- Levels of Interferon-Gamma Increase after Treatment for Latent Tuberculosis Infection in a High-Transmission Setting
- Gene Expression Profile of High IFN-γ Producers Stimulated with Leishmania braziliensis Identifies Genes Associated with Cutaneous Leishmaniasis
- Circulating Interferon-Gamma Levels Are Associated with Low Body Weight in Newly Diagnosed Kenyan Non-Substance Using Tuberculosis Individuals
- 1. Introduction
- 2.1. Study Design and Participants
- 2.2. Physical Measurements
- 2.3. Vital Signs
- 2.4. Laboratory Diagnosis of TB
- 2.5. Haematological Enumerations
- 2.6. Plasma Preparation
- 2.7. Cytokine Measurements
- 2.8. Statistical Analysis
- 2.9. Ethical Considerations
- 3.1. Baseline Characteristics of the Study Participants
- Serum IFN-γ levels predict the therapeutic effect of mesenchymal stem cell transplantation in active rheumatoid arthritis
Levels of Interferon-Gamma Increase after Treatment for Latent Tuberculosis Infection in a High-Transmission Setting
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Gene Expression Profile of High IFN-γ Producers Stimulated with Leishmania braziliensis Identifies Genes Associated with Cutaneous Leishmaniasis
The initial response to Leishmania parasites is essential in determining disease development or resistance. In vitro, a divergent response to Leishmania, characterized by high or low IFN-γ production has been described as a potential tool to predict both vaccine response and disease susceptibility in vivo.
We identified uninfected and healthy individuals that were shown to be either high- or low IFN-γ producers (HPs and LPs, respectively) following stimulation of peripheral blood cells with Leishmania braziliensis. Following stimulation, RNA was processed for gene expression analysis using immune gene arrays.
Both HPs and LPs were shown to upregulate the expression of CXCL10, IFI27, IL6 and LTA. Genes expressed in HPs only (CCL7, IL8, IFI44L and IL1B) were associated with pathways related to IL17 and TREM 1 signaling.
In LPs, uniquely expressed genes (for example IL9, IFI44, IFIT1 and IL2RA) were associated with pathways related to pattern recognition receptors and interferon signaling. We then investigated whether the unique gene expression profiles described here could be recapitulated in vivo, in individuals with active Cutaneous Leishmaniasis or with subclinical infection.
Indeed, using a set of six genes (TLR2, JAK2, IFI27, IFIT1, IRF1 and IL6) modulated in HPs and LPs, we could successfully discriminate these two clinical groups. Finally, we demonstrate that these six genes are significantly overexpressed in CL lesions.
Upon interrogation of the peripheral response of naive individuals with diverging IFN-γ production to L. braziliensis, we identified differences in the innate response to the parasite that are recapitulated in vivo and that discriminate CL patients from individuals presenting a subclinical infection.
Control and development of Cutaneous Leishmaniasis (CL) are dependent on the host immunological response. One of the key molecules in determining elimination of Leishmania parasites from the infected host cell is the cytokine interferon gamma (IFN-γ).
The aim of this study was to investigate which immune response genes are associated with the production of IFN-γ in the context of Leishmania infection. We identified individuals that are high- or low IFN-γ producers upon stimulation of their peripheral blood cells with Leishmania parasites.
We then determined the immune gene expression profile of these individuals and we identified a set of genes that are differentially expressed comparing high- and low IFN-γ producers. The expression of these genes was also evaluated in patients with CL and in individuals with a subclinical Leishmania infection (SC).
In this setting, the overall pattern of expression of this particular gene combination discriminated the CL patients x from SC individuals. Understanding the initial response to Leishmania may lead to the identification of markers that are associated with development of CL.
Citation: Carneiro MW, Fukutani KF, Andrade BB, Curvelo RP, Cristal JR, Carvalho AM, et al. (2016) Gene Expression Profile of High IFN-γ Producers Stimulated with Leishmania braziliensis Identifies Genes Associated with Cutaneous Leishmaniasis. PLoS Negl Trop Dis 10(11): e0005116. https://doi.org/10.1371/journal.pntd.0005116
Editor: Mary Ann McDowell, University of Notre Dame, UNITED STATES
Received: May 24, 2016; Accepted: October 18, 2016; Published: November 21, 2016
Copyright: © 2016 Carneiro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant 302464/2009-3 to M.
