- Email: [email protected]
Combination of flow cytometry and qPCR to study the immune response of american cutaneous leishmaniasis patients
Accepted Manuscript Combination of flow cytometry and qPCR to study the immune response of american cutaneous leishmaniasis patients Maria Carolina Accioly Brelaz-de-Castro, Thays Miranda de Almeida, Amanda Ferreira de Almeida, Marina de Assis Souza, Andresa Pereira de Oliveira Mendes, Rafael de Freitas e Silva, Maria Edileuza Felinto de Brito, Valéria Rêgo Alves Pereira PII:
S0882-4010(18)30992-6
DOI:
10.1016/j.micpath.2018.07.044
Reference:
YMPAT 3084
To appear in:
Microbial Pathogenesis
Received Date: 29 May 2018 Revised Date:
28 June 2018
Accepted Date: 31 July 2018
Please cite this article as: Brelaz-de-Castro MCA, de Almeida TM, de Almeida AF, de Assis Souza M, de Oliveira Mendes AP, Silva RdFe, de Brito MEF, Pereira ValéRêAlves, Combination of flow cytometry and qPCR to study the immune response of american cutaneous leishmaniasis patients, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.07.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Title: Combination of flow cytometry and qPCR to study the immune response of
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american cutaneous leishmaniasis patients
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Short running title: Immune response study of dermal leishmaniasis
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Maria Carolina Accioly Brelaz-de-Castroa,b,+, Thays Miranda de Almeidaa, Amanda
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Ferreira de Almeidaa, Marina de Assis Souzaa, Andresa Pereira de Oliveira Mendesa,
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Rafael de Freitas e Silvaa,c, Maria Edileuza Felinto de Britoa, Valéria Rêgo Alves
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Pereiraa.
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a-Departamento
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(CPqAM/FIOCRUZ), Recife, PE, Brasil;
Imunologia,
Centro
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Pesquisas
Aggeu
Magalhães
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b- Universidade Federal de Pernambuco, Núcleo de Enfermagem, Vitória de Santo
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Antão, PE – Brasil
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c- Universidade de Pernambuco, Departamento de Ciências Naturais e Exatas,
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Garanhuns, PE – Brasil
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+
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Pesquisa Aggeu Magalhães/FIOCRUZ, Av. Professor Moraes Rego s/n, Cidade
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Universitária, Campus da UFPE, 50670-420, Recife, PE, Brazil, CEP. Phone: 55-81-
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2101-263, Fax: 55-81-21012640. E-mail: [email protected]
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Corresponding author. Mailing address: Departamento de Imunologia, Centro de
ACKNOWLEDGEMENTS:
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We thank the platform PDTIS/Flow Cytometry (RPT08F)-FIOCRUZ. This work
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was supported by the Coordination of Improvement of Higher Level Personnel
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(CAPES). The authors declare that there is no conflict of interest.
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Abstract
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American Cutaneous leishmaniasis (ACL) is a public health problem. The
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immunological response is mainly dependent on T cell cytokine responses and might
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influence disease presentation, susceptibility and development. The understanding of
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the host immune response role in the control and in the pathology of leishmaniasis is
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relevant and has implications on diagnosis, follow-up and vaccine development. In this
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study, the differences in the immune response and T cell profile of patients before
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ACCEPTED MANUSCRIPT treatment was investigated through flow cytometry and real time PCR in peripheral
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blood mononuclear cells after different antigenic stimulations. Among the main findings
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are the significant presence of TNF and IFN-γ gene expression after 24 hours of in vitro
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stimulation, and 48h later the presence of CD4+ T and CD8+ T cells producing IL-10
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and IL-4. This may be due to the differences in cytokine release over time and the
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presence of cells other than lymphocytes influencing the mRNA transcript detection.
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Evaluation of the immune response of individuals with leishmaniasis or other diseases
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should associate different technologies and times points for a clear and more reliable
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assessment of the immune response. This would help in the design of vaccine strategies/
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immunotherapies.
Keywords: American Cutaneous leishmaniasis, Immune response, T lymphocytes, Flow cytometry, qPCR, cytokines, Leishmania braziliensis.
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1) Introduction
Leishmaniasis is considered a public health problem, being endemic in many
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countries and with associated social and economic burdens (1, 2, 3, 4). Cutaneous
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leishmaniasis has a distribution in more than 97 countries with an estimated of 0.7 to 1.3
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million new cases each year (3). American Cutaneous Leishmaniasis (ACL) has a
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diverse clinical presentation, associated with the immunological response of the patient,
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epidemiological patterns, which may differ according to the phlebotomine sandfly
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species involved in transmission, population susceptibility, and its level of exposure
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(5,6,7).
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More than 20 species of Leishmania protozoa are responsible for the disease in
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humans, among them, Leishmania (L). Viannia (V.) braziliensis is the main etiological
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agent in Pernambuco, and other Brazilian states (1, 2, 5, 8). Human infection with L.
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braziliensis can present itself with varied clinical forms, ranging from asymptomatic to
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cutaneous and mucocutaneous forms of leishmaniasis. Cutaneous leishmaniasis is the
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most common type, characterized as a single or a few ulcers with elevated borders (2, 7,
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9). Host’s immunological status is also a key factor that acts by dramatically influencing
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disease presentation (5,6,7,10).
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ACCEPTED MANUSCRIPT Susceptibility or resistance to leishmaniasis is dependent on T cell responses (11), being
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cytokine produced by CD4+ T cells responsible for the disease cured or aggravated
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profile (9, 10, 12), whereas CD8+ T cells are usually related to the healing process and
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immunological protection (12, 13, 14) but some authors also describe them associated
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to tissue injury (15, 16). These T cells produce relevant cytokines for activation of
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macrophage and monocytes, and they undergo a differentiation process before
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becoming specific T cells. In general, Th1 cytokine production of IFN-γ, TNF and IL-
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12, has been associated with infection control (1, 11, 17), while Th2 cytokines, such as
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IL-4, IL-10 and TGF-β, have been enrolled in parasite multiplication and disease
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progression (1, 18). These later cytokines are also able to inhibit T cell differentiation
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into a Type 1 profile, thus, acting as a counterbalance to the Th1 proinflammatory effect
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(6,9,17,19).
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Therefore, the type of immune response can influence disease susceptibility and
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development. Even though there are studies that helped to clarify the immune
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mechanisms of ACL development and pathology (11, 12, 20, 21, 22), no clear data is
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available about what is the immune response necessary to control the disease and which
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is the best tool to evaluate this response. Also, only some studies associate different
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methods to evaluate the immune response in these patients (21, 23, 24, 25). Since
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cytokines act in a multifaceted way, analysis of cytokine profiles through different tools
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are powerful and a useful approach to understand the response, and activation of the
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immune system (26).
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Towards a personalized treatment, assessment of the host’s immunological
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status may help to identify resistant or susceptible patients, thus, increasing the chances
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of success of chemotherapy. For this immune evaluation, different factors may
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influence somehow, such as the specie of the parasite that is infecting the host and the
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antigen that is being used to evaluate it (20, 27). In this sense, few studies have helped
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to clarify the immunological status of individuals infected by L. braziliensis in Brazil.
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For this task, combination of flow cytometry (FC) and quantitative real-time PCR
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(qPCR) have become important tools in the investigation of the immune response.
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qPCR allows quantification of transcripts and small changes in gene expression,
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and it is easy to perform and provides accuracy, rapid and reliable results (28). In
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ACCEPTED MANUSCRIPT parallel, complementary results can be achieved with FC which detects cytokines
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produced by different immune cells and can differentiate T cell profiles (29).
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Combining these two technologies provides means to better characterize the
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immunological status of L. braziliensis infected patients. In this context, this work was
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designed to combine flow cytometry and qPCR to characterize differences in the
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cytokine and T cell profile of cells from L. braziliensis infected patients, prior to
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chemotherapy, stimulated with distinct antigens from L. braziliensis.
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2) Materials and Methods 2.1
Ethical aspects and Study Group
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CPqAM/FIOCRUZ Research Ethics Committee (Recife, Brazil) have approved
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the experimental protocols for this research (CAEE number 00082.0.095.000-09).
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Selected individuals were invited to participate in the research and signed the “Term of
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Free and Informed Consent”. The selection of these individuals was based on criteria
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such as: being more than 12 years old, having confirmed diagnosis by the
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CPqAM/Fiocruz Leishmaniasis Referee Center and being active lesion carriers. Our
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study group consisted of 11 patients, from Pernambuco rural endemic areas, with one to
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four lesions and a disease with a mean development of 50 days (15 days – 3 months).
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Patients were submitted to blood collection prior to chemotherapy treatment (Before
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Treatment – BT group) with Glucantime®. The control group consisted of 5 healthy
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individuals, from non-endemic areas, without previous history of ACL and no prior
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blood transfusion. Patients and controls (CT group) had a similar age range (controls
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28.91±14.27/ patients 27.4±13.28). The major characteristics and test results of the
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study group are summarized in Table 1.
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2.2
Leishmania Antigenic Fractions
Promastigote forms of L. (V.) braziliensis (MHOM/BR/75/M2903), cultured in
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vitro, were expanded in Schneider’s medium (Sigma, St. Louis, MO) supplemented
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with 10% of fetal calf serum (Cultilab, Campinas, SP, Brazil) and 1% of antibiotics
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(100UI/ml penicillin and 100 µg/ml streptomycin; Sigma, St. Louis, MO) until they
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reached the exponential phase. Afterwards, they were sedimented by centrifugation at
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800 x g for 15min at 4oC and three times washed with phosphate-buffered saline (PBS;
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pH 7.2). Proteases inhibitors such as 0.1mM methyl-phenyl-fluoride and 2mM
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ACCEPTED MANUSCRIPT ethylenediaminetetraacetic acid (Sigma, St. Louis, MO), pepstatin A 0.001M (Sigma,
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St. Louis, MO), were added right after ultrasonicate (100w/10cycle/30s with 2pulses/s).
