of 1, 2.5 or 5 caused a similar expression of miR-132 (Fig. 3D). These results demonstrate
Please cite this article in press as: Das Gupta, M., et al., Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.005
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Fig. 1. Cytokine release confirms activation of human immune cells. Monocytes and DCs were stimulated with lipopolysaccharide (LPS, 1 g/ml) or live A. fumigatus germ tubes with a multiplicity of infection (MOI) of 1. Levels of IL-6 and TNF-␣ were quantified in culture supernatants after 4 and 6 h (monocytes) or 6 and 9 h (DCs) by ELISA assays [log (pg/ml)]. Data are presented as mean + SEM with n = 2.
Fig. 2. Expression of immune relevant miRNAs in human monocytes and DCs. (A) and (B) Monocytes were stimulated for 2, 4 and 6 h and (C) and (D) DCs for 4, 6 and 9 h with lipopolysaccharide (LPS, 1 g/ml) or living A. fumigatus germ tubes with a multiplicity of infection (MOI) of 1. Expression of (A) and (C) miR-132 and (B) and (D) miR-155 was quantified by RT-qPCR and expression levels were calculated relative to the expression at 0 h. Data are presented as mean + SEM with n = 3. Statistical analysis was performed by t-test comparing expression levels of two conditions as indicated or compared to the expression at 0 h. * p < 0.1; ** p < 0.05.
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Fig. 3. Correlation of miR-132 expression and fungal morphology and fungal MOI. (A) and (B) Correlation of miR-132 expression and fungal morphology is shown over time. Immune cells were stimulated with inactivated A. fumigatus morphologies (resting conidia, germ tubes or hyphae) with a multiplicity of infection (MOI) of 1. (C) and (D) Correlation of miR-132 expression and fungal MOI is shown over time. Immune cells were stimulated with inactivated A. fumigatus germ tubes with MOIs of 1, 2.5 or 5. Expression of miR-132 was quantified by RT-qPCR and expression levels were calculated relative to the expression at 0 h. Data are presented as mean + SEM with n = 3. Statistical analysis was performed by t-test comparing expression levels of two conditions as indicated or compared to the expression at 0 h. * p < 0.1; ** p < 0.05.
that in monocytes, up-regulation of miR-132 increases with the amount of fungus used for immune cell stimulation. Taken together, induction of miR-132 and miR-155 expression was dependent on the immune cell type (monocytes or DCs) and the applied stimulus (A. fumigatus or LPS). In monocytes, miR-132 expression was also dependent on fungal morphology and fungal MOI. Accumulating evidence suggests that miR-155 expression is induced by a variety of stimuli such as tumour necrosis factor, interferon-, Helicobacter pylori and Epstein–Barr virus (O’Neill et al., 2011). Our results complement this concept as we show that miR-155 is up-regulated in DCs by both, LPS and as a new finding, by A. fumigatus. In consequence, miR-155 might not be a specific component of one of the PRR pathways, but rather a general feature of DCs responding to a broad range of pathogens. In contrast, miR-132 expression in monocytes and DCs was exclusively induced by A. fumigatus, but not by LPS. Living (Fig. 2) and inactivated (Fig. 3) fungal morphologies revealed comparable results, thus, a fungal cell wall structure rather than a factor secreted by live fungus might be responsible for miR-132 induction. A. fumigatus cell wall molecules activate TLR2 and TLR4 (Braedel et al., 2004) as well as dectin-1 signalling pathways (Mezger et al., 2008), whereas the ultrapure LPS preparation used in our study activates TLR4 only. Therefore, our observations suggest an association between miR-132 expression and the activation of TLR2 and/or dectin-1 but not TLR4 in DCs. A recent publication with the monocytic THP-1 cell line supports the association between miR-132 expression and TLR2 activation. Nahid et al. describe the induction of miR-132 after TLR2 ligation and hypothesize a feedback regulatory mechanism of miR-132 on the TLR2 signalling