BN and fellowship to MWC and KFF), Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) (fellowship to KFF) and Coordenação de Apoio à Pesquisa e Ensino Superior (CAPES) (fellowship to JRC). AB, MBN, and CIdO are senior researchers at CNPq.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cutaneous Leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a broad spectrum of clinical manifestations, ranging from localized CL to Mucosal Leishmaniasis (rev. in ).
A hallmark feature of the immunological response in localized CL is a strong Th1 type immune response to soluble Leishmania antigen (SLA), demonstrated by a positive delayed type hypersensitivity (DTH) reaction to the Leishmania skin test, as well as lymphocyte proliferation and high levels of IFN-γ and TNF-α [2–4].
Since T-cell-mediated immunity plays a central role in the host’s response to intracellular parasites, in vitro experimental settings have been used to address the initial lymphocyte response to Leishmania: PBMCs from naive volunteers stimulated with L. major produce mainly IFN-γ and this effect is regulated by IL-10 and IL-12 .
Using an in vitro priming system with Leishmania amazonensis antigen, Pompeu et al. showed that cells from naïve individuals produce either high or low amounts of IFN-γ .
These two patterns of in vitro anti-Leishmania response correlated with the in vivo post-vaccination response: low in vitro IFN-γ producers exhibit a delayed response to vaccination with SLA, whereas an accelerated immune reaction vaccine is observed in those who were high IFN-γ producers . Upon stimulation with L.
amazonensis, high IFN-γ producers also secrete more TNF , more IL-12 and less IL-13 . These results indicate that a low IFN-γ response in vitro accompanies a slower IFN-γ production in vivo and authors suggested that in vitro responses could be used to predict, for example, the pace of post vaccination responses.
IFN-γ, produced primarily by T cells and natural killer cells, is an important mediator of macrophage activation and intracellular pathogen killing, including Leishmania. We previously demonstrated that PBMCs from healthy uninfected individuals respond differently to Leishmania stimulation (secreting either high or low amounts of IFN-γ).
In this study, we aimed at characterizing the immune gene signature that parallels these two responses.
Further, we investigated whether such in vitro responses had in vivo equivalents by probing the gene expression of CL patients and that of individuals presenting a subclinical infection which is associated with absence of lesions, a positive Leishmania skin test (LST) , and lower levels of both IFN-γ and TNF .
We expand the current knowledge in the field by identifying genes that are expressed in association with the capacity to produce IFN-γ upon stimulation with Leishmania braziliensis. The immune signature associated with IFN-γ production also discriminates CL patients from individuals with subclinical infection.
Peripheral Blood Mononuclear Cells (PBMCs) were obtained from healthy uninfected individuals (n = 9) recruited in the city of Salvador (Bahia state, Brazil), where L. braziliensis transmission in not endemic (S1 Table).
These individuals had negative serology results for leishmaniasis, negative serology for Chagas’ disease, hepatitis and human immunodeficiency virus. CL patients and individuals presenting a subclinical (SC) infection were recruited from the area of Jiquiriça (Bahia state, Brazil), where L. braziliensis transmission is endemic (S2 Table).
Patients with active CL (n = 5) were diagnosed the presence of a typical clinical leishmaniasis lesion, a positive Leishmania skin test (LST) and documentation of parasites in culture or by histopathology. SC individuals (n = 8) were identified in the same endemic area, following a medical interview.
These individuals had no history of past CL (absence of scars consistent with CL or Mucosal Leishmaniasis in the skin, nose and soft palate) and a positive LST to Leishmania.
This research was conducted with the approval of the ethical committee of Centro de Pesquisas Gonçalo Moniz (CPqGM), Fundação Oswaldo Cruz (FIOCRUZ) (Salvador, Bahia, Brazil; 177/2008) and Comissão Nacional de Ética em Pesquisa (Brazilian National Ethics Committee, Brazil), and written informed consent was obtained from each participant.No minors participated in the study.
L. braziliensis promastigotes (strain MHOM/BR/01/BA788) were grown in Schneider medium (Sigma), supplemented with 100 U/ml of penicillin, 100 ug/ml of streptomycin and 10% heat-inactivated fetal calf serum (all from Invitrogen).