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The parasitic suspension was centrifuged at 10,000 x g for 10 min at 4oC. The resultant
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supernatant was removed and submitted to a new centrifugation at 100,000 x g for 1 hr
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at the same temperature. The resultant supernatant was the soluble antigenic fraction
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(SOL), while the sediment was the insoluble antigenic fraction (INS). Both were
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submitted to protein determination according to Brito et al. (2001) (30). The antigens
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were stored at -20oC until use. The electrophoretic profile of soluble and insoluble
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antigens of L. (V.) braziliensis is shown in Brelaz-de-Castro et al.(2012) (12).
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2.3
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Forty milliliters of peripheral blood in heparinized tubes were collected, diluted
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in a proportion of 2:1 (v/v) in PBS pH 7.2 and then added to a Ficoll-Hypaque
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(Amersham Biosciences, Uppsala, Sweden) solution. After centrifugation at 400 x g for
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30 min at 20oC, a peripheral blood mononuclear cells (PBMCs) ring was obtained. Cells
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were washed twice with PBS pH 7.2 and submitted to centrifugation (300 x g for 15
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min at 20oC). PBMCs were resuspended with 2 ml of RPMI 1640 medium (Cultilab,
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Campinas, SP, Brazil) supplemented with 10% of fetal calf serum (Cultilab, Campinas,
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SP, Brazil) and 1% of antibiotics (100 UI/ml penicillin and 100µg/ml streptomycin-
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Sigma, St. Louis, MO). Cell viability was evaluated with Trypan blue dye (Sigma, St.
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Louis, MO), cells were counted in a Neubauer chamber and cell concentration was
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adjusted.
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Blood sampling and peripheral blood mononuclear cells isolation
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2.4
Flow Cytometry
Peripheral blood mononuclear cells (PBMCs) (4x106 cells/tube) from each
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patient/control were individually incubated with Leishmania soluble (SOL, 1.25µg/ml)
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or insoluble (INS, 2.25µg/ml) antigenic fractions (37ºC/ 5% CO2), for 48 hours, by
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means of previous kinetic assay. Positive controls consisted of cells stimulated with
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phytohemagglutinin (PHA, 10µg/ml) 4 hours prior to the end of the incubation period.
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Brefeldin A (10µg/ml) (Sigma, St. Louis, MO) was added to all plates 4 hours prior to
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the end of the incubation period. After the incubation, ethylene-diamine-tetra-acetic acid
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ACCEPTED MANUSCRIPT (EDTA, 20 mM) (Sigma, St. Louis, MO) was added to the culture and then incubated
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for 10 minutes to detach cells. Cells were then washed with PBS with 0.5% bovine
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serum albumin (Sigma, St. Louis, MO) and 0.1% sodium azide (Sigma, St. Louis, MO),
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a solution called PBS-W, centrifuged (370 x g, 7 min, RT) and transferred to
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polystyrene tubes. The tubes contained monoclonal antibodies anti-CD4 or anti-CD8,
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both labeled with fluorescein isothiocyanate (FITC) (BD Bioscience, San Jose, CA),
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and the cells were incubated for 30 min at RT. Then, cells were fixed with 1%
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paraformaldehyde in PBS, washed by centrifugation with PBS-W (400 x g, 5 minutes)
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and permeabilized with PBS plus 0.5% of saponin. Subsequently, the cells were washed
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with PBS-W by centrifugation (400 x g, 5 minutes) and then incubated with cytokine-
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specific antibodies against IFN-γ, TNF, IL-10 (Miltenyi Biotec, Bergisch Gladbach,
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Germany) and IL-4 (BD Bioscience, San Jose, CA), all labeled with phycoerythrin
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(PE), for 30 minutes, RT. After this time, they were washed by centrifugation with
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PBS-W, (400 x g, 5 minutes) and resuspended with 1% paraformaldehyde in PBS.
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Samples were then analyzed (20.000 events within the electronics area compatible with
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the lymphocyte/sample) through flow cytometry (FACSCalibur-BD, San Jose, CA)
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using the software CELLQuestProTM (BD Bioscience, San Jose, CA) for acquisition and
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analysis of data. The analyses were done first delimitating the lymphocytic region in a
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Forward Scatter (FSC) versus Side Scatter (SSC) graph. Based on this region the
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fluorescence graphs (FITCxPE) were constructed, delimitating the quadrants for
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analyses. The quadrant limits were based on the negative population and single stained
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controls. The values considered were the percentage of the quadrant region for each
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quadrant.
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2.5
Cell culture, RNA isolation and cDNA synthesis
PBMCs (106 per well/ ml), from the same individuals which was performed flow
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cytometry, were incubated with Leishmania soluble (SOL, 1.25µg/ml) and insoluble
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(INS, 2.25µg/ml) antigenic fractions for 24 hours (37ºC/ 5% CO2) in a 24 well plate
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(TPP Trasadingen, Switzerland). This time interval for cell culture was used since RNA
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lifetime is shorter when compared for cytokine production, and accumulation for
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release. Total RNA was isolated from PBMC using Trizol solution (Life Technologies
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Carlsbad, CA), according to the manufacturer’s recommendations. RNA purity was
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verified by agarose gel electrophoresis followed by ethidium bromide staining. OD260/280
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determination (above 1.8) and RNA quantification were done using a NanoDrop™
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Spectrophotometer (Thermo Scientific, Waltham, Massachusetts) and the RNA was
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kept at -80°C until ready to use. RNA samples from patients and controls were adjusted to 100ng/ul, and the
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Reverse Transcription PCR reaction (RT) was carried out with the “TaqMan® Reverse
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Transcription Reagents” kit (Applied Biosystems, Foster City, CA. Catalog
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#N8080234). RT-PCR was performed in a final volume of 50µl, containing TaqMan RT
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Buffer 1x, MgCl2 5.5mM, dNTPs mix 500µM, random hexamers 2.5µM, RNase
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inhibitor 0.4 U/µL, multiscribe reverse transcriptase enzyme 3.125 U/µL and 12.375µL
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of the sample. The cycling conditions were as follows: 10 minutes at 25ºC, 30 minutes
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at 48 ºC and 5 minutes at 95 ºC. Negative controls were included in each run. After this
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stage, samples were stored at -20 ºC until its utilization in the real time PCR assays
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(qPCR).
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Relative quantitative Real-Time PCR
To quantify the mRNA expression of the cytokines TNF (Assay ID:00174128_m1),
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IFN-γ (Assay ID: Hs99999041_m1), IL-10 (Assay ID: Hs00174086_m1) and IL-4
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(Assay ID:99999030_m1) in the samples, the real time TaqMan® PCR was used (those
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kits do not provide primers sequences). A housekeeping gene 18S was simultaneously
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processed in Real-time TaqMan® PCR for each sample, as an endogenous control. This
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was done to normalize the process due to the variability in the number of cells in the
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original samples, for differences in extraction efficiency, for mRNA degradation in the
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starting material, and for reverse transcriptase reaction efficiency. qPCR was carried out
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using 1µg of cDNA as the starting material in a total volume of 20 µl with TaqMan®
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Universal PCR Master Mix (Applied Biosystems, Foster City, CA) . All reactions were
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performed in the Model ABI PRISM 7500 Sequence Detector (PE Applied
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Biosystems). The relative quantification was calculated by the 2-∆∆CT method. In this
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method, the fold change in the sample gene, given by delta-delta Ct (∆∆Ct), is
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determined by the following equation: ∆∆CT = ∆CT (sample) - ∆CT (calibrator) and
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∆CT is the CT of the target gene (sample or calibrator) subtracted from the Ct of the
30
housekeeping gene, 18S. Using this method, a positive fold change would represent up-
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regulation of the sample gene compared to the calibrator; whereas a negative fold
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change would represent down-regulation of the sample gene compared to the calibrator.
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2.7
Statistical Analysis
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The statistical analyses were performed with GraphPad Prism 5.1 (GraphPad
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Prism 5.1 Software, San Diego, CA, USA), using nonparametric tests. To detect
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differences between groups the Mann–Whitney U-test was used. The results were
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analyzed considering the value of p≤0.05 statistically significant (based on the two-
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tailed p value).
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3) Results
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3.1
Patients with active lesions presented a higher proportion of CD4+ T
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cells producing IL-4 and IL-10 48h after antigenic stimulation.
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Figure 1 shows the medium cytokines (IFN-γ, TNF, IL-4 and IL-10) percentage
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levels produced by T cells 48h after PBMC culture with the soluble (SOL) and
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insoluble (INS) antigenic fractions of L (V.) braziliensis, and with the mitogen PHA. Under PHA stimulus, all the groups produced cytokines, with significant difference
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observed in the production of TNF by lymphocytes (CTxBT p= 0.005) and specifically
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by CD8+ T cells (CTxBT p= 0.022). After stimulation with the Leishmania antigens
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CD8+ T cells were the main responsible for the production of IFN-γ and CD4+ T cells
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were the main producers of IL-10, IL-4 and TNF. Overall, the soluble antigen fraction
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stimulated IL-10 significant production by lymphocytes (BTxCT p= 0.041), and
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specifically CD4+ T cells to produce IL-10 (BTxCT p=0.0007) and IL-4 (BTxCT
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p=0.003), and CD8+ T cells to produce TNF (CTxBT p=0.019), IL-4 (BTxCT p=0.028)
23
in a significant manner when compared to controls. The insoluble antigenic fraction
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stimulated a significant production of IL-10 (BTxCT p=0.004) and IL-4 (BTxCT
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p=0.006) by patients CD4+ T cells and IL-10 (BTxCT p=0.033) and IL-4 (BTxCT
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p=0.043) by patients CD8+ T, when comparing to controls.