cascade, mediated by miR-132 targeting IRAK4 (Nahid et al., 2013). Originally, miR-132 was discovered as a critical regulator for neuronal development and function (Vo et al., 2005). Other targets of miR-132 include molecules involved in inflammation and immune response, e.g. acetylcholinesterase in the murine brain (Shaked et al., 2009) and the p300 transcriptional co-activator in primary lymphatic endothelial cells (Lagos et al., 2010). However, the role of miR-132 in human monocytes and DCs is still poorly understood. It is obvious that more detailed and functional studies are highly warranted to define specific miR-132 target pathways and to identify additional miRNAs relevant for the cellular immune response against A. fumigatus. Our data, showing specific up-regulation of miR-132 in human monocytes and DCs provide an important first insight to a novel aspect of the human immune response against A. fumigatus. Ethics statement This study, using whole blood specimens obtained from human healthy volunteer donors, was approved by the Ethical Committee of the University Hospital of Würzburg. Informed consent was written and provided by all study participants. Data analysis was conducted anonymously. Acknowledgements This study was supported by research funding from the SanderStiftung (Project 2007.102.1), the IZKF Würzburg, project A127 and
Please cite this article in press as: Das Gupta, M., et al., Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.005
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the CRC TR124 (Pathogenic Fungi and their Human Host: Networks of Interaction). Work in the laboratory of OK and KH was supported by the Federal Ministry of Science and Technology (BMBF 03Z2JN21 to OK Germany) within the Unternehmen Region programme. References Aimanianda, V., Bayry, J., Bozza, S., Kniemeyer, O., Perruccio, K., Elluru, S.R., Clavaud, C., Paris, S., Brakhage, A.A., Kaveri, S.V., Romani, L., Latge, J.P., 2009. Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature 460, 1117–1121. Bartel, D.P., 2009. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233. Bozza, S., Gaziano, R., Spreca, A., Bacci, A., Montagnoli, C., di Francesco, P., Romani, L., 2002. Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus. J. Immunol. 168, 1362–1371. Braedel, S., Radsak, M., Einsele, H., Latge, J.P., Michan, A., Loeffler, J., Haddad, Z., Grigoleit, U., Schild, H., Hebart, H., 2004. Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4. Br. J. Haematol. 125, 392–399. Ceppi, M., Pereira, P.M., Dunand-Sauthier, I., Barras, E., Reith, W., Santos, M.A., Pierre, P., 2009. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells. Proc. Natl. Acad. Sci. USA 106, 2735–2740. Diamond, R.D., Huber, E., Haudenschild, C.C., 1983. Mechanisms of destruction of Aspergillus fumigatus hyphae mediated by human monocytes. J. Infect. Dis. 147, 474–483.
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Lagos, D., Pollara, G., Henderson, S., Gratrix, F., Fabani, M., Milne, R.S., Gotch, F., Boshoff, C., 2010. miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator. Nat. Cell Biol. 12, 513–519. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. Mezger, M., Bonin, M., Kessler, T., Gebhardt, F., Einsele, H., Loeffler, J., 2009. Toll-like receptor 3 has no critical role during early immune response of human monocyte-derived dendritic cells after infection with the human cytomegalovirus strain TB40E. Viral Immunol. 22, 343–351. Mezger, M., Kneitz, S., Wozniok, I., Kurzai, O., Einsele, H., Loeffler, J., 2008. Proinflammatory response of immature human dendritic cells is mediated by dectin-1 after exposure to Aspergillus fumigatus germ tubes. J. Infect. Dis. 197, 924–931. Morton, C.O., Varga, J.J., Hornbach, A., Mezger, M., Sennefelder, H., Kneitz, S., Kurzai, O., Krappmann, S., Einsele, H., Nierman, W.C., Rogers, T.R., Loeffler, J., 2011. The temporal dynamics of differential gene expression in Aspergillus fumigatus interacting with human immature dendritic cells in vitro. PLoS ONE 6, e16016. Nahid, M.A., Yao, B., Dominguez-Gutierrez, P.R., Kesavalu, L., Satoh, M., Chan, E.K., 2013. Regulation of TLR2-mediated tolerance and cross-tolerance through IRAK4 modulation by miR-132 and miR-212. J. Immunol. 190, 1250–1263. O’Neill, L.A., Sheedy, F.J., McCoy, C.E., 2011. MicroRNAs: the fine-tuners of Toll-like receptor signalling. Nat. Rev. Immunol. 11, 163–175. Segal, B.H., 2009. Aspergillosis. N. Engl. J. Med. 360, 1870–1884. Shaked, I., Meerson, A., Wolf, Y., Avni, R., Greenberg, D., Gilboa-Geffen, A., Soreq, H., 2009. MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase. Immunity 31, 965–973. Vo, N., Klein, M.E., Varlamova, O., Keller, D.M., Yamamoto, T., Goodman, R.H., Impey, S., 2005. A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc. Natl. Acad. Sci. USA 102, 16426–16431.
