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2832 Virginie Tardif et al. DOI: 10.1002/eji.201343600 Eur. J. Immunol. 2013. 43: 2832–2844 Carbon monoxide decreases endosome–lysosome fusion and inhibits soluble antigen presentation by dendritic cells to T cells Virginie Tardif ∗ 1,2,3 , Sebasti´ an A. Riquelme ∗ 4 , S´ everine Remy 1,2,3 , Leandro J. Carre˜ no 4 , es 5 , Thomas Simon 1,2,3 , Marcelo Hill 1,2,3 , C´ edric Louvet 1,2,3 , Claudia A. Riedel 5 , Claudia M. Cort´ Philippe Blancou 1,2,3 , Jean-Marie Bach 6 , Christine Chauveau 1,2,3 , Susan M. Bueno 4 , Ignacio Anegon ∗∗ 1,2,3 and Alexis M. Kalergis ∗∗ 1,2,3,4,7 1 INSERM, UMR 1064, Nantes, France 2 CHU Nantes, ITUN, Nantes, France 3 Facult´ e de M´ edecine, Universit´ e de Nantes, Nantes, France 4 Millenium Institute of Immunology and Immunotherapy, Facultad de Ciencias Biol´ ogicas, Pontificia Universidad Cat´ olica de Chile, Santiago, Chile 5 Millenium Institute of Immunology and Immunotherapy, Facultad de Ciencias Biol´ ogicas, Universidad Andr´ es Bello, Santiago, Chile 6 ONIRIS, Nantes, France 7 Departamento de Reumatolog´ ıa, Facultad de Medicina, Pontificia Universidad Cat´ olica de Chile, Santiago, Chile Heme oxygenase-1 (HO-1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe 2 + , and biliverdin. We have pre- viously shown that either induction of HO-1 or treatment with exogenous CO inhibits LPS-induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen- specific inflammation. Here, we evaluated the capacity of HO-1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS-treated DCs. We observed that HO-1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA-derived peptides, bead-bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO-1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO-1 and CO can inhibit the ability of LPS-treated DCs to present exogenous soluble antigens to na¨ ıve T cells by blocking antigen trafficking at the level of late endosome– lysosome fusion. Keywords: Antigen processing · Carbon monoxide · Cross-presentation · Endocytosis · Heme oxygenase-1 � Additional supporting information may be found in the online version of this article at the publisher’s web-site ∗ These authors contributed equally to this work. Correspondence: Dr. Alexis M. Kalergis ∗∗ These authors share senior co-authorship. e-mail: akalergis@bio.puc.cl � 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu C

2833 Eur. J. Immunol. 2013. 43: 2832–2844 Antigen processing Introduction by CO when DCs internalized soluble antigens, causing an accu- mulation of OVA in degradative late endosomal compartments. In summary, our data suggest that CO inhibits presentation of exoge- Heme oxygenase-1 (HO-1) is one of the three isoforms of the HO nous soluble antigens to CD8 + and CD4 + na¨ ıve T cells by blocking enzyme [1,2] and catabolyzes heme into carbon monoxide (CO), Fe 2 + , and biliverdin [1]. HO-1 expression is induced by agents normal antigen trafficking in LPS-treated DCs. involved in oxidative stress, such as oxygen-derived free radicals, pro-inflammatory cytokines, and inflammatory stimuli [3], hav- Results ing a protective effect in a variety of experimental inflammatory models [3–8]. HO-1 and CO prevent DCs from presenting soluble Consistent with this notion, it has been shown that both induc- antigens to T cells tion of HO-1 expression by drugs, such as cobalt protoporphyrin (CoPP) and hemin, or the overexpression of HO-1 by gene transfer can contribute to reducing inflammatory damage during disorders To evaluate whether CO can modulate the presentation of solu- ble antigens on MHC class I (MHC-I) molecules to CD8 + T cells, involving detrimental immune responses, such as organ transplan- DCs were treated with tricarbonyldichlororuthenium (II) dimer tation and autoimmunity, which usually arise after dendritic cell (Ru(CO) 3 Cl 2 ) 2 (CORM 2 ) or CO gas to increase intracellular CO (DC) activation [5,7,9–14]. levels, washed, and then pulsed either with soluble OVA protein, Interestingly, several studies have suggested that the function particulate OVA (OVA adsorbed to 3 µ m polystyrene beads), or of DCs can be modulated by HO-1 activity [3, 6, 15]. Because OVA peptide SIINFEKL (OVA 257–264 , which binds to H-2K b to con- DCs are key