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The inflammatory versus regulatory cytokine balance was evaluated through the
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IFN-γ/IL-10 ratio between patients and controls. Patients presented a lower IFN-γ/IL-10
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ratio when compared to controls (Table 2). CD4/CD8 ratio was also evaluated between
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patients and controls, with patients presenting CD4 predominance when compared to
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controls (table 2).
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3.2
qPCR revealed IFN-γ and TNF significant production 24h after PBMC culture
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Figure 2 shows the medium mRNA cytokine (IFN-γ, TNF, IL-4 and IL-10)
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expression 24h after PBMC culture with the soluble (SOL) and insoluble (INS)
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antigenic fractions of L (V.) braziliensis, and compares patients and controls samples.
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Under PHA stimulus, both groups of patients and controls expressed mRNA for the
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cytokines, with significant difference observed in the production of IFN-γ by patients
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(∆CT=13.06± 1.15) when compared to controls (∆CT=14.72±1.070) (p=0.045). The
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soluble antigenic fraction (1.25µg/ml) stimulated patients and controls cells to produce
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cytokines, with statically significance observed in the production of IFN-γ (Patients
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∆CT=14.76±1.20; Controls ∆CT=19.56± 1.58; p<0.001) and
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∆CT=13.25±2.53; Controls ∆CT=16.26±0.650; p=0.028).
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fraction (2.5µg/ml) also stimulated patients and controls cells to produce cytokines, and
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it was observed a significant production of IFN-γ (Patients ∆CT=12.17±2.94; Controls
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∆CT= 19.24± 1.77; p=0.001) and TNF (Patients ∆CT=11.49±2.33; Controls ∆CT=
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14.96± 0.562, p=0.013).
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4) Discussion
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Leishmaniasis is a group of diseases where the development of lesions and their
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resolution is closely linked to the development of an appropriate immunological
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response. For this, T cells have a major role, acting through direct cytotoxicity (CD8+ T
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cells) and/or producing several cytokines and chemokines. Cytokines can react in
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cascades with other cytokines and lead to a higher or lower production or secretion of
27
further cytokines, acting in a very complex manner. Therefore, the analysis of cytokine
28
profiles is useful to understand the response and activation of the immune system. In
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this study, we described the in vitro production of cytokines and T cell profiles from
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ACL patients after PBMC culture with the soluble and insoluble antigenic fractions of L
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ACCEPTED MANUSCRIPT 1
(V.) braziliensis. They were analyzed through qPCR and Flow Cytometry, the last one
2
also evidencing the population that produced it. Flow cytometry analysis demonstrated the presence of the cytokines IFN-γ, TNF,
4
IL-4 and IL-10, being IL-10 and IL-4 significantly expressed by patients T cells when
5
compared to healthy controls under different circumstances of stimulation. CD4+ T cells
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were the main responsible for IL-4 and IL-10 production, and CD8+ T cells for IFN-γ,
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but also producing IL-4 and IL-10 significantly. When considering the CD4/CD8 ratio,
8
we observed an expansion of CD4+ T cells when compared to controls. This may
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account for the increased proportion of IL-10 and IL-4 observed, since CD4+ T cells
10
were their main producers. Previous studies by our group also demonstrated similar
11
results, and it was shown that this type 2 response later evolved to a Th1 profile after
12
patients’ clinical cure, however still with the presence of IL-10 (6,12). This result may
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suggest a follow-up of the patients during treatment, in order to better characterize the
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time point in which a type 1 response is taking place.
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The development and maintenance of a protective immune response seems to be
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associated with a Type 1 cytokine predominant profile, however with an appropriated
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modulation. When considering the IFN-γ/IL-10 ratio among PBMCs from patients and
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controls stimulated with the soluble and insoluble L (V.) braziliensis antigens, it was
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observed that patients presented a higher proportion of T CD4+ and CD8+ T cells
20
producing IL-10, while the controls presented higher proportion of T CD4+ and CD8+ T
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cells producing IFN-γ. As remarked in other studies, CD4+ and CD8+ T cells proportion
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and cytokine balance is important for lesion progression or healing (12, 30, 31, 32).
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qPCR is a common method to detect cytokine transcripts in cells; in our study we
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observed mRNA expression of the cytokines IFN-γ, TNF, IL-4 and IL-10, being IFN-γ
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significant expressed by patients when compared to healthy controls under all
26
stimulation, and TNF under soluble and insoluble stimulus. Although the Th1 immune
27
response seems to be beneficial, the control of an exacerbated immune response is
28
essential for cure. The production of IL-10, initially related mainly to parasite survival
29
and macrophage deactivation, seems to represent a necessary counterbalance for disease
30
resolution (33). IFN-γ and TNF production is also not associated with the absence of IL-
31
10 (2). IL-10 is the cytokine that is most strongly associated with lesions development,
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ACCEPTED MANUSCRIPT and is mainly released by monocytes and CD4+CD25+ T lymphocytes (21, 34, 35, 36).
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Therefore, the balance between a pathological and protective immune response seems to
3
be given by regulatory T cells (21). Cells with regulatory functions have been
4
frequently described in Leishmania infections, and the existence of concomitant
5
immunity is discussed (37, 38). Nevertheless, conclusive role of Treg cells in
6
suppression of immunity in patients and on the mechanisms involved in the disease
7
development is yet to be defined.
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Trying to understand the immune response mechanisms in America cutaneous
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Leishmaniasis, we studied the production of cytokines through qPCR and flow
10
cytometry aiming to better understand its production and release. These technologies are
11
usually performed in different time points, according to what was established by
12
different research groups and antigen being used. Usually, leishmaniasis flow cytometry
13
studies range from ex vivo analysis to culture evaluations after different time points (4h,
14
24h, 48h to 96 hours) (7, 12, 21). qPCR transcripts are measured from 4h, 24h, 72h up
15
to 15 days after culture (39, 40, 41). However, cytokine secretion is usually a process of
16
hours or days, controlled by autocrine and paracrine regulation and feedback loops, with
17
secretory activity dropping down to normal without continuous stimulation. Also,
18
lymphocytes have no reservoirs for cytokines (26). Therefore, the initial activation of
19
transcription and biosynthesis in lymphocytes is strongly related to its consecutive
20
secretion and may be the reason for the absence of complete agreement of the different
21
technologies used in our study (26). Since there is no definitive immune profile
22
established for this disease, qPCR and flow cytometry are useful tools to understand the
23
development of the patients’ immune responses and help on its monitoring.
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Leishmania active infections has been related to type 1 and 2 cytokine profiles,
25
depending on the antigen used, the time of disease evolution and the method reported
26
(9, 18, 19, 42, 43). Previous works by our group, with cells stimulated with the L. (V.)
27
braziliensis soluble antigen, reported the presence of significant Th1 cytokines however
28
still with the significant presence of IL-10 (44) also seen in other studies with L. (V.)
29
braziliensis antigens (9, 18, 19, 45). Our study measured cytokines through ELISA, in
30
patients with longer disease evolution time. Additional studies with the same antigens
31
used in this article but with patients with shorter evolution times (<60 days) and
32
analyzed only through flow cytometry, demonstrated T CD4+ and type 2 cytokines
12
ACCEPTED MANUSCRIPT predominance profile (6, 12) that have been also described by other groups (9, 21, 42,
2
46). Therefore, this Th1/Th2 profile seems to be an oversimplification of what is
3
happening inside the host and it seems that regulation of the inflammatory response
4
rather than biased Th1/Th2 response defines the outcomes of infection with Leishmania
5
Viannia species (39, 44, 47). Other T cell subsets have important roles in susceptibility
6
and resistance to leishmaniasis, such as Treg and Th17 cells. All this emphasizes the
7
need to study T cell response regarding its magnitude, quality and through different
8
technologies.
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Given that the qPCR mRNA examination at 24 h only reflects a snapshot, it can be
10
assumed that if a low gene expression values and a high cytokine levels are detected for
11
a distinct data pair, this may reflect that the gene expression had already declined due to
12
an effectual level of cytokines reached in the cell culture supernatant at an earlier point.
13
Also since this technology identifies the presence of cytokines transcripts, but not the
14
cells containing the transcripts, we may be analyzing transcripts from cells other than
15
lymphocytes, such as monocyte, that largely contribute to IFN-γ and TNF secretion and
16
type 1 or 2 cytokine differentiation (2, 48). Therefore, the different cytokines profiles
17
observed in our patients when using flow cytometry and qPCR may be due to the
18
differences in cytokine release over time and the presence of cells other than
19
lymphocytes influencing the detection of the mRNA transcript.
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In this study, we described the significant presence of TNF and IFN-γ transcripts at
21
24h of infection, and 48h later the presence of CD4+ T cells producing IL-10 and IL-4,
22
and of CD8+ T cells producing IL10, IL-4 and TNF, after the soluble and insoluble
23
antigen of L. (V.) braziliensis were used for PBMC stimulation. These antigens, as seen
24
in previous studies from our group (6, 12, 22, 44, 49), allow the study of a more specific
25
immune response, since they come from L. braziliensis, the predominant species in the
26
region (4, 8). The understanding of the role of the host’s immune response in the control
27
and in the pathology of leishmaniasis through different technologies and with different
28
antigens is highly relevant and has implications for the control of leishmaniasis, since
29
there is not an established immunological profile associated with a cure. Therefore, as
30
evidenced by our findings, studies aiming to evaluate the immune response of
31
individuals with leishmaniasis or other diseases should consider associate different
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ACCEPTED MANUSCRIPT 1
technologies and times point, such as flow cytometry and qPCR - to obtain a clear a
2
more reliable evaluation of the immune response developed by patients.
5) References 1. Ameen M. 2010. Cutaneous leishmaniasis: advances in disease pathogenesis, diagnostics and therapeutics. Clin Exp Dermatol 2010; 35(7):699-705. doi: 10.1111/j.1365-2230.2010.03851.x.
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2. Carvalho LP, Passos S, Schriefer A, Carvalho EM. Protective and pathologic immune responses in human tegumentary leishmaniasis. Front Immunol 2012; 4;3:301. doi: 10.3389/fimmu.2012.00301.
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3. WHO. World Health Organization. http://www.who.int/leishmaniasis/en/ accessed 10 March 2018.
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5. de Brito ME, Andrade MS, Dantas-Torres F, et al. Cutaneous leishmaniasis in northeastern Brazil: a critical appraisal of studies conducted in State of Pernambuco. Rev Soc Bras Med Trop 2012; 45(4):425-9.
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6. Brelaz MC, de Oliveira AP, de Almeida AF, et al. Leishmania (Viannia) braziliensis antigenic fractions: the immune response characterization of patients at the initial phase of disease. Parasite Immunol 2012; 34(4):236-9. doi: 10.1111/j.1365-3024.2012.01351.x.
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7. Macedo AB, Sánchez-Arcila JC, Schubach AO, et al. Multifunctional CD4⁺ T cells in patients with American cutaneous leishmaniasis. Clin Exp Immunol 2012; 167(3):505-13. doi: 10.1111/j.1365-2249.2011.04536.x.
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12. Brelaz-de-Castro MC, de Almeida AF, de Oliveira AP, de Assis-Souza M, da Rocha LF, Pereira VR. Cellular immune response evaluation of cutaneous leishmaniasis patients cells stimulated with Leishmania (Viannia) braziliensis antigenic fractions before and after clinical cure. Cell Immunol 2012; 279(2):180-6. doi: 10.1016/j.cellimm.2012.11.006.
13. Ferraz R, Cunha CF, Gomes-Silva A, Schubach AO, Pimentel MI, Lyra MR, et al. Apoptosis and frequency of total and effector CD8+ T lymphocytes from cutaneous leishmaniasis patients during antimonial therapy. BMC Infect Dis. 2015; 15:74. doi: 10.1186/s12879-015-0799-x.
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14. Castro Gomes CM, Sousa MG, Menezes JP, et al. Immunopathological characterization of human cutaneous leishmaniasis lesions caused by Leishmania (Viannia) spp. in Amazonian Brazil. Parasitol Res 2017. doi: 10.1007/s00436-017-5403-4.
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15. Santos CS, Boaventura V, Ribeiro Cardoso C, et al. CD8(+) granzyme B(+)mediated tissue injury vs. CD4(+)IFNγ(+)-mediated parasite killing in human cutaneous leishmaniasis. J Invest Dermatol 2013; 133(6):1533-40. doi:10.1038/jid.2013.4.
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16. Faria DR, Souza PE, Durães FV, et al. Recruitment of CD8(+) T cells expressing granzyme A is associated with lesion progression in human cutaneous leishmaniasis. Parasite Immunol 2009; 31(8):432-9. doi: 10.1111/j.13653024.2009.01125.x.
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ACCEPTED MANUSCRIPT 21. Salhi A, Rodrigues V Jr, Santoro F, et al. Immunological and Genetic Evidence for a Crucial Role of IL-10 in Cutaneous Lesions in Humans Infected with Leishmania braziliensis. J Immunol 2008; 180(9): 6139 - 6148.
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22. de Assis Souza M, de Castro MC, de Oliveira AP, et al. Cytokines and NO in American tegumentary leishmaniasis patients: profiles in active disease, after therapy and in self-healed individuals. Microb Pathog 2013; 57:27-32. doi: 10.1016/j.micpath.2013.02.004
23. Chamakh-Ayari R, Bras-Gonçalves R, Bahi-Jaber N, et al. In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis. PLoS One 2014; 9(5):e92708. doi: 10.1371/journal.pone.0092708.
12 13 14 15
24. Gomes CM, Ávila LR, Pinto SA, et al. Leishmania braziliensis amastigotes stimulate production of IL-1β, IL-6, IL-10 and TGF-β by peripheral blood mononuclear cells from nonendemic area healthy residents. Parasite Immunol 2014; 36(5):225-31. doi: 10.1111/pim.12109.
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25. Silverman JM, Clos J, Horakova E, et al. Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells. J Immunol 2010; 185(9):5011-22. doi: 10.4049/jimmunol.1000541.
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26. Radke L, López Hemmerling DA, Lubitz A, Giese C, Frohme M. Induced cytokine response of human PMBC-cultures: correlation of gene expression and secretion profiling and the effect of cryopreservation. Cell Immunol 2012; 272(2):144-53. doi: 10.1016/j.cellimm.2011.10.018.
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27. Silva RF, Ferreira LF, Hernandes MZ, et al. Combination of In Silico Methods in the Search for Potential CD4(+) and CD8(+) T Cell Epitopes in the Proteome of Leishmania braziliensis. Front Immunol 2016; 7:327. doi: 10.3389/fimmu.2016.00327.
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30. Brito ME, Mendonça MG, Gomes YM, Jardim ML, Abath FG. Dynamics of the antibody response in patients with therapeutic or spontaneous cure of American cutaneous leishmaniasis. Trans R Soc Trop Med Hyg 2001; 95(2):203-6.
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31. Bottrel RL, Dutra WO, Martins FA, et al. 2001. Flow Cytometric Determination of Cellular Sources and Frequencies of Key Cytokine-Producing Lymphocytes
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28. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29(9):e45. 29. Freer, G. Intracellular Staining and Detection of Cytokines by FluorescenceActivated Flow Cytometry. Methods Mol Biol 2014; 1172:221-34. doi: 10.1007/978-1-4939-0928-5_20.
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ACCEPTED MANUSCRIPT Directed against Recombinant LACK and Soluble Leishmania Antigen in Human Cutaneous Leishmaniasis. Infect Immun 69(5): 3232–3239.
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32. Toledo VP, Mayrink W, Gollob KJ, et al. Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment. Mem Inst Oswaldo Cruz 2001; 96(1):89-98.
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33. Antonelli LR, Dutra WO, Almeida RP, Bacellar O, Carvalho EM, Gollob KJ. Activated inflammatory T cells correlate with lesion size in human cutaneous leishmaniasis. Immunol Lett 2005; 101: 226-30.
34. Campanelli AP, Roselino AM, Cavassani KA, et al. CD4+CD25+ T cells in skin lesions of patients with cutaneous leishmaniasis exhibit phenotypic and functional characteristics of natural regulatory T cells. J Infect Dis 2006; 193(9):1313-22.
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35. Bourreau E, Ronet C, Darcissac E, Lise MC, Sainte Marie D, Clity E et al. Intralesional regulatory T-cell suppressive function during human acute and chronic cutaneous leishmaniasis due to Leishmania guyanensis. Infect Immun 2009; 77(4):1465-74. doi: 10.1128/IAI.01398-08.
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36. Bhattacharya P, Ghosh S, Ejazi SA, et al. Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani. PLoS Negl Trop Dis 2016; 10(2):e0004422. doi: 10.1371/journal.pntd.0004422.
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37. Belkaid, Y. Regulatory T cells and infection: a dangerous necessity. Nat Rev Immunol. 2007; 7(11): 875-88.
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38. Falcão CS, de Moura TR, Clarencio J, Brodskyn C, Barral A, de Oliveira CI. The presence of Tregs does not preclude immunity to reinfection with Leishmania braziliensis. Int. J. Parasitol 2012; 42(8):771-80. doi: 10.1016/j.ijpara.2012.05.006.
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40. Ettinger NA, Wilson ME. Macrophage and T-cell gene expression in a model of early infection with the protozoan Leishmania chagasi. PLoS Negl Trop Dis 2008; 2(6):e252. doi: 10.1371/journal.pntd.0000252.
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41. Novoa R, Bacellar O, Nascimento M, et al. IL-17 and Regulatory Cytokines (IL10 and IL-27) in L. braziliensis Infection. Parasite Immunol 2011; 33(2):132-6. doi: 10.1111/j.1365-3024.2010.01256.x.
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39. Díaz YR, Rojas R, Valderrama L, Saravia NG. T-bet, GATA-3, and Foxp3 expression and Th1/Th2 cytokine production in the clinical outcome of human infection with Leishmania (Viannia) species. J Infect Dis 2010; 202(3):406-15. doi: 10.1086/653829.
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42. Ajdary S, Alimohammadian MH, Eslami MB, Kemp K, Kharazmi A. Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection. Infect Immun 2000; 68(4): 1760-64.
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43. Leopoldo PT, Machado PR, Almeida RP, et al. Differential effects of antigens from L. braziliensis isolates from disseminated and cutaneous leishmaniasis on in vitro cytokine production. BMC Infect Dis 2006; 6:75.
8 9 10 11
44. Reis LC, Brito ME, Souza MA, Medeiros AC, Silva CJ, Luna CF et al. Cellular Immune Response Profile in Patients With American Tegumentary Leishmaniasis Prior and Post Chemotherapy Treatment. J Clin Lab Anal 2009; 23(1):63-9. doi: 10.1002/jcla.20291.
12 13 14
45. Telino E, De Luca PM, Matos DC, et al. In vitro responses of human peripheral blood mononuclear cells to whole-cell, particulate and soluble extracts of Leishmania promastigotes. Clin Exp Immunol. 2005; 143: 338–44.
