Portal de Periódicos da CAPES

Revisão Acesso aberto Produção Nacional Revisado por pares

BIBTEX RIS

Inflammasome Activation by CD8+ T Cells from Patients with Cutaneous Leishmaniasis Caused by Leishmania braziliensis in the Immunopathogenesis of the Disease

2020; Elsevier BV; Volume: 141; Issue: 1 Linguagem: Inglês

10.1016/j.jid.2020.05.106

1523-1747

Thiago M. Cardoso, Jonilson Berlink Lima, Ícaro Bonyek-Silva, Sara Nunes, Daniel F. Feijó, Hugo Leonardo de Almeida, Juliana Silva, Aldina Barral, Viviane Boaventura, Valéria M. Borges, Dario S. Zamboni, Lucas P. Carvalho, Edgar M. Carvalho, Natália Machado Tavares, Cláudia Brodskyn,

Eosinophilic Disorders and Syndromes

Cutaneous leishmaniasis (CL) is characterized by an inflammatory response mainly mediated by CD4+ T cells producing IFN-γ, which are responsible for macrophage activation and intracellular Leishmania braziliensis parasite killing. We recently showed the importance of CD8+ T cells in the pathogenesis of human CL as skin lesions from patients with CL present higher frequencies of CD8+ T cells, contributing to the inflammatory response (Cardoso et al., 2015Cardoso T.M. Machado Á. Costa D.L. Carvalho L.P. Queiroz A. Machado P. et al.Protective and pathological functions of CD8+ T cells in Leishmania braziliensis infection.Infect Immun. 2015; 83: 898-906Crossref PubMed Scopus (61) Google Scholar, Santos et al., 2013Santos C.D.S. Boaventura V. Ribeiro Cardoso C. Tavares N. Lordelo M.J. Noronha A. et al.CD8(+) granzyme B(+) -mediated tissue injury vs. CD4(+)IFNγ(+)-mediated parasite killing in human cutaneous leishmaniasis.J Invest Dermatol. 2013; 133: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Moreover, lesion size was found to positively correlate with the frequency of CD8+ T cells‒expressing granzyme B. The cytotoxic response mediated by CD8+ T cells was not found to be linked to decreased parasite load in human macrophages infected in vitro (Cardoso et al., 2015Cardoso T.M. Machado Á. Costa D.L. Carvalho L.P. Queiroz A. Machado P. et al.Protective and pathological functions of CD8+ T cells in Leishmania braziliensis infection.Infect Immun. 2015; 83: 898-906Crossref PubMed Scopus (61) Google Scholar, Santos et al., 2013Santos C.D.S. Boaventura V. Ribeiro Cardoso C. Tavares N. Lordelo M.J. Noronha A. et al.CD8(+) granzyme B(+) -mediated tissue injury vs. CD4(+)IFNγ(+)-mediated parasite killing in human cutaneous leishmaniasis.J Invest Dermatol. 2013; 133: 1533-1540Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Recently, some authors have stated that CD8+ T cells mediate tissue damage through the activation of NLRP3, leading to IL-1β secretion in L. braziliensis‒infected mouse models and cells obtained from human CL skin lesions (Novais et al., 2017Novais F.O. Carvalho A.M. Clark M.L. Carvalho L.P. Beiting D.P. Brodsky I.E. et al.CD8+T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1β production.PLoS Pathog. 2017; 13e1006196Crossref PubMed Scopus (90) Google Scholar). Here, we evaluated the ability of CD8+ T cells to induce inflammasome expression and activation in cells from patients with CL and establish correlations with healing time. This study was approved by the Ethics and Research Committee from the Federal University of Bahia, Brazil, and the National Commission of Ethics in Research (39324114.0.0000.5577). All individuals were volunteer adults and provided written informed consent. A detailed description of the methods is presented in the Supplementary Materials and Methods. Increased expression of NLRP3, AIM2, and CASP-1/5 was observed by qPCR in CL skin lesions compared with healthy skin samples (Figure 1a). Increased CASP-1/5 expression was also observed in PBMCs obtained from patients with CL stimulated with L. (Viannia) braziliensis after 6 and 12 hours of culture (Figure 1b). To determine the role of CD8+ T cells in increased inflammasome expression, these cells were cocultured with L. braziliensis‒infected macrophages. After 12 hours of coculture, increased expression of NLRP3, AIM2, and CASP-1/5 was observed compared with uninfected cell cultures (Figure 1c). The activation of these molecules contributes to the pathophysiology of several diseases, including leishmaniasis (Ferraz et al., 2015Ferraz R. Cunha C.F. Gomes-Silva A. Schubach A.O. Pimentel M.I.F. Lyra M.R. et al.Apoptosis and frequency of total and effector CD8+ T lymphocytes from cutaneous leishmaniasis patients during antimonial therapy.BMC Infect Dis. 2015; 15: 74Crossref PubMed Scopus (12) Google Scholar, Hyman and Yuan, 2012Hyman B.T. Yuan J. Apoptotic and non-apoptotic roles of caspases in neuronal physiology and pathophysiology.Nat Rev Neurosci. 