players in regulating adaptive immunity and T-cell stitute the cognate ligand for the TCR expressed by OT-I CD8 + activation, the effect of HO-1 on regulating their function can be ıve OT-I CD8 + T cells). Next, treated DCs were used to prime na¨ highly relevant for modulating the adaptive immune response. We T cells. As shown in Fig. 1A and B, an increase of CO levels induced have previously shown that immature human, rat, and mouse DCs by CORM 2 treatment led to a significant reduction in the capacity express HO-1 and that their expression drastically decreases as a of DCs to cross-present soluble OVA protein to OT-I T cells, as result of DC maturation [10,15]. Also, we and others have shown compared with untreated DCs (even at different DC:T cell ratios, that overexpression of HO-1 by rat and humans DCs inhibits LPS- Supporting Information Fig. 1A). HO-1 induction in DCs by CoPP induced maturation and the pro-inflammatory function of these treatment also led to a reduction on antigen presentation equiv- cells [6,16,17]. alent to the CO-driven one (Fig. 1A and B). We confirmed the Interestingly, in several models, CO mimics the effects of HO-1 effects of the CORM 2 -mediated CO by treating DCs with CO gas [7, 10, 15, 18], indicating that HO-1 acts via generation of CO. We have recently shown that the principal mediator of the effects either in the presence or absence of soluble OVA and LPS (Fig. 1C, of HO-1 induction in DC maturation in vivo and in vitro is CO left panel). [15,18]. Thus, the exposure of DCs to CO seems to be an appro- Remarkably, CO did not inhibit cross-presentation when the priate approach to mimic the immunomodulatory effects of HO-1 antigen was internalized by phagocytosis as latex bead-bound OVA [18]. However, despite the effects of CO on DC maturation and (Fig. 1F and G), at various OVA and BSA ratios bound to the beads. inflammation, whether CO can directly affect antigen presentation These results suggest that CO blocked cross-presentation only by DCs to T cells remains unknown. when DCs internalize antigens via endocytosis, but not through phagocytosis. Here, we examined the effect of HO-1 and CO on the ability of Importantly, CORM 2 and CO gas treatment failed to block acti- LPS-treated DCs to present protein-derived antigens on class I and II MHC molecules to CD8 + and CD4 + T cells. We found that HO-1 vation of OT-I T cells in response to DCs pulsed with the antigenic and CO treatment inhibited the ability of DCs to activate CD4 + and OVA peptide at various concentrations (Fig. 1C (right panel), D, CD8 + T cells in response to soluble antigens, both OVA and the and E) even at different DC:T cell ratios (Supporting Information Fig. 1B). These data rule out an unspecific CO inhibitory effect on Ag85B antigen from Mycobacterium tuberculosis . However, HO-1 the antigen presentation capacity of DCs. and CO failed to inhibit presentation to T cells when DCs were loaded either with antigens as large particles or small peptides. Remarkably, CO did not block the activation of CD8 + T cells when Presentation of cytoplasmatic antigens on MHC-I is antigen was endogenously expressed by DCs, suggesting that CO not inhibited by CO was specifically impairing cross-presentation of endocytosed anti- gens. As antigen uptake as endocytosis and phagocytosis were not impaired by HO-1 and CO, the data suggest that inhibition tar- We tested whether CO can modulate MHC-I-restricted presen- geted the processing/trafficking of soluble antigens internalized tation of endogenous antigens, which also involves proteasomal only by endocytosis. Because an efficient late endosome–lysosome activity [23,24]. DCs were transfected with 2 µ g mRNA encoding fusion is required to generate both class I and class II MHC epitopes OVA SIINFEKL and treated with CORM 2 to promote CO produc- from soluble antigens [19–22], we evaluated whether CO could tion. One hour after transfection, DCs were induced to mature with suppress this process. Consistently, we observed that Rab7 + -late LPS and used to stimulate OT-I T cells. As shown in Fig. 1H and I, endosome-Lamp1 + lysosomes fusion was significantly inhibited CORM 2 treatment did not diminish the capacity of DCs to present � 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu C