CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway

Copyright: © 2015 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Acknowledgments We are grateful to the Second Core Laboratory of Research Core Facility at the National Taiwan University Hospital for confocal microscopy service and providing ultracentrifuge. We thank Dr. William E. Goldman (University of North Carolina, Chapel Hill, NC) for kindly providing WT and ags1-null mutant of H. capsulatum G186A. Funding: This work is supported by research grants 101-2320-B-002-030-MY3 from the Ministry of Science and Technology (http://www.most.gov.tw) and AS-101-TP-B06-3 from Academia Sinica (http://www.sinica.edu.tw) to BAWH. GDB is funded by research grant 102705 from Welcome Trust (http://www.wellcome.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway

Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection

Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Positively Modulates Matrix Metalloproteinase-9 Production in Alveolar Macrophages upon Toll-Like Receptor 7 Signaling and Influenza Virus Infection

Influenza A virus (IAV) infection causes significant morbidity and mortality worldwide. Matrix metalloproteinase-9 (MMP-9) degrades extracellular matrix and is involved in the pathology of influenza. It has been reported that MMP-9 mediates neutrophil migration in IAV infection. Whether alveolar macrophages, the first immune cells that encounter IAV, produce MMP-9, and the mechanism of its regulation have never been investigated. As Toll-like receptor 7 (TLR7) is one of the receptors in innate immune cells that recognize IAV, we used TLR7 agonists and IAV to stimulate alveolar macrophage MH-S cells, primary macrophages, and bone marrow neutrophils. Results showed that MMP-9 expression in macrophages is inducible by TLR7 agonists and IAV, yet, MMP-9 production by neutrophils is not inducible by either one of them. We hypothesized that MMP-9 production in macrophages is mediated through TLR7-NF-κB pathway and used microarray to analyze TLR7 agonist-induced NF-κB-related genes. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), a positive regulator of NF-κB, is amongst the top highly induced genes. By use of MALT1 inhibitor (z-VRPR-fmk) and alveolar macrophages from MALT1-deficient mice, we found that MMP-9 production is MALT1-dependent. While MALT1 can act as a paracaspase in lymphocytes through degrading various signaling proteins, we discovered that MALT1 functions to reduce a negative regulator of NF-κB, cylindromatosis (CYLD), in alveolar macrophages. IAV-induced MMP-9, TNF, and IL-6 in lungs of MALT1-deficient mice are significantly lower than in wild-type mice after intratracheal infection. MALT1-deficient mice also have less body weight loss and longer survival after infection. Taken together, we demonstrated a novel role of MALT1 in regulating alveolar macrophage MMP-9 production whose presence exacerbates the severity of influenza

Immunization with Apoptotic Phagocytes Containing Histoplasma capsulatum Activates Functional CD8+ T Cells To Protect against Histoplasmosis ▿

We have previously revealed the protective role of CD8+ T cells in host defense against Histoplasma capsulatum in animals with CD4+ T cell deficiency and demonstrated that sensitized CD8+ T cells are restimulated in vitro by dendritic cells that have ingested apoptotic macrophage-associated Histoplasma antigen. Here we show that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently activated functional CD8+ T cells whose contribution was equal to that of CD4+ T cells in protection against Histoplasma challenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8+ T cell but not the CD4+ T cell response to pulmonary Histoplasma infection. In mice subcutaneously immunized with viable Histoplasma yeasts whose CD8+ T cells are protective against Histoplasma challenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killed Histoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8+ T cell activation. Taken together, our results revealed that phagocyte apoptosis in the Histoplasma-infected host is associated with CD8+ T cell activation and that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently evokes a protective CD8+ T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8+ T cell as well as CD4+ T cell responses to Histoplasma infection

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/β or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1−/− mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1−/− macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response

Clustering of CR3 and Dectin-1 on lipid rafts is required for their collaboration in cytokine production and signaling activation.

<p>(A and C) Macrophages were stimulated with or without (0 min) HK <i>H</i>. <i>capsulatum</i> for 15 or 30 min. Cells were fixed and stained for CR3 (red) and Dectin-1 (green) (A), or for p-Syk (green) (C). Cells were viewed under confocal microscope. Lipid raft was identified by staining with cholera toxin B (CTB) (violet). Nuclear compartment was stained by DAPI (blue). Arrowheads in the DIC/DAPI fields point to <i>H</i>. <i>capsulatum</i> yeasts. The intensity of different fluorochromes along the white arrow in the merged images is shown in the histogram on the right. (B) Macrophages from WT mice were stimulated with or without (control) HK <i>H</i>. <i>capsulatum</i> for 30 min. Cell lysates were subjected to sucrose gradient ultracentrifugation. The fractions were collected and subjected to Western blotting and probed with anti-CD11b, anti-Dectin-1 and anti-flotillin-1 antibodies. (D and E) Macrophages were untreated or treated with methyl-β-cyclodextrin (MβCD, 10 mM) for 30 min. To reconstitute cholesterol, MβCD-treated cells were cultured in medium containing water-soluble cholesterol (MβCD-CHO, 400 μg/ml) for 1 h. After washing, macrophages were stimulated with HK <i>H</i>. <i>capsulatum</i> for 6 h (D), or 30 min (E). The concentrations of TNF and IL-6 in culture supernatants were analyzed by ELISA. Mean ± SD are shown (n = 10 from 4 independent experiments) (D). Cell lysates were analyzed by Western blotting by using antibodies against p-Syk, Syk, and β-actin. Data are representative of 2 (A and C) and 3 (B and E) independent experiments with similar results. ** <i>p</i> ≦ 0.01, *** <i>p</i> ≦ 0.001. NS, not significant [one-way ANOVA with Tukey post-hoc analysis (D)].</p

Micro-Western Array to screen for signaling molecules involved in <i>H</i>. <i>capsulatum</i>-induced macrophage response.

<p>Macrophages from WT mice were stimulated with or without (0 min) HK <i>H</i>. <i>capsulatum</i> for 15, 30, 60, 90, and 120 min. (A) Heat map chart shows MWA results. Protein abundance was normalized against the mean of β-actin and GAPDH. Black color indicates no change, while red and green indicate increase and decrease, respectively, of the levels of protein compared to unstimulated control. Proteins below the level of detection are in grey. (B) Cell lysates were subjected to Western blotting. Beta-actin was used as an internal control. Data shown are representative of 3 independent experiments with similar results.</p

CR3 and Dectin-1 collaborate to intensify Syk activation and subsequent cytokine response in macrophage.

<p>(A and B) Macrophages from WT, <i>Itgam</i>^-/-, <i>Clec7a</i>^-/- and <i>Itgam</i>^-/-<i>Clec7a</i>^-/- mice were stimulated with or without (0 h) heat-killed (HK) <i>H</i>. <i>capsulatum</i>. (A) Supernatants were collected at 6, 12, and 24 h after stimulation, and the concentrations of TNF and IL-6 in the supernatants were quantified by ELISA (n = 6 from 3 independent experiments). (B) Cell lysates were collected at 30 min after stimulation and analyzed by Western blotting using antibodies against p-Syk and Syk. The intensity of p-Syk was normalized against the corresponding Syk. Data shown in the lower panel are relative intensity of p-Syk (n = 3). (C and G) Macrophages from WT mice were treated with Syk inhibitors (SkyI, 10 μM; Bay 61-3606, 3 μM) starting at 1 h prior to stimulation with HK <i>H</i>. <i>capsulatum</i> (C) or with the combination of iC3b-coated beads (iC3b-LB) and depleted zymosan (dZ) (G). Culture supernatants were harvested 6 h later and analyzed for cytokine production (n = 5 from 2 independent experiments). (D) Fetal liver-derived macrophages (FLDMs) from <i>Syk</i>^+/+, <i>Syk</i>^<i>-/-</i> and <i>Syk</i>^<i>+/-</i> embryos were stimulated with HK <i>H</i>. <i>capsulatum</i> for 6 h. TNF levels in the culture supernatants were evaluated (n = 9 from 3 independent experiments). (E) Macrophages from WT, <i>Itgam</i>^-/-, and <i>Clec7a</i>^-/- mice were stimulated with iC3b-LB, dZ, uncoated Latex beads (LB), or the combination of iC3b-LB and dZ. Culture supernatants were collected 6 h later and analyzed for cytokine production (n = 6 from 3 independent experiments). (F) Macrophages from WT mice were stimulated with or without iC3b-LB, dZ, LB, or the combination of iC3b-LB and dZ for 30 min. Cell lysates were subjected to Western blotting. Data are representative of 3 independent experiment with similar results. The bars represent the mean ± SD. In (A),* represent comparisons with WT and # with <i>Itgam</i>^-/-<i>Clec7a</i>^-/-. * or # <i>p</i> ≦ 0.05, ** or ## <i>p</i> ≦ 0.01, *** or ### <i>p</i> ≦ 0.001. NS, not significant [one-way ANOVA with Tukey post-hoc test analysis (A, B, D and E); 2-tailed <i>t</i>-test (C and G)].</p

AP-1, but not NF-κB, mediates the collaborative cytokine response upon CR3 and Dectin-1 ligation.