2012; 13: 395-406Crossref PubMed Scopus (193) Google Scholar, Novais et al., 2015Novais F.O. Carvalho L.P. Passos S. Roos D.S. Carvalho E.M. Scott P. et al.Genomic profiling of human Leishmania braziliensis lesions identifies transcriptional modules associated with cutaneous immunopathology.J Invest Dermatol. 2015; 135: 94-101Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, Santos et al., 2018Santos D. Campos T.M. Saldanha M. Oliveira S.C. Nascimento M. Zamboni D.S. et al.IL-1β production by intermediate monocytes is associated with immunopathology in cutaneous leishmaniasis.J Invest Dermatol. 2018; 138 ([published correction appears in J Invest Dermatol 2014 Nov;134(11):2850]): 1107-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). CASP-1 is responsible for converting proinflammatory cytokines, such as IL-1β and IL-18, into their mature and active form, thereby favoring cytokine release and inflammation (Howard et al., 1997Howard A.D. Kostura M.J. Thornberry N. Ding G.J. Weidner J. Salley J.P. et al.IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31- kDa IL-1 alpha.J Immunol. 1997; 147: 2964-2969Google Scholar, Place and Kanneganti, 2018Place D.E. Kanneganti T.D. Recent advances in inflammasome biology.Curr Opin Immunol. 2018; 50: 32-38Crossref PubMed Scopus (187) Google Scholar). Our results showed elevated expression of inflammasome components NLRP3 and AIM2 in patients with CL skin lesions, which could explain the upregulation of inflammatory CASP genes observed herein. Concurrently, some reports have described NLR activation by different species of Leishmania in vitro and in vivo (Charmoy et al., 2016Charmoy M. Hurrell B.P. Romano A. Lee S.H. Ribeiro-Gomes F. Riteau N. et al.The Nlrp3 inflammasome, IL-1β, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL / 6 mice.Eur J Immunol. 2016; 46: 897-911Crossref PubMed Scopus (88) Google Scholar, Lima-Junior et al., 2013Lima-Junior D.S. Costa D.L. Carregaro V. Cunha L.D. Silva A.L.N. Mineo T.W.P. et al.Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania.Nat Med. 2013; 19: 909-915Crossref PubMed Scopus (241) Google Scholar, Santos et al., 2018Santos D. Campos T.M. Saldanha M. Oliveira S.C. Nascimento M. Zamboni D.S. et al.IL-1β production by intermediate monocytes is associated with immunopathology in cutaneous leishmaniasis.J Invest Dermatol. 2018; 138 ([published correction appears in J Invest Dermatol 2014 Nov;134(11):2850]): 1107-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) To validate our expression data, IL-1β and CASP-1 levels were measured in biopsied cell cultures for 12 hours. Increased production of IL-1β (Figure 2a) and CASP-1 (Figure 2b) was seen in the supernatant of CL biopsy cultures compared with that of healthy controls. To confirm our results regarding inflammasome activation by CD8+ T cells, L. braziliensis‒infected macrophages were cocultured with CD8+ T cells from patients with CL for 12 hours, followed by the assessment of IL-1β and CASP-1 secretion. We found increased levels of IL-1β and CASP-1 in autologous cocultures (CD8+ T cells + macrophages from patients with CL) in contrast to cocultured uninfected macrophages (Figures 2c–d). In addition, L. braziliensis‒infected macrophages alone (Figures 2g–h) or those cocultured with CD4+ T cells did not induce CASP-1 or IL-1β secretion compared with cocultured uninfected macrophages (Figures 2e–f). It is well-known that during the cytolytic process, CD8+ T cells release perforin and granzyme-mediating apoptosis in target cells (Li et al., 2014Li X.Y. Li Z. An G.J. Liu S. Lai Y.D. Co-expression of perforin and granzyme B genes induces apoptosis and inhibits the tumorigenicity of laryngeal cancer cell line Hep-2.Int J Clin Exp Pathol. 2014; 7: 978-986PubMed Google Scholar). Accordingly, we hypothesized that perforin released by activated CD8+ T cells binds to the surface of target cells, triggering potassium ion imbalance, thereby leading to inflammasome activation. To test this, we inhibited granzyme B and perforin during the coculturing of L. braziliensis‒infected macrophages and CD8+ T cells or added a hyperosmotic medium containing 50 μM potassium chloride or sodium chloride. The inhibition of granzyme B and perforin reduced active CASP-1 secretion levels and YVAD-FLICA–positive cells during infected macrophages and CD8+ T-cell cocultures (Figures 2g–h). This reduction suggested that CD8+ T cells may induce potassium efflux and, consequently, inflammasome activation. In contrast, in cocultures of CD4+ T cells and infected macrophages, no significant differences were found in CASP-1 and IL-1β production. In addition, increases in extracellular potassium ion by potassium chloride were shown to reduce the activation of CASP-1 in CD14+ cells (Figure 2h). These results indirectly suggest that the cytolytic process in the membranes of target cells caused by perforin do indeed induce a potassium ion efflux. Potassium efflux is a remarkable signal involved in the activation of the NLRP3 inflammasome and inflammatory caspases (Muñoz-Planillo et al., 2013Muñoz-Planillo R. Kuffa P. Martínez-Colón G. Smith B.L. Rajendiran T.M. Núñez G. K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter.Immunity. 2013; 38: 1142-1153Abstract Full Text Full Text PDF PubMed Scopus (1114) Google Scholar). Recent work described the value of inflammasome platforms in predicting treatment outcome in CL (Amorim et al., 2019Amorim C.F. Novais F.O. Nguyen B.T. Misic A.M. Carvalho L.P. Carvalho E.M. et al.Variable gene expression and parasite load predict treatment outcome in cutaneous leishmaniasis.Sci Transl Med. 2019; 11: eaax4204Crossref PubMed Scopus (29) Google Scholar). To investigate whether CASP-1 and IL-1β production could be related to poor disease prognosis, we correlated the production of these proteins in the supernatant of infected macrophage and CD8+ T-cell cocultures with the healing time of each patient. CASP-1 but not IL-1β production presented a positive correlation with healing time (Figures 2i and j). The fact that CASP-1 secretion is linked to healing time supports the role of CD8+-dependent inflammasome activation in worsening CL outcomes. Indeed, recent work demonstrates that patients presenting the higher expression of genes associated with cytotoxic T cells presented a poorer response to treatment in the healing of CL lesions (Amorim et al., 2019Amorim C.F. Novais F.O. Nguyen B.T. Misic A.M. Carvalho L.P. Carvalho E.M. et al.Variable gene expression and parasite load predict treatment outcome in cutaneous leishmaniasis.Sci Transl Med. 2019; 11: eaax4204Crossref PubMed Scopus (29) Google Scholar). This study investigated the role of CD8+ T cells obtained from patients with CL in the activation of the inflammasome pathway. The increased inflammasome component expression observed in patients with CL indicates that this process may be related to tissue damage (Santos et al., 2018Santos D. Campos T.M. Saldanha M. Oliveira S.C. Nascimento M. Zamboni D.S. et al.IL-1β production by intermediate monocytes is associated with immunopathology in cutaneous leishmaniasis.J Invest Dermatol. 2018; 138 ([published correction appears in J Invest Dermatol 2014 Nov;134(11):2850]): 1107-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Recent reports have highlighted the modulation of inflammasome activation as a possible target for treating tissue injury in this disease (Carvalho et al., 2020Carvalho A.M. Novais F.O. Paixão C.S. De Oliveira C.I. Machado P.R. Scott P. et al.Glyburide, a NLRP3 inhibitor, decreases inflammatory response and is a candidate to reduce pathology in Leishmania braziliensis infection.J Infect Dis. 2020; 140 (246–9.e2)Google Scholar). Further studies will be crucial in determining methods of modulating the inflammasome to design therapeutic targets against CL. No datasets were generated or analyzed during this study. Thiago Marconi Cardoso: http://orcid.org/0000-0002-0074-9789 Jonilson B. Lima: http://orcid.org/0000-0002-1403-4768 Ícaro Bonyek-Silva: http://orcid.org/0000-0002-0346-9360 Sara Nunes: http://orcid.org/0000-0001-8546-7598 Daniel Feijó: http://orcid.org/0000-0002-3552-8847 Hugo Almeida: http://orcid.org/0000-0002-6584-228X Juliana Silva: http://orcid.org/0000-0002-5546-932X Aldina Barral: http://orcid.org/0000-0002-7177-464X Viviane Boaventura: http://orcid.org/0000-0002-7241-6844 Valéria M. Borges: http://orcid.org/0000-0002-2775-5409 Dario S. Zamboni: http://orcid.org/0000-0002-7856-7512 Lucas Pedreira de Carvalho: http://orcid.org/0000-0001-5033-1666 Edgar M. Carvalho: http://orcid.org/0000-0002-2697-8002 Natalia M. Tavares: http://orcid.org/0000-0002-4026-679X Cláudia Brodskyn: http://orcid.org/0000-0003-3367-5365 The authors state no conflict of interest. This study was funded by Fundação de Apoio a Pesquisa do Estado da Bahia (FAPESB), Bahia, Brazil, grant number 05/2015 and Conselho Nacional de Pesquisa (CNPq) grant number 401379/2014-0. DF received a CNPq fellowship (BJT_2014), and HA received a CNPq fellowship for undergraduate students. AB, VMB, EMC, LPC, DSZ, and CB are senior investigators of CNPq. Conceptualization: TMC, JBL CB; Data Curation: TMC, NMT, CB; Formal Analysis: TMC, NMT, CB; Funding Acquisition: DF, CB; Investigation: TMC, IBS, SN, DF, HA, JBL; Methodology: TMC, JBL, DF, HA, JS; Project Administration: CB; Resources: AB, VB, VMB, DSZ, LPC, EMC, CB; Supervision: CB; Validation: TMC, NMT, CB; Visualization: TMC, JBL, NMT, CB; Writing - Original Draft Preparation: JBL, CB; Writing - Review and Editing: JBL, NMT, CB Patient enrollment was performed in epidemic areas in the municipalities of Corte de Pedra and Jequiriça, located in the state of Bahia (Brazil). Patients with cutaneous leishmaniasis (CL) presented typical ulcerative skin lesions, and diagnoses were made on the basis of parasite detection by culture aspirate histopathology or the presence of a typical CL lesion plus leishmanin skin test positivity. Blood and tissue specimens were obtained before patients received treatment with antimoniate-N-methyl-glucantime. Biopsies were obtained from the borders of skin lesions of patients with CL (n = 10), and skin samples were collected from healthy subjects (n = 7) submitted to elective plastic surgery. PBMCs were isolated using Ficoll-Hypaque gradients (GE Healthcare, Uppsala, Sweden). PBMC fractions were collected and washed twice with 1 × PBS at 300g for 10 minutes. A representative portion of these cells was labeled with APC-conjugated mAb α-CD14 (clone 61D3) (Sigma Aldrich, St. Louis, MO). Flow cytometry was employed to determine monocyte frequency in each sample and adjusted to 1 × 106 CD14+ cells per well to obtain differentiated macrophages at identical proportions. For macrophage differentiation, cells were incubated at 37 °C under 5% carbon dioxide for 2 hours to achieve adhesion. Then, PBMCs were washed to remove any nonadherent cells and maintained in culture with complete RPMI (supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM nonessential amino acids) for 7 days to induce macrophage differentiation. Leishmania (Viannia) braziliensis (strain Ba788) promastigotes were cultured in Schneider's Insect medium (Sigma Aldrich) (supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin). Cultured macrophages from patients with CL and healthy subjects were infected with L. braziliensis (1:10 macrophage-to-promastigote ratio). After 2 hours of infection, cells were washed to remove any noninternalized promastigotes, followed by coculturing with CD8+ and CD4+ T cells (5:1 cell-to-macrophage ratio) for variable durations as indicated in figure legends. CD8+ and CD4+ T-cell isolation was performed in PBMCs using a magnetic bead system (Dynabeads Untouched Human CD8+ T Cells and Untouched Human CD4+ T Cells Dynabeads) in accordance with manufacturer's instructions. The reagent 3,4-dichloroisocumarin (Sigma