Please cite this article in press as: Das Gupta, M., et al., Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.005
ARTICLE IN PRESS
IJMM-50813; No. of Pages 5
International Journal of Medical Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm
Short Communication
Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells Mithun Das Gupta a,1 , Mirjam Fliesser a,1 , Jan Springer a , Tanja Breitschopf a , Hannes Schlossnagel a , Anna-Lena Schmitt a , Oliver Kurzai b , Kerstin Hünniger b , Hermann Einsele a , Jürgen Löffler a,∗ a
University Hospital Wuerzburg, Medical Clinic and Polyclinic II, Wuerzburg, Germany Septomics Research Centre, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology–Hans Knoell Institute, Jena, Germany b
a r t i c l e
i n f o
Article history: Received 3 February 2014 Received in revised form 10 April 2014 Accepted 19 April 2014 Keywords: Aspergillus fumigatus Dendritic cells Microrna Monocytes
a b s t r a c t Aspergillus fumigatus is responsible for severe and often fatal infections in immunocompromised patients. The human immune response against this pathogenic mould is still not fully understood. Recently, microRNAs (miRNAs) have been characterized as regulators of inflammation and immune response in various diseases. MiRNAs specifically bind to mRNA target sequences, thereby leading to gene silencing by target degradation and/or translational repression. To investigate the possible role of miRNAs during A. fumigatus infection, we studied the expression of two major immune relevant miRNAs, miR-132 and miR-155, in human monocytes and dendritic cells (DCs). Both cell types are crucial for the immune response against A. fumigatus. Here, we demonstrate for the first time that miR-132 and miR-155 are differentially expressed in monocytes and DCs upon stimulation with A. fumigatus or bacterial lipopolysaccharide (LPS). Interestingly, miR-132 was induced by A. fumigatus but not by LPS in both cell types. Our data suggest that miR-132 may be a relevant regulator of the immune response directed against A. fumigatus. © 2014 Elsevier GmbH. All rights reserved.
Introduction Aspergillus fumigatus is the major pathogen causing invasive aspergillosis (IA), one of the most fatal infections in patients with acute leukaemia and recipients of allogeneic hematopoietic stem-cell transplants (Segal, 2009). Diagnosis of IA is difficult and although antifungal agents are widely applied in the clinic, every second patient dies from this infection. A better characterization of the human immune response directed against A. fumigatus may help to develop novel treatment strategies as well as improve diagnosis of IA. Monocytes are important for the innate immune response against A. fumigatus, for example by damaging fungal hyphae (Diamond et al., 1983). Dendritic cells (DCs) are antigen presenting cells that bridge innate and adaptive immunity. DCs initiate a T-helper cell response against A. fumigatus, which is important
∗ Corresponding author. Tel.: +49 931 201 36412; fax: +49 931 201 36409. E-mail address: loeffler [email protected] (J. Löffler). 1 These authors contributed equally to the manuscript.