15 16 17
46. Rocha PN, Almeida RP, Bacellar O, et al. Down-regulation of Th1 type of response in early human American cutaneous leishmaniasis. J Infect Dis 1999; 180(5):1731-4.
18 19
47. Nylén, S., Gautam, S. Immunological perspectives of leishmaniasis. J Glob Infect Dis 2010; 2(2):135-46. doi: 10.4103/0974-777X.62876.
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48. Sachdeva N, Asthana D. Cytokine quantitation: technologies and applications. Front Biosci 2007; 12:4682-95.
22 23 24 25
49. Souza MA, Castro MC, Oliveira AP, et al. American Tegumentary Leishmaniasis: Cytokines and Nitric Oxide in Active Disease and After Clinical Cure, With or Without Chemotherapy. Scand J Immunol 2012; 76(2):175-80. doi: 10.1111/j.1365-3083.2012.02717.x.
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Tables and figures captions
2
MST1
Age
#
(years)
lesions
1
43
1
N/D
-
2
26
1
10mm
-
3
13
1
10mm
-
4
32
1
11.5mm
5
21
1
14mm
6
27
7
15
8
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10 11
PCR3
Evolution times
Microscopy
+
+
2 months
+
2 months
+
+
3 months
-
+
N/D
3 months
-
+
+
1 month
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+
1
10mm
-
-
+
1 month
4
12mm
-
+
+
1 month
2
16mm
-
-
+
15 days
23
1
14mm
+
-
+
3 months
45
1
9mm
N/D
-
+
1 month
44
1
10mm
+
+
N/D
1 month
43
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9
IFI2
Direct
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Patient
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Table 1. Test results and characteristics of the ACL patients
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MST: Montenegro skin test (Positive ≥ 5mm); 2IFI: Indirect Immunofluorescence;
5
3
PCR: Polymerase chain reaction; +: Positive; -: Negative; N/D: Not done.
6
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ACCEPTED MANUSCRIPT Table 2: CD4+ T/CD8+ T cell ratio in cultures stimulated 48h with Leishmania soluble and insoluble antigenic fractions
2.71
CT CD4+/CD8+ ratio
1.49
BT CD4+ IFN-γ/IL-10 ratio
0.78 (2.23-0.24)
BT CD8+ IFN-γ/IL-10 ratio
2.35 (7.51-0.68)
CT CD4+ IFN-γ/IL-10 ratio
3.52 (7.56-0.42)
CT CD8+ IFN-γ/IL-10 ratio
2.08 (9.7-0.45)
3
2.51 1.5
0.71 (1.19-0.45) 0.95 (1.45-0.46)
1.95 (3.83-0.40)
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BT CD4+/CD8+ ratio
Insoluble Antigen
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Soluble Antigen
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1 2
4.83 (6.89-1.96)
Figure 1: Cytokine expression by (A) T cells, (B) T CD4+ cells, and (C) T CD8+
5
cells after in vitro stimulation with PHA (10µg/ml), Leishmania soluble (1.25µg/ml) and
6
insoluble (2.25µg/ml) antigenic fractions. The results are expressed as mean values ±
7
SD. Differences between groups were considered significant with a P value less than
8
0.05 and are represented by the symbol “*” (difference between patients and controls).
9
BT= Before Treatment/ CT= Controls.
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Figure 2: Patients and controls cytokines mRNA expression 24h after PBMC
12
culture with the mitogen PHA or with the soluble (SOL, 1.25µg/ml) and insoluble
13
(INS, 2.5µg/ml) antigenic fractions of L (V.) braziliensis, following normalization with
14
the reference gene (18S). The results are expressed as mean values ± SD. Differences
15
between groups were considered significant with a P value less than 0.05 and are
16
represented by the symbol “*” (difference between patients and controls). BT= Before
17
Treatment/ CT= Controls.
18
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S0882-4010(18)30992-6
DOI:
10.1016/j.micpath.2018.07.044
Reference:
YMPAT 3084
To appear in:
Microbial Pathogenesis
Received Date: 29 May 2018 Revised Date:
28 June 2018
Accepted Date: 31 July 2018
Please cite this article as: Brelaz-de-Castro MCA, de Almeida TM, de Almeida AF, de Assis Souza M, de Oliveira Mendes AP, Silva RdFe, de Brito MEF, Pereira ValéRêAlves, Combination of flow cytometry and qPCR to study the immune response of american cutaneous leishmaniasis patients, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.07.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT Title: Combination of flow cytometry and qPCR to study the immune response of
2
american cutaneous leishmaniasis patients
3
Short running title: Immune response study of dermal leishmaniasis
4
Maria Carolina Accioly Brelaz-de-Castroa,b,+, Thays Miranda de Almeidaa, Amanda
5
Ferreira de Almeidaa, Marina de Assis Souzaa, Andresa Pereira de Oliveira Mendesa,
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Rafael de Freitas e Silvaa,c, Maria Edileuza Felinto de Britoa, Valéria Rêgo Alves
7
Pereiraa.
8
a-Departamento
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(CPqAM/FIOCRUZ), Recife, PE, Brasil;
Imunologia,
Centro
de
Pesquisas
Aggeu
Magalhães
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de
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b- Universidade Federal de Pernambuco, Núcleo de Enfermagem, Vitória de Santo
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Antão, PE – Brasil
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c- Universidade de Pernambuco, Departamento de Ciências Naturais e Exatas,
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Garanhuns, PE – Brasil
14
+
15
Pesquisa Aggeu Magalhães/FIOCRUZ, Av. Professor Moraes Rego s/n, Cidade
16
Universitária, Campus da UFPE, 50670-420, Recife, PE, Brazil, CEP. Phone: 55-81-
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2101-263, Fax: 55-81-21012640. E-mail: [email protected]
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Corresponding author. Mailing address: Departamento de Imunologia, Centro de
ACKNOWLEDGEMENTS:
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We thank the platform PDTIS/Flow Cytometry (RPT08F)-FIOCRUZ. This work
20
was supported by the Coordination of Improvement of Higher Level Personnel
21
(CAPES). The authors declare that there is no conflict of interest.
22
Abstract
23
American Cutaneous leishmaniasis (ACL) is a public health problem. The
24
immunological response is mainly dependent on T cell cytokine responses and might
25
influence disease presentation, susceptibility and development. The understanding of
26
the host immune response role in the control and in the pathology of leishmaniasis is
27
relevant and has implications on diagnosis, follow-up and vaccine development. In this
28
study, the differences in the immune response and T cell profile of patients before
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ACCEPTED MANUSCRIPT treatment was investigated through flow cytometry and real time PCR in peripheral
2
blood mononuclear cells after different antigenic stimulations. Among the main findings
3
are the significant presence of TNF and IFN-γ gene expression after 24 hours of in vitro
4
stimulation, and 48h later the presence of CD4+ T and CD8+ T cells producing IL-10
5
and IL-4. This may be due to the differences in cytokine release over time and the
6
presence of cells other than lymphocytes influencing the mRNA transcript detection.
7
Evaluation of the immune response of individuals with leishmaniasis or other diseases
8
should associate different technologies and times points for a clear and more reliable
9
assessment of the immune response. This would help in the design of vaccine strategies/
12
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immunotherapies.
Keywords: American Cutaneous leishmaniasis, Immune response, T lymphocytes, Flow cytometry, qPCR, cytokines, Leishmania braziliensis.
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1) Introduction
Leishmaniasis is considered a public health problem, being endemic in many
15
countries and with associated social and economic burdens (1, 2, 3, 4). Cutaneous
16
leishmaniasis has a distribution in more than 97 countries with an estimated of 0.7 to 1.3
17
million new cases each year (3). American Cutaneous Leishmaniasis (ACL) has a
18
diverse clinical presentation, associated with the immunological response of the patient,
19
epidemiological patterns, which may differ according to the phlebotomine sandfly
20
species involved in transmission, population susceptibility, and its level of exposure
21
(5,6,7).
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More than 20 species of Leishmania protozoa are responsible for the disease in
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humans, among them, Leishmania (L). Viannia (V.) braziliensis is the main etiological
24
agent in Pernambuco, and other Brazilian states (1, 2, 5, 8). Human infection with L.
25
braziliensis can present itself with varied clinical forms, ranging from asymptomatic to
26
cutaneous and mucocutaneous forms of leishmaniasis. Cutaneous leishmaniasis is the
27
most common type, characterized as a single or a few ulcers with elevated borders (2, 7,
28
9). Host’s immunological status is also a key factor that acts by dramatically influencing
29
disease presentation (5,6,7,10).
3
ACCEPTED MANUSCRIPT Susceptibility or resistance to leishmaniasis is dependent on T cell responses (11), being
2
cytokine produced by CD4+ T cells responsible for the disease cured or aggravated
3
profile (9, 10, 12), whereas CD8+ T cells are usually related to the healing process and
4
immunological protection (12, 13, 14) but some authors also describe them associated
5
to tissue injury (15, 16). These T cells produce relevant cytokines for activation of
6
macrophage and monocytes, and they undergo a differentiation process before
7
becoming specific T cells. In general, Th1 cytokine production of IFN-γ, TNF and IL-
8
12, has been associated with infection control (1, 11, 17), while Th2 cytokines, such as
9
IL-4, IL-10 and TGF-β, have been enrolled in parasite multiplication and disease
10
progression (1, 18). These later cytokines are also able to inhibit T cell differentiation
11
into a Type 1 profile, thus, acting as a counterbalance to the Th1 proinflammatory effect
12
(6,9,17,19).