Aldrich) was used as a pharmacological inhibitor of granzyme B. The neutralizing antibody (clone δG9) was used to block perforin. In order to confirm inflammasome activation caused by the influx of potassium, a complete RPMI medium with a high concentration of potassium chloride (50 μM) was used for inhibition purposes, with sodium chloride employed as a negative control. Total RNA from skin lesions and cell culture lysates was extracted using an miRNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed to synthesize cDNA using 0.5 μg of total RNA, M-MLV reverse transcriptase, and random primers (Invitrogen, Carlsbad, CA). Relative qPCR reactions were performed (StepOnePlus AB, Applied Biosystems, Foster City, CA) on 96-well microtiter plates using SYBR Green Master Mix (Applied Biosystems). Forward and reverse primer sequences are: for Caspase 1 forward (5′-GCTGAGGTTGACATCACAGGCA-3′) reverse (5′-TGCTGTCAGAGGTCTTGTGCTC-3′); Caspase 5 forward (5′-TGTAAAACGACGGCCAGT-3′) reverse (5′-CAGGAAACAGCTATGCACC-3′); NLRP 3 forward (5′-GATCTTCGCTGCGATCAACA-3′) reverse (5′-GGGATTCGAAACACGTGCATTA-3′); AIM2 forward (5′-CAACAAGACTTGAACACAACGAG-3′) reverse (5′-CTCTCAATGACTGTGCTGGGTA-3′); and β-actin forward (5′-CACCATTGGCAATGAGCGGTTC-3′) reverse (5′-AGGTCTTTGCGGATGTCCACGT-3′). All primers were purchased from Applied Biosystems. Samples were amplified in duplicate, and relative expression was calculated as mean comparative computer tomography method for each gene using StepOne Software v2.0.2 (Applied Biosystems). β-actin gene was used as an internal control. All reagents were used in accordance with the manufacturer's recommendations. To measure cytokine production in cocultures, supernatants were collected after 12 hours of culturing. IL-1β (Human IL-1β and/or IL-1F2 DuoSet ELISA Development System; R&D Systems, Minneapolis, MI) and active CASP-1 (Human CASP-1/ICE Quantikine ELISA Kit; R&D Systems) concentrations were determined by ELISA sandwich assays in accordance with the manufacturer's instructions. IL-1 β and CASP-1 were measured in the supernatants of patients with CL biopsy cultures after 72 hours of incubation, as previously described by Santos et al., 2018Santos D. Campos T.M. Saldanha M. Oliveira S.C. Nascimento M. Zamboni D.S. et al.IL-1β production by intermediate monocytes is associated with immunopathology in cutaneous leishmaniasis.J Invest Dermatol. 2018; 138 ([published correction appears in J Invest Dermatol 2014 Nov;134(11):2850]): 1107-1115Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar. Briefly, biopsy samples were cultured in complete RPMI media without stimuli at 37 °C, 5% carbon dioxide for 72 hours. Supernatants were collected and stored at −70 °C until the time of analysis by ELISA (R&D Systems) in accordance with the manufacturer's instructions. After 12 hours of coculturing, cells were stained with FAM-FLICA FITC reagents in accordance with manufacturer's instructions to determine CASP-1 activation in infected MØ (iMØ) and MØ (FAM-FLICA Caspase-1 Assay Kit, Immunochemistry Technologies, Bloomington, MN). In addition, cells were labeled with phycoerythrin anti-human CD3 mouse antibody (clone SP34-2), phycoerythrin-Cy5–conjugated mAb α-CD8 (clone RPA-T8), and APC-conjugated mAb α-CD14 (clone 61D3) (Sigma Aldrich). A total of 105 gated events from each sample were acquired on a FACS Canto II cytometer (BD-Bioscience Pharmingen, San Jose, CA) and analyzed using FlowJo TreeStar software. To analyze relative gene expression in skin lesions and PBMC cultures and cocultures, Mann–Whitney U test was used. The Mann–Whitney U test was also used to assess differences in IL-1β and active CASP-1 production by ELISA in all experiments. Comparison of CASP-1 and FLICA positive cells of multiple groups in the presence or absence of different inhibitors were analyzed by Kruskal–Wallis test, followed by Dunn's multiple comparisons post-test. Pearson's coefficient testing was performed to assess correlations between patients with CL healing time and CASP-1 or IL-1β production. All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA). P-values < 0.05 were considered significant.