PBMCs from healthy individuals (n = 9) were obtained from heparinized venous blood layered over a Ficoll-Hypaque gradient (GE Healthcare). Cells were washed and resuspended in RPMI1640 supplemented with 10% human AB serum, 100 IU/ml of penicillin and 100μg/ml of streptomycin (all from Invitrogen).
PBMCs (3×106/ml) were placed in the wells of a 24-well plate at 500 μl per well. L. braziliensis stationary phase promastigotes were added to the cultures at a parasite/cell ratio 1:1. Control cultures were maintained in medium only. Cultures were performed in triplicate and maintained at 37°C/5% CO2.
After 72h, IFN-γ levels in culture supernatants were determined by ELISA (R&D Systems), following manufacturer´s instructions.
PBMCs obtained from previously defined HPs (n = 3) and LPs (n = 3) were stimulated with L. braziliensis promastigotes for 72h, as described above. After stimulation, total RNA was obtained using Trizol (Invitrogen), according to manufacturer's instructions. RNA (500 ng) was suspended in 50μl DEPC-treated water and stored at –70°C until use.
cDNA was synthesized from DNAse-treated RNA by reverse transcription using RT2 First Strand kit (Qiagen), following manufacturer´s instructions. cDNA obtained from cultures stimulated with L.
braziliensis or from control cultures (maintained in the absence of stimulus) was then employed in PCR array analysis using RT2 Real-Time SYBR Green PCR Master Mix (Qiagen) and the following human RT2 Profiler PCR arrays: Th1 & Th2 responses, Toll- Receptor Signaling Pathway, Interferon & receptors and Chemokines & Receptors (Qiagen), following manufacturer´s instructions. Reactions were performed on ABI 7500 Sequence Analyzer (Applied Biosystems). Fold changes in gene expression between L. braziliensis-stimulated and control cultures were calculated using the RT2 Profiler PCR array data analysis tool, the ΔΔCt method, after normalization to housekeeping genes, determined by the manufacturer. A gene was considered differentially expressed when fold change was above or below 2, compared to control cultures, and p300pg/ml whereas LPs were defined as presenting IFN-γ levels
Circulating Interferon-Gamma Levels Are Associated with Low Body Weight in Newly Diagnosed Kenyan Non-Substance Using Tuberculosis Individuals
Although interferon-gamma, interleukin-10, and adiponectin are key immunopathogenesis mediators of tuberculosis, their association with clinical manifestations of early stage disease is inconclusive.
We determined interferon-gamma, interleukin-10, and adiponectin levels in clinically and phenotypically well-characterised non-substance using new pulmonary tuberculosis patients () and controls () from Kenya. Interferon-gamma levels () and interferon-gamma to interleukin-10 () and interferon-gamma to adiponectin () ratios were elevated in tuberculosis cases.
Correlation analyses in tuberculosis cases showed associations of interferon-gamma levels with body weight (; ), body mass index (; ), hip girth (; ), and plateletcrit (; ); interferon-gamma to interleukin-10 ratio with diastolic pressure (; ); and interferon-gamma to adiponectin ratio with body weight (; ), body mass index (; ), and plateletcrit (; ).
Taken together, our results suggest mild-inflammation in early stage infection characterised by upregulation of circulating interferon-gamma production in newly infected TB patients.
Tuberculosis (TB) due to Mycobacterium tuberculosis is an important cause of infectious disease burden in the world. An estimated 9.0 million cases, 5.7 million new cases, and 1.5 million deaths were attributable to TB in 2013 .
A vast majority of new TB cases occur in developing countries particularly among individuals living in crowded and informal settlements and those with underlying conditions such as HIV, diabetes, malnutrition, smoking, and alcohol abuse . Sub-Saharan Africa accounts for the highest burden of TB comprising 2.8 million cases including 2.3 million new cases and 230,000 deaths .
Kenya is among the high TB burdened countries with annual incidence of 110,000 cases, 90,000 new infections, and over 4,000 deaths .
While molecular mechanisms underlying TB-related burden are poorly understood, host inflammatory responses mediate pathogenesis of the disease . Previous studies showed elevated plasma IFN-γ with lower IL-10 levels in first-time TB smear positive patients .