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Therefore, the type of immune response can influence disease susceptibility and
14
development. Even though there are studies that helped to clarify the immune
15
mechanisms of ACL development and pathology (11, 12, 20, 21, 22), no clear data is
16
available about what is the immune response necessary to control the disease and which
17
is the best tool to evaluate this response. Also, only some studies associate different
18
methods to evaluate the immune response in these patients (21, 23, 24, 25). Since
19
cytokines act in a multifaceted way, analysis of cytokine profiles through different tools
20
are powerful and a useful approach to understand the response, and activation of the
21
immune system (26).
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Towards a personalized treatment, assessment of the host’s immunological
23
status may help to identify resistant or susceptible patients, thus, increasing the chances
24
of success of chemotherapy. For this immune evaluation, different factors may
25
influence somehow, such as the specie of the parasite that is infecting the host and the
26
antigen that is being used to evaluate it (20, 27). In this sense, few studies have helped
27
to clarify the immunological status of individuals infected by L. braziliensis in Brazil.
28
For this task, combination of flow cytometry (FC) and quantitative real-time PCR
29
(qPCR) have become important tools in the investigation of the immune response.
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qPCR allows quantification of transcripts and small changes in gene expression,
31
and it is easy to perform and provides accuracy, rapid and reliable results (28). In
4
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2
produced by different immune cells and can differentiate T cell profiles (29).
3
Combining these two technologies provides means to better characterize the
4
immunological status of L. braziliensis infected patients. In this context, this work was
5
designed to combine flow cytometry and qPCR to characterize differences in the
6
cytokine and T cell profile of cells from L. braziliensis infected patients, prior to
7
chemotherapy, stimulated with distinct antigens from L. braziliensis.
9
2) Materials and Methods 2.1
Ethical aspects and Study Group
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CPqAM/FIOCRUZ Research Ethics Committee (Recife, Brazil) have approved
11
the experimental protocols for this research (CAEE number 00082.0.095.000-09).
12
Selected individuals were invited to participate in the research and signed the “Term of
13
Free and Informed Consent”. The selection of these individuals was based on criteria
14
such as: being more than 12 years old, having confirmed diagnosis by the
15
CPqAM/Fiocruz Leishmaniasis Referee Center and being active lesion carriers. Our
16
study group consisted of 11 patients, from Pernambuco rural endemic areas, with one to
17
four lesions and a disease with a mean development of 50 days (15 days – 3 months).
18
Patients were submitted to blood collection prior to chemotherapy treatment (Before
19
Treatment – BT group) with Glucantime®. The control group consisted of 5 healthy
20
individuals, from non-endemic areas, without previous history of ACL and no prior
21
blood transfusion. Patients and controls (CT group) had a similar age range (controls
22
28.91±14.27/ patients 27.4±13.28). The major characteristics and test results of the
23
study group are summarized in Table 1.
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Leishmania Antigenic Fractions
Promastigote forms of L. (V.) braziliensis (MHOM/BR/75/M2903), cultured in
26
vitro, were expanded in Schneider’s medium (Sigma, St. Louis, MO) supplemented
27
with 10% of fetal calf serum (Cultilab, Campinas, SP, Brazil) and 1% of antibiotics
28
(100UI/ml penicillin and 100 µg/ml streptomycin; Sigma, St. Louis, MO) until they
29
reached the exponential phase. Afterwards, they were sedimented by centrifugation at
30
800 x g for 15min at 4oC and three times washed with phosphate-buffered saline (PBS;
31
pH 7.2). Proteases inhibitors such as 0.1mM methyl-phenyl-fluoride and 2mM
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2
St. Louis, MO), were added right after ultrasonicate (100w/10cycle/30s with 2pulses/s).
3
The parasitic suspension was centrifuged at 10,000 x g for 10 min at 4oC. The resultant
4
supernatant was removed and submitted to a new centrifugation at 100,000 x g for 1 hr
5
at the same temperature. The resultant supernatant was the soluble antigenic fraction
6
(SOL), while the sediment was the insoluble antigenic fraction (INS). Both were
7
submitted to protein determination according to Brito et al. (2001) (30). The antigens
8
were stored at -20oC until use. The electrophoretic profile of soluble and insoluble
9
antigens of L. (V.) braziliensis is shown in Brelaz-de-Castro et al.(2012) (12).
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2.3
12
Forty milliliters of peripheral blood in heparinized tubes were collected, diluted
13
in a proportion of 2:1 (v/v) in PBS pH 7.2 and then added to a Ficoll-Hypaque
14
(Amersham Biosciences, Uppsala, Sweden) solution. After centrifugation at 400 x g for
15
30 min at 20oC, a peripheral blood mononuclear cells (PBMCs) ring was obtained. Cells
16
were washed twice with PBS pH 7.2 and submitted to centrifugation (300 x g for 15
17
min at 20oC). PBMCs were resuspended with 2 ml of RPMI 1640 medium (Cultilab,
18
Campinas, SP, Brazil) supplemented with 10% of fetal calf serum (Cultilab, Campinas,
19
SP, Brazil) and 1% of antibiotics (100 UI/ml penicillin and 100µg/ml streptomycin-
20
Sigma, St. Louis, MO). Cell viability was evaluated with Trypan blue dye (Sigma, St.
21
Louis, MO), cells were counted in a Neubauer chamber and cell concentration was
22
adjusted.
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Blood sampling and peripheral blood mononuclear cells isolation
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2.4
Flow Cytometry
Peripheral blood mononuclear cells (PBMCs) (4x106 cells/tube) from each
26
patient/control were individually incubated with Leishmania soluble (SOL, 1.25µg/ml)
27
or insoluble (INS, 2.25µg/ml) antigenic fractions (37ºC/ 5% CO2), for 48 hours, by
28
means of previous kinetic assay. Positive controls consisted of cells stimulated with
29
phytohemagglutinin (PHA, 10µg/ml) 4 hours prior to the end of the incubation period.
30
Brefeldin A (10µg/ml) (Sigma, St. Louis, MO) was added to all plates 4 hours prior to
31
the end of the incubation period. After the incubation, ethylene-diamine-tetra-acetic acid
6
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2
for 10 minutes to detach cells. Cells were then washed with PBS with 0.5% bovine
3
serum albumin (Sigma, St. Louis, MO) and 0.1% sodium azide (Sigma, St. Louis, MO),
4
a solution called PBS-W, centrifuged (370 x g, 7 min, RT) and transferred to
5
polystyrene tubes. The tubes contained monoclonal antibodies anti-CD4 or anti-CD8,
6
both labeled with fluorescein isothiocyanate (FITC) (BD Bioscience, San Jose, CA),
7
and the cells were incubated for 30 min at RT. Then, cells were fixed with 1%
8
paraformaldehyde in PBS, washed by centrifugation with PBS-W (400 x g, 5 minutes)
9
and permeabilized with PBS plus 0.5% of saponin. Subsequently, the cells were washed
10
with PBS-W by centrifugation (400 x g, 5 minutes) and then incubated with cytokine-
11
specific antibodies against IFN-γ, TNF, IL-10 (Miltenyi Biotec, Bergisch Gladbach,
12
Germany) and IL-4 (BD Bioscience, San Jose, CA), all labeled with phycoerythrin
13
(PE), for 30 minutes, RT. After this time, they were washed by centrifugation with
14
PBS-W, (400 x g, 5 minutes) and resuspended with 1% paraformaldehyde in PBS.
15
Samples were then analyzed (20.000 events within the electronics area compatible with
16
the lymphocyte/sample) through flow cytometry (FACSCalibur-BD, San Jose, CA)
17
using the software CELLQuestProTM (BD Bioscience, San Jose, CA) for acquisition and
18
analysis of data. The analyses were done first delimitating the lymphocytic region in a
19
Forward Scatter (FSC) versus Side Scatter (SSC) graph. Based on this region the
20
fluorescence graphs (FITCxPE) were constructed, delimitating the quadrants for
21
analyses. The quadrant limits were based on the negative population and single stained
22
controls. The values considered were the percentage of the quadrant region for each
23
quadrant.
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2.5
Cell culture, RNA isolation and cDNA synthesis
PBMCs (106 per well/ ml), from the same individuals which was performed flow
26
cytometry, were incubated with Leishmania soluble (SOL, 1.25µg/ml) and insoluble
27
(INS, 2.25µg/ml) antigenic fractions for 24 hours (37ºC/ 5% CO2) in a 24 well plate
28
(TPP Trasadingen, Switzerland). This time interval for cell culture was used since RNA
29
lifetime is shorter when compared for cytokine production, and accumulation for
30
release. Total RNA was isolated from PBMC using Trizol solution (Life Technologies
31
Carlsbad, CA), according to the manufacturer’s recommendations. RNA purity was
32
verified by agarose gel electrophoresis followed by ethidium bromide staining. OD260/280
7
ACCEPTED MANUSCRIPT 1
determination (above 1.8) and RNA quantification were done using a NanoDrop™
2
Spectrophotometer (Thermo Scientific, Waltham, Massachusetts) and the RNA was
3
kept at -80°C until ready to use. RNA samples from patients and controls were adjusted to 100ng/ul, and the
5
Reverse Transcription PCR reaction (RT) was carried out with the “TaqMan® Reverse
6
Transcription Reagents” kit (Applied Biosystems, Foster City, CA. Catalog
7
#N8080234). RT-PCR was performed in a final volume of 50µl, containing TaqMan RT
8
Buffer 1x, MgCl2 5.5mM, dNTPs mix 500µM, random hexamers 2.5µM, RNase
9
inhibitor 0.4 U/µL, multiscribe reverse transcriptase enzyme 3.125 U/µL and 12.375µL
10
of the sample. The cycling conditions were as follows: 10 minutes at 25ºC, 30 minutes
11
at 48 ºC and 5 minutes at 95 ºC. Negative controls were included in each run. After this
12
stage, samples were stored at -20 ºC until its utilization in the real time PCR assays
13
(qPCR).