to overcome the infection (Bozza et al., 2002). A. fumigatus is recognized on the cell surface of monocytes and DCs by toll-like receptors (TLR2 and TLR4) and the c-type lectin receptor dectin-1 (Segal, 2009). Downstream of these receptors, an intricate regulatory system coordinates the inflammatory response of the immune cells. Recent reports have identified microRNAs (miRNAs) as important regulators of TLR signalling (O’Neill et al., 2011). MiRNAs are short, approximately 22 nucleotide long RNA sequences that bind to complementary sequences in the 3 -untranslated region (3 -UTR) of multiple target mRNAs, thereby leading to gene silencing by translational repression and/or mRNA target degradation (Bartel, 2009). Upon activation of TLRs, the expression of certain miRNAs is up-regulated in immune cells. These miRNAs may then modulate TLR signalling by targeting downstream molecules or TLR expression itself. Among others, miR-132 and miR-155 have been characterized as “fine-tuners of TLR signaling” (O’Neill et al., 2011). Briefly, miR-132 was shown to modulate peptidoglycan-induced TLR2 signalling by targeting IL-1R-associated kinase 4 (IRAK4) downstream of TLR2 in the human monocytic THP-1 cell line (Nahid et al., 2013). In human DCs, miR-155 has been shown to target
http://dx.doi.org/10.1016/j.ijmm.2014.04.005 1438-4221/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Das Gupta, M., et al., Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.005
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TGF-beta activated kinase 1, an adaptor in the TLR/IL1 signalling cascade, thereby controlling the intensity of the DC inflammatory response (Ceppi et al., 2009). However, gene regulation by miRNAs is highly complex and miRNA profiles may vary between different cell types and pathogenic stimuli. Very little is known so far about the possible role of miRNAs during fungal infections and about miRNA regulation in primary human immune cells responding to fungal pathogens like A. fumigatus. Here, to our knowledge for the first time, we present data on the regulation of miR-132 and miR-155 in primary human monocytes and DCs after stimulation with the pathogenic mould A. fumigatus. We stimulated both cell types with different A. fumigatus morphologies (conidia, germ tubes, hyphae) or with the bacterial TLR4 ligand ultrapure lipopolysaccharide (LPS). Our data demonstrate that miR-132 is specifically induced in human monocytes and DCs by stimulation with A. fumigatus but not by LPS, indicating a unique role of miR-132 in the response of human primary innate immune cells during Aspergillus infection. Materials and methods Cell culture and fungus Human monocytes were isolated from peripheral blood mononuclear cells of healthy volunteers by magnetic-activated cell sorting using microbeads conjugated to anti-human CD14 monoclonal antibodies according to manufacturer’s instructions (Miltenyi Biotec). Monocytes were either used immediately or differentiated into DCs by five days incubation with GM-CSF and IL-4 as described before (Mezger et al., 2009). Experiments were performed with 5 × 106 monocytes or 1 × 106 DCs per condition in 1 ml RPMI 1640 medium (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich) and 120 g/ml gentamicin (Merck). Conidia of the A. fumigatus isolate ATCC 46645 (American Type Culture Collection) were prepared as previously described (Morton et al., 2011). Germ tubes and hyphae were grown by incubating conidia in RPMI 1640 medium without FCS at 200 rpm and room temperature overnight followed by incubation at 37 ◦ C until the respective fungal morphology was microscopically visible. Hyphae had an approximate length of 50–100 M. In order to maintain stable conditions over time, experiments with different A. fumigatus morphologies or different multiplicities of infection (MOIs) were performed with fungus inactivated by incubation in 70% ethanol for 30 min. Immune cells were stimulated with A. fumigatus conidia, germ tubes or hyphae with a multiplicity of infection (MOI) of 1 (if not indicated otherwise) or with 1 g/ml ultrapure LPS (Invivogen) or left untreated (ctrl). Co-cultures with monocytes (5 × 106 /ml) were performed for 2, 4 and 6 h and co-cultures with DCs (1 × 106 /ml) for 4, 6 and 9 h, respectively. Cytokine quantification To confirm activation of immune cells, the pro-inflammatory cytokines interleukin 6 (IL-6) and tumour necrosis factor ␣ (TNF-␣) were quantified in culture supernatants of immune cells cultivated for 4 and 6 h (monocytes) or for 6 and 9 h (DCs) with ultrapure LPS (1 g/ml) and A. fumigatus germ tubes (MOI = 1), respectively. Enzyme-linked immunosorbent assays (ELISA) were performed using Quantikine Colorimetric Sandwich ELISA assays (R&D Systems) according to manufacturer’s instructions. MiRNA quantification RNA was extracted using a total RNA isolation kit according to manufacturer’s instructions (mirVana miRNA Isolation Kit,
Ambion). 10 ng of total RNA was constantly used for the miRNA specific reverse transcription (RT). Subsequent quantification of these specific miRNAs was performed by quantitative real-time PCR (qPCR) with TaqMan probes as described by the manufacturer (MicroRNA RT Kit and miRNA-specific TaqMan MicroRNA Assays, both Applied Biosystems). For qPCR, three technical replicates were amplified. Data analysis was performed by the 2Ct method (Livak and Schmittgen, 2001), calculating expression levels as fold changes relative to the expression at 0 h. Statistical analysis was performed by paired t-test using GraphPad Prism.