<p>FLDMs from <i>Syk</i>^+/+, <i>Syk</i>^-/- and <i>Syk</i>^<i>+/-</i> embryos (A) and macrophages from WT, <i>Itgam</i>^-/-, <i>Clec7a</i>^-/- and <i>Itgam</i>^-/-<i>Clec7a</i>^-/- mice (B and C) were stimulated with or without (0 min) HK <i>H</i>. <i>capsulatum</i> for 30 and 60 min. Cell lysates were analyzed by Western blotting. The blot shown in (A) is the same one shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004985#ppat.1004985.g004" target="_blank">Fig 4B</a>. Macrophages from WT mice (D and E), and <i>Itgam</i>^-/- and <i>Clec7a</i>^-/- mice (F) were stimulated with or without iC3b-LB, dZ, LB, or the combination of iC3b-LB and dZ for 30 min (E) or 60 min (D and F). Cell lysates were analyzed by Western blotting. Data are representative of at least 3 independent experiments (A-F). (G and H) Macrophages from WT mice were transfected with small interfering RNA (siRNA) against c-Fos or c-Jun, followed by stimulation with HK <i>H</i>. <i>capsulatum</i> 48 h later. Cell lysates and culture supernatants were collected at 6 h after stimulation. Silencing of c-Fos and c-Jun was confirmed by Western blotting (G). TNF and IL-6 levels in culture supernatants were quantified by ELISA and are presented as the mean ± SD of relative levels of TNF and IL-6 (n = 3 from 3 independent experiments) (H). ** <i>p</i> ≦ 0.01, *** <i>p</i> ≦ 0.001 [2-tailed <i>t</i>-test].</p

Cathepsin B and K⁺ efflux both function as signals 2 in <i>H</i>. <i>capsulatum</i>-stimulated inflammasome activation.

<p>Wild type BMDCs were pretreated with (A) cathepsin B activity inhibitor (Ca-074 ME, 25 μM), phagosome acidification inhibitor (bafilomycin A1 (BA1), 250 nM), (B) extracellular KCl (50 mM) and NaCl (50 mM), K⁺ channel inhibitor (glibenclamide (Gliben), 50 μM), (C) ROS inhibitor (apocynin (Apo), 10 μM) and ROS scavenger (N-acetyl cysteine (Nac), 20 mM) for 1 h before stimulation with <i>H</i>. <i>capsulatum</i>. Cell-free supernatants were harvested at 18 h after stimulation. Secreted IL-1β was quantified by ELISA (n = 3). (D) BMDCs were treated with DQ ovalbumin for 1 h before stimulation with (<i>Hc</i>) or without (ctrl) <i>H</i>. <i>capsulatum</i> at MOI of 1. Cells were collected at 1 h after stimulation before cytospun on microscope slides. Cells were fixed and stained for CD11c (red). The nuclei were stained by Hoechst reagent (blue). Cells were viewed under confocal microscope. White arrows point to ingested-<i>H</i>. <i>capsulatum</i> yeasts in DIC/Hoechst field. (E) BMDCs (2 × 10⁶) were treated with or without Ca-074 ME (25 μM) and bafilomycin A1 (250 nM) before stimulation by <i>H</i>. <i>capsulatum</i> at MOI of 20 for 18 h. Cell lysates and supernatants were analyzed for cathepsin B activity (n = 3). (F) Wild type BMDCs were treated with Ca-074 ME (10 and 25 μM) before stimulation with <i>H</i>. <i>capsulatum</i>. Cell lysates and supernatants were analyzed for inflammasome components by Western blotting. Error bars indicate standard deviation of the mean. One representative of three (A, B and C) and two (D, E and F) independent experiments is presented. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 [2-tailed <i>t</i>-test (A, B and C); one-way ANOVA with Tuckey post-hoc analysis (E)].</p