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Relative quantitative Real-Time PCR
To quantify the mRNA expression of the cytokines TNF (Assay ID:00174128_m1),
16
IFN-γ (Assay ID: Hs99999041_m1), IL-10 (Assay ID: Hs00174086_m1) and IL-4
17
(Assay ID:99999030_m1) in the samples, the real time TaqMan® PCR was used (those
18
kits do not provide primers sequences). A housekeeping gene 18S was simultaneously
19
processed in Real-time TaqMan® PCR for each sample, as an endogenous control. This
20
was done to normalize the process due to the variability in the number of cells in the
21
original samples, for differences in extraction efficiency, for mRNA degradation in the
22
starting material, and for reverse transcriptase reaction efficiency. qPCR was carried out
23
using 1µg of cDNA as the starting material in a total volume of 20 µl with TaqMan®
24
Universal PCR Master Mix (Applied Biosystems, Foster City, CA) . All reactions were
25
performed in the Model ABI PRISM 7500 Sequence Detector (PE Applied
26
Biosystems). The relative quantification was calculated by the 2-∆∆CT method. In this
27
method, the fold change in the sample gene, given by delta-delta Ct (∆∆Ct), is
28
determined by the following equation: ∆∆CT = ∆CT (sample) - ∆CT (calibrator) and
29
∆CT is the CT of the target gene (sample or calibrator) subtracted from the Ct of the
30
housekeeping gene, 18S. Using this method, a positive fold change would represent up-
31
regulation of the sample gene compared to the calibrator; whereas a negative fold
32
change would represent down-regulation of the sample gene compared to the calibrator.
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2.7
Statistical Analysis
3
The statistical analyses were performed with GraphPad Prism 5.1 (GraphPad
4
Prism 5.1 Software, San Diego, CA, USA), using nonparametric tests. To detect
5
differences between groups the Mann–Whitney U-test was used. The results were
6
analyzed considering the value of p≤0.05 statistically significant (based on the two-
7
tailed p value).
8
3) Results
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3.1
Patients with active lesions presented a higher proportion of CD4+ T
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cells producing IL-4 and IL-10 48h after antigenic stimulation.
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Figure 1 shows the medium cytokines (IFN-γ, TNF, IL-4 and IL-10) percentage
13
levels produced by T cells 48h after PBMC culture with the soluble (SOL) and
14
insoluble (INS) antigenic fractions of L (V.) braziliensis, and with the mitogen PHA. Under PHA stimulus, all the groups produced cytokines, with significant difference
16
observed in the production of TNF by lymphocytes (CTxBT p= 0.005) and specifically
17
by CD8+ T cells (CTxBT p= 0.022). After stimulation with the Leishmania antigens
18
CD8+ T cells were the main responsible for the production of IFN-γ and CD4+ T cells
19
were the main producers of IL-10, IL-4 and TNF. Overall, the soluble antigen fraction
20
stimulated IL-10 significant production by lymphocytes (BTxCT p= 0.041), and
21
specifically CD4+ T cells to produce IL-10 (BTxCT p=0.0007) and IL-4 (BTxCT
22
p=0.003), and CD8+ T cells to produce TNF (CTxBT p=0.019), IL-4 (BTxCT p=0.028)
23
in a significant manner when compared to controls. The insoluble antigenic fraction
24
stimulated a significant production of IL-10 (BTxCT p=0.004) and IL-4 (BTxCT
25
p=0.006) by patients CD4+ T cells and IL-10 (BTxCT p=0.033) and IL-4 (BTxCT
26
p=0.043) by patients CD8+ T, when comparing to controls.
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The inflammatory versus regulatory cytokine balance was evaluated through the
28
IFN-γ/IL-10 ratio between patients and controls. Patients presented a lower IFN-γ/IL-10
29
ratio when compared to controls (Table 2). CD4/CD8 ratio was also evaluated between
9
ACCEPTED MANUSCRIPT 1
patients and controls, with patients presenting CD4 predominance when compared to
2
controls (table 2).
3
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3.2
qPCR revealed IFN-γ and TNF significant production 24h after PBMC culture
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Figure 2 shows the medium mRNA cytokine (IFN-γ, TNF, IL-4 and IL-10)
7
expression 24h after PBMC culture with the soluble (SOL) and insoluble (INS)
8
antigenic fractions of L (V.) braziliensis, and compares patients and controls samples.
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Under PHA stimulus, both groups of patients and controls expressed mRNA for the
10
cytokines, with significant difference observed in the production of IFN-γ by patients
11
(∆CT=13.06± 1.15) when compared to controls (∆CT=14.72±1.070) (p=0.045). The
12
soluble antigenic fraction (1.25µg/ml) stimulated patients and controls cells to produce
13
cytokines, with statically significance observed in the production of IFN-γ (Patients
14
∆CT=14.76±1.20; Controls ∆CT=19.56± 1.58; p<0.001) and
15
∆CT=13.25±2.53; Controls ∆CT=16.26±0.650; p=0.028).
16
fraction (2.5µg/ml) also stimulated patients and controls cells to produce cytokines, and
17
it was observed a significant production of IFN-γ (Patients ∆CT=12.17±2.94; Controls
18
∆CT= 19.24± 1.77; p=0.001) and TNF (Patients ∆CT=11.49±2.33; Controls ∆CT=
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14.96± 0.562, p=0.013).
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4) Discussion
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Leishmaniasis is a group of diseases where the development of lesions and their
23
resolution is closely linked to the development of an appropriate immunological
24
response. For this, T cells have a major role, acting through direct cytotoxicity (CD8+ T
25
cells) and/or producing several cytokines and chemokines. Cytokines can react in
26
cascades with other cytokines and lead to a higher or lower production or secretion of
27
further cytokines, acting in a very complex manner. Therefore, the analysis of cytokine
28
profiles is useful to understand the response and activation of the immune system. In
29
this study, we described the in vitro production of cytokines and T cell profiles from
30
ACL patients after PBMC culture with the soluble and insoluble antigenic fractions of L
10
ACCEPTED MANUSCRIPT 1
(V.) braziliensis. They were analyzed through qPCR and Flow Cytometry, the last one
2
also evidencing the population that produced it. Flow cytometry analysis demonstrated the presence of the cytokines IFN-γ, TNF,
4
IL-4 and IL-10, being IL-10 and IL-4 significantly expressed by patients T cells when
5
compared to healthy controls under different circumstances of stimulation. CD4+ T cells
6
were the main responsible for IL-4 and IL-10 production, and CD8+ T cells for IFN-γ,
7
but also producing IL-4 and IL-10 significantly. When considering the CD4/CD8 ratio,
8
we observed an expansion of CD4+ T cells when compared to controls. This may
9
account for the increased proportion of IL-10 and IL-4 observed, since CD4+ T cells
10
were their main producers. Previous studies by our group also demonstrated similar
11
results, and it was shown that this type 2 response later evolved to a Th1 profile after
12
patients’ clinical cure, however still with the presence of IL-10 (6,12). This result may
13
suggest a follow-up of the patients during treatment, in order to better characterize the
14
time point in which a type 1 response is taking place.
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The development and maintenance of a protective immune response seems to be
16
associated with a Type 1 cytokine predominant profile, however with an appropriated
17
modulation. When considering the IFN-γ/IL-10 ratio among PBMCs from patients and
18
controls stimulated with the soluble and insoluble L (V.) braziliensis antigens, it was
19
observed that patients presented a higher proportion of T CD4+ and CD8+ T cells
20
producing IL-10, while the controls presented higher proportion of T CD4+ and CD8+ T
21
cells producing IFN-γ. As remarked in other studies, CD4+ and CD8+ T cells proportion
22
and cytokine balance is important for lesion progression or healing (12, 30, 31, 32).
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qPCR is a common method to detect cytokine transcripts in cells; in our study we
24
observed mRNA expression of the cytokines IFN-γ, TNF, IL-4 and IL-10, being IFN-γ
25
significant expressed by patients when compared to healthy controls under all
26
stimulation, and TNF under soluble and insoluble stimulus. Although the Th1 immune
27
response seems to be beneficial, the control of an exacerbated immune response is
28
essential for cure. The production of IL-10, initially related mainly to parasite survival
29
and macrophage deactivation, seems to represent a necessary counterbalance for disease
30
resolution (33). IFN-γ and TNF production is also not associated with the absence of IL-
31
10 (2). IL-10 is the cytokine that is most strongly associated with lesions development,
11
ACCEPTED MANUSCRIPT and is mainly released by monocytes and CD4+CD25+ T lymphocytes (21, 34, 35, 36).
2
Therefore, the balance between a pathological and protective immune response seems to
3
be given by regulatory T cells (21). Cells with regulatory functions have been
4
frequently described in Leishmania infections, and the existence of concomitant
5
immunity is discussed (37, 38). Nevertheless, conclusive role of Treg cells in
6
suppression of immunity in patients and on the mechanisms involved in the disease
7
development is yet to be defined.
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Trying to understand the immune response mechanisms in America cutaneous
9
Leishmaniasis, we studied the production of cytokines through qPCR and flow
10
cytometry aiming to better understand its production and release. These technologies are
11
usually performed in different time points, according to what was established by
12
different research groups and antigen being used. Usually, leishmaniasis flow cytometry
13
studies range from ex vivo analysis to culture evaluations after different time points (4h,
14
24h, 48h to 96 hours) (7, 12, 21). qPCR transcripts are measured from 4h, 24h, 72h up
15
to 15 days after culture (39, 40, 41). However, cytokine secretion is usually a process of
16
hours or days, controlled by autocrine and paracrine regulation and feedback loops, with
17
secretory activity dropping down to normal without continuous stimulation. Also,
18
lymphocytes have no reservoirs for cytokines (26). Therefore, the initial activation of
19
transcription and biosynthesis in lymphocytes is strongly related to its consecutive
20
secretion and may be the reason for the absence of complete agreement of the different
21
technologies used in our study (26). Since there is no definitive immune profile
22
established for this disease, qPCR and flow cytometry are useful tools to understand the
23
development of the patients’ immune responses and help on its monitoring.