Results and discussion In this study, we investigated the expression of two major immune relevant miRNAs, miR-132 and miR-155 in human monocytes and DCs after stimulation with A. fumigatus or the bacterial TLR 4 ligand ultrapure LPS. Initially, to confirm activation of immune cells by these two stimuli, we quantified the cytokines IL-6 and TNF-␣ in cell culture supernatants. In both cell types, IL-6 and TNF-␣ were up-regulated compared to untreated control cells after co-incubation for 4 and 6 h (monocytes) and 6 and 9 h (DCs) with LPS or A. fumigatus (Fig. 1A–D), thus confirming immune cell activation in our experimental setup. Expression of miR-155 and miR-132 was measured in monocytes and DCs co-cultivated for 2, 4 and 6 h (monocytes) or for 4, 6 and 9 h (DCs) with A. fumigatus germ tubes or LPS, respectively. To further characterize specificity of miR-132 regulation, monocytes and DCs were additionally co-cultivated with different fungal morphologies (conidia, germ tubes or hyphae) and different multiplicities of infection (MOI 1, 2.5 or 5). In monocytes, A. fumigatus germ tubes induced the expression of miR-132 (Fig. 2A) but not of miR-155 (Fig. 2B). In contrast, stimulation with LPS only enhanced the expression of miR-155, but not of miR-132 (Fig. 2A and B). In DCs, miR-132 expression was again increased by A. fumigatus germ tubes, whereas LPS did not induce miR-132 expression (Fig. 2C). MiR-155 was enhanced in DCs by LPS and, in contrast to monocytes, also by A. fumigatus germ tubes (Fig. 2D). Taken together, expression of miR-132 was specifically induced by A. fumigatus but not by LPS in monocytes as well as in DCs. Therefore, we focused our subsequent experiments on miR-132 expression kinetics. A. fumigatus conidia possess a layer of surface hydrophobins that prevent the recognition by immune cells. Upon germination, this rodlet layer is lost and the fungus is recognized by immune cells (Aimanianda et al., 2009). Therefore, the immune response against A. fumigatus is dependent on the morphology of the fungus. Our study supports these data for miRNA expression by stimulating monocytes and DCs with different A. fumigatus morphologies. In both cell types, conidia were unable to induce miR-132 expression, whereas both germ tubes and hyphae increased miR-132 levels (Fig. 3A and B). In DCs, expression of miR-132 was comparable between germ tubes and hyphae (Fig. 3B). Interestingly, monocytes stimulated with hyphae showed significantly higher miR-132 levels compared to germ tubes (Fig. 3A). Furthermore, in monocytes, miR-132 levels increased over time whereas in DCs, expression was already at a maximum level after 4 h (Fig. 3A and B). To evaluate the dependency of miR-132 up-regulation on the immune stimulatory capacity of the fungus, we tested the relevance of the multiplicity of infection (MOI) for miR-132 induction. Higher MOIs (= higher numbers of fungal cells per immune cell) possess a stronger immune-stimulatory capacity. We observed markedly increased miR-132 expression levels in monocytes stimulated with higher MOIs (Fig. 3C), whereas in DCs, MOIs of 1, 2.5 or 5 caused a similar expression of miR-132 (Fig. 3D). These results demonstrate
Please cite this article in press as: Das Gupta, M., et al., Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.005
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Fig. 1. Cytokine release confirms activation of human immune cells. Monocytes and DCs were stimulated with lipopolysaccharide (LPS, 1 g/ml) or live A. fumigatus germ tubes with a multiplicity of infection (MOI) of 1. Levels of IL-6 and TNF-␣ were quantified in culture supernatants after 4 and 6 h (monocytes) or 6 and 9 h (DCs) by ELISA assays [log (pg/ml)]. Data are presented as mean + SEM with n = 2.
Fig. 2. Expression of immune relevant miRNAs in human monocytes and DCs. (A) and (B) Monocytes were stimulated for 2, 4 and 6 h and (C) and (D) DCs for 4, 6 and 9 h with lipopolysaccharide (LPS, 1 g/ml) or living A. fumigatus germ tubes with a multiplicity of infection (MOI) of 1. Expression of (A) and (C) miR-132 and (B) and (D) miR-155 was quantified by RT-qPCR and expression levels were calculated relative to the expression at 0 h. Data are presented as mean + SEM with n = 3. Statistical analysis was performed by t-test comparing expression levels of two conditions as indicated or compared to the expression at 0 h. * p < 0.1; ** p < 0.05.
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Fig. 3. Correlation of miR-132 expression and fungal morphology and fungal MOI. (A) and (B) Correlation of miR-132 expression and fungal morphology is shown over time. Immune cells were stimulated with inactivated A. fumigatus morphologies (resting conidia, germ tubes or hyphae) with a multiplicity of infection (MOI) of 1. (C) and (D) Correlation of miR-132 expression and fungal MOI is shown over time. Immune cells were stimulated with inactivated A. fumigatus germ tubes with MOIs of 1, 2.5 or 5. Expression of miR-132 was quantified by RT-qPCR and expression levels were calculated relative to the expression at 0 h. Data are presented as mean + SEM with n = 3. Statistical analysis was performed by t-test comparing expression levels of two conditions as indicated or compared to the expression at 0 h. * p < 0.1; ** p < 0.05.
that in monocytes, up-regulation of miR-132 increases with the amount of fungus used for immune cell stimulation. Taken together, induction of miR-132 and miR-155 expression was dependent on the immune cell type (monocytes or DCs) and the applied stimulus (A. fumigatus or LPS). In monocytes, miR-132 expression was also dependent on fungal morphology and fungal MOI. Accumulating evidence suggests that miR-155 expression is induced by a variety of stimuli such as tumour necrosis factor, interferon-, Helicobacter pylori and Epstein–Barr virus (O’Neill et al., 2011). Our results complement this concept as we show that miR-155 is up-regulated in DCs by both, LPS and as a new finding, by A. fumigatus. In consequence, miR-155 might not be a specific component of one of the PRR pathways, but rather a general feature of DCs responding to a broad range of pathogens. In contrast, miR-132 expression in monocytes and DCs was exclusively induced by A. fumigatus, but not by LPS. Living (Fig. 2) and inactivated (Fig. 3) fungal morphologies revealed comparable results, thus, a fungal cell wall structure rather than a factor secreted by live fungus might be responsible for miR-132 induction. A. fumigatus cell wall molecules activate TLR2 and TLR4 (Braedel et al., 2004) as well as dectin-1 signalling pathways (Mezger et al., 2008), whereas the ultrapure LPS preparation used in our study activates TLR4 only. Therefore, our observations suggest an association between miR-132 expression and the activation of TLR2 and/or dectin-1 but not TLR4 in DCs. A recent publication with the monocytic THP-1 cell line supports the association between miR-132 expression and TLR2 activation. Nahid et al. describe the induction of miR-132 after TLR2 ligation and hypothesize a feedback regulatory mechanism of miR-132 on the TLR2 signalling
cascade, mediated by miR-132 targeting IRAK4 (Nahid et al., 2013). Originally, miR-132 was discovered as a critical regulator for neuronal development and function (Vo et al., 2005). Other targets of miR-132 include molecules involved in inflammation and immune response, e.g. acetylcholinesterase in the murine brain (Shaked et al., 2009) and the p300 transcriptional co-activator in primary lymphatic endothelial cells (Lagos et al., 2010). However, the role of miR-132 in human monocytes and DCs is still poorly understood. It is obvious that more detailed and functional studies are highly warranted to define specific miR-132 target pathways and to identify additional miRNAs relevant for the cellular immune response against A. fumigatus. Our data, showing specific up-regulation of miR-132 in human monocytes and DCs provide an important first insight to a novel aspect of the human immune response against A. fumigatus. Ethics statement This study, using whole blood specimens obtained from human healthy volunteer donors, was approved by the Ethical Committee of the University Hospital of Würzburg. Informed consent was written and provided by all study participants. Data analysis was conducted anonymously. Acknowledgements This study was supported by research funding from the SanderStiftung (Project 2007.102.1), the IZKF Würzburg, project A127 and
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Please cite this article in press as: Das Gupta, M., et al., Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells. Int. J. Med. Microbiol. (2014), http://dx.doi.org/10.1016/j.ijmm.2014.04.005