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Leishmania active infections has been related to type 1 and 2 cytokine profiles,
25
depending on the antigen used, the time of disease evolution and the method reported
26
(9, 18, 19, 42, 43). Previous works by our group, with cells stimulated with the L. (V.)
27
braziliensis soluble antigen, reported the presence of significant Th1 cytokines however
28
still with the significant presence of IL-10 (44) also seen in other studies with L. (V.)
29
braziliensis antigens (9, 18, 19, 45). Our study measured cytokines through ELISA, in
30
patients with longer disease evolution time. Additional studies with the same antigens
31
used in this article but with patients with shorter evolution times (<60 days) and
32
analyzed only through flow cytometry, demonstrated T CD4+ and type 2 cytokines
12
ACCEPTED MANUSCRIPT predominance profile (6, 12) that have been also described by other groups (9, 21, 42,
2
46). Therefore, this Th1/Th2 profile seems to be an oversimplification of what is
3
happening inside the host and it seems that regulation of the inflammatory response
4
rather than biased Th1/Th2 response defines the outcomes of infection with Leishmania
5
Viannia species (39, 44, 47). Other T cell subsets have important roles in susceptibility
6
and resistance to leishmaniasis, such as Treg and Th17 cells. All this emphasizes the
7
need to study T cell response regarding its magnitude, quality and through different
8
technologies.
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Given that the qPCR mRNA examination at 24 h only reflects a snapshot, it can be
10
assumed that if a low gene expression values and a high cytokine levels are detected for
11
a distinct data pair, this may reflect that the gene expression had already declined due to
12
an effectual level of cytokines reached in the cell culture supernatant at an earlier point.
13
Also since this technology identifies the presence of cytokines transcripts, but not the
14
cells containing the transcripts, we may be analyzing transcripts from cells other than
15
lymphocytes, such as monocyte, that largely contribute to IFN-γ and TNF secretion and
16
type 1 or 2 cytokine differentiation (2, 48). Therefore, the different cytokines profiles
17
observed in our patients when using flow cytometry and qPCR may be due to the
18
differences in cytokine release over time and the presence of cells other than
19
lymphocytes influencing the detection of the mRNA transcript.
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In this study, we described the significant presence of TNF and IFN-γ transcripts at
21
24h of infection, and 48h later the presence of CD4+ T cells producing IL-10 and IL-4,
22
and of CD8+ T cells producing IL10, IL-4 and TNF, after the soluble and insoluble
23
antigen of L. (V.) braziliensis were used for PBMC stimulation. These antigens, as seen
24
in previous studies from our group (6, 12, 22, 44, 49), allow the study of a more specific
25
immune response, since they come from L. braziliensis, the predominant species in the
26
region (4, 8). The understanding of the role of the host’s immune response in the control
27
and in the pathology of leishmaniasis through different technologies and with different
28
antigens is highly relevant and has implications for the control of leishmaniasis, since
29
there is not an established immunological profile associated with a cure. Therefore, as
30
evidenced by our findings, studies aiming to evaluate the immune response of
31
individuals with leishmaniasis or other diseases should consider associate different
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technologies and times point, such as flow cytometry and qPCR - to obtain a clear a
2
more reliable evaluation of the immune response developed by patients.
5) References 1. Ameen M. 2010. Cutaneous leishmaniasis: advances in disease pathogenesis, diagnostics and therapeutics. Clin Exp Dermatol 2010; 35(7):699-705. doi: 10.1111/j.1365-2230.2010.03851.x.
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2. Carvalho LP, Passos S, Schriefer A, Carvalho EM. Protective and pathologic immune responses in human tegumentary leishmaniasis. Front Immunol 2012; 4;3:301. doi: 10.3389/fimmu.2012.00301.
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4. Karimkhani C, Wanga V, Coffeng LE, Naghavi P, Dellavalle RP, Naghavi M. Global burden of cutaneous leishmaniasis: a cross-sectional analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis 2016;16(5):584-91. doi: 10.1016/S1473-3099(16)00003-7
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5. de Brito ME, Andrade MS, Dantas-Torres F, et al. Cutaneous leishmaniasis in northeastern Brazil: a critical appraisal of studies conducted in State of Pernambuco. Rev Soc Bras Med Trop 2012; 45(4):425-9.
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6. Brelaz MC, de Oliveira AP, de Almeida AF, et al. Leishmania (Viannia) braziliensis antigenic fractions: the immune response characterization of patients at the initial phase of disease. Parasite Immunol 2012; 34(4):236-9. doi: 10.1111/j.1365-3024.2012.01351.x.
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7. Macedo AB, Sánchez-Arcila JC, Schubach AO, et al. Multifunctional CD4⁺ T cells in patients with American cutaneous leishmaniasis. Clin Exp Immunol 2012; 167(3):505-13. doi: 10.1111/j.1365-2249.2011.04536.x.
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10. Santos SC, Brodskyn CI. The Role of CD4 and CD8 T Cells in Human Cutaneous Leishmaniasis. Front Public Health 2014; 2:165. doi: 10.3389/fpubh.2014.00165.
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ACCEPTED MANUSCRIPT 11. Scott P, Novais FO. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. Nat Rev Immunol 2016;16(9):581-92. doi: 10.1038/nri.2016.72.
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12. Brelaz-de-Castro MC, de Almeida AF, de Oliveira AP, de Assis-Souza M, da Rocha LF, Pereira VR. Cellular immune response evaluation of cutaneous leishmaniasis patients cells stimulated with Leishmania (Viannia) braziliensis antigenic fractions before and after clinical cure. Cell Immunol 2012; 279(2):180-6. doi: 10.1016/j.cellimm.2012.11.006.
13. Ferraz R, Cunha CF, Gomes-Silva A, Schubach AO, Pimentel MI, Lyra MR, et al. Apoptosis and frequency of total and effector CD8+ T lymphocytes from cutaneous leishmaniasis patients during antimonial therapy. BMC Infect Dis. 2015; 15:74. doi: 10.1186/s12879-015-0799-x.
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14. Castro Gomes CM, Sousa MG, Menezes JP, et al. Immunopathological characterization of human cutaneous leishmaniasis lesions caused by Leishmania (Viannia) spp. in Amazonian Brazil. Parasitol Res 2017. doi: 10.1007/s00436-017-5403-4.
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Tables and figures captions
2
MST1
Age
#
(years)
lesions
1
43
1
N/D
-
2
26
1
10mm
-
3
13
1
10mm
-
4
32
1
11.5mm
5
21
1
14mm
6
27
7
15
8
EP
10 11
PCR3
Evolution times
Microscopy
+
+
2 months
+
2 months
+
+
3 months
-
+
N/D
3 months
-
+
+
1 month
M AN U
+
1
10mm
-
-
+
1 month
4
12mm
-
+
+
1 month
2
16mm
-
-
+
15 days
23
1
14mm
+
-
+
3 months
45
1
9mm
N/D
-
+
1 month
44
1
10mm
+
+
N/D
1 month
43
AC C
9
IFI2
Direct
SC
Patient
RI PT
Table 1. Test results and characteristics of the ACL patients
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MST: Montenegro skin test (Positive ≥ 5mm); 2IFI: Indirect Immunofluorescence;
5
3
PCR: Polymerase chain reaction; +: Positive; -: Negative; N/D: Not done.
6
2
ACCEPTED MANUSCRIPT Table 2: CD4+ T/CD8+ T cell ratio in cultures stimulated 48h with Leishmania soluble and insoluble antigenic fractions
2.71
CT CD4+/CD8+ ratio
1.49
BT CD4+ IFN-γ/IL-10 ratio
0.78 (2.23-0.24)
BT CD8+ IFN-γ/IL-10 ratio
2.35 (7.51-0.68)
CT CD4+ IFN-γ/IL-10 ratio
3.52 (7.56-0.42)
CT CD8+ IFN-γ/IL-10 ratio
2.08 (9.7-0.45)
3
2.51 1.5
0.71 (1.19-0.45) 0.95 (1.45-0.46)
1.95 (3.83-0.40)
M AN U
BT CD4+/CD8+ ratio
Insoluble Antigen
RI PT
Soluble Antigen
SC
1 2
4.83 (6.89-1.96)
Figure 1: Cytokine expression by (A) T cells, (B) T CD4+ cells, and (C) T CD8+
5
cells after in vitro stimulation with PHA (10µg/ml), Leishmania soluble (1.25µg/ml) and
6
insoluble (2.25µg/ml) antigenic fractions. The results are expressed as mean values ±
7
SD. Differences between groups were considered significant with a P value less than
8
0.05 and are represented by the symbol “*” (difference between patients and controls).
9
BT= Before Treatment/ CT= Controls.
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Figure 2: Patients and controls cytokines mRNA expression 24h after PBMC
12
culture with the mitogen PHA or with the soluble (SOL, 1.25µg/ml) and insoluble
13
(INS, 2.5µg/ml) antigenic fractions of L (V.) braziliensis, following normalization with
14
the reference gene (18S). The results are expressed as mean values ± SD. Differences
15
between groups were considered significant with a P value less than 0.05 and are
16
represented by the symbol “*” (difference between patients and controls). BT= Before
17
Treatment/ CT= Controls.
18
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