In contrast, increased plasma IL-10 levels in the presence of lower concentrations of IFN-γ were detected in chronic TB patients . In addition, previous studies showed a higher IFN-γ to IL-10 ratio in treatment-naive newly infected TB patients [6, 7], suggesting that M.
tuberculosis infections are characterised by an early burst in the proinflammatory cytokine response followed by an increased anti-inflammatory cytokine response in the chronic phases of disease.
The inflammatory cytokine response is linked to development of clinical manifestations of TB. For instance, higher levels of IFN-γ and IL-10 are associated with lower body weight and wasting in newly infected TB patients [8, 9]. Whilst a higher IFN-γ to IL-10 ratio is associated with protection and TB cure , a lower IFN-γ to IL-10 ratio is linked to TB relapse [6, 11].
Furthermore, increased plasma adiponectin levels are associated with severe TB characterised by extensive pulmonary lesions . Although adiponectin promotes IL-10 release and impairs IFN-γ secretion in human macrophages , the magnitude of IFN-γ and IL-10 to adiponectin production as an indicator of inflammatory response and clinical manifestations in TB is largely unknown.
Increasing evidence indicate that host response to TB is ly to be compounded by underlying patient factors including malnutrition, coinfections, and alcohol and substance consumption.
For example, plasma levels of IFN-γ increase in acute HIV infection  and decrease in chronic HIV infection , suggesting that stage of HIV infection determines inflammatory cytokine responses.
In addition, circulating adiponectin levels are decreased as IFN-γ and IL-10 levels are elevated in individuals with malnutrition and obesity [16–19], indicating that malnutrition promotes alterations in inflammatory cytokine production. Substance use also influences adiponectin, IL-10, and IFN-γ production.
For instance, marijuana components induce IFN-γ and suppress IL-10 production [20, 21] while alcohol increases IFN-γ and IL-10 levels .
In addition, reduced IFN-γ and IL-10 levels were found in opiate addicts  while low levels of adiponectin were reported in cocaine, opiate, cigarette, and alcohol users [24–27].
Taken together, underlying disease conditions and substance consumption are key factors promoting increased dysregulation in the inflammatory response in TB [28, 29]. To our knowledge, however, no study has examined cytokine levels and their clinical correlates in phenotypically and clinically well-characterised newly infected pulmonary TB patients. As such, the present study determined circulating IFN-γ, IL-10, and adiponectin levels and their association with clinical manifestations of early stage disease in non-substance using newly diagnosed pulmonary TB cases.
2.1. Study Design and Participants
This cross-sectional study was conducted among consenting new pulmonary TB patients and controls at Bomu Hospital, a social enterprise facility in Mombasa, a coastal city in Kenya.
Substance and drug using individuals  and those presenting with underlying conditions such as HIV and diabetes including retreatment TB cases were excluded from the study.
Newly diagnosed pulmonary TB patients were defined as individuals with a first case of TB positive sputum smear while controls comprised individuals with TB negative sputum smears and presenting with no evidence of illness. A total of 13 newly diagnosed pulmonary TB cases and 14 controls were recruited in this study.
The sample size was calculated the formula of  and plasma IFN-γ concentrations previously determined in TB patients and controls . Chest X-rays were taken on all study participants on the day of TB diagnosis and independently interpreted by two radiologists.
2.2. Physical Measurements
Anthropometric measures of the study participants were obtained by trained clinicians. Body weight was measured to the nearest 0.1 kg in light clothes, and height was measured to the nearest 1.0 cm in an upright posture. Waist circumference (WC) was assessed to the nearest 0.1 cm at smallest diameter between the iliac crest and lower rib.
Hip circumference (HC) was measured to the nearest 0.1 cm around the maximum circumference of the buttocks. Middle upper arm circumference (MUAC) was measured midway to nearest 0.1 cm amid the tip of acromion and olecranon process.
Body mass index (BMI, kg/m2) was calculated as weight (kg)/height (m) while waist-to-hip ratio was calculated as WC (cm)/HC (cm).
2.3. Vital Signs
Axillary temperature was obtained using a digital thermometer. Systolic and diastolic blood pressures were measured to the nearest 2 mmHg after a 10-minute rest in a sitting position using a digital blood pressure machine. Pulse rate was determined after a 10-minute rest in the sitting position using fingertip heart rate monitor.
2.4. Laboratory Diagnosis of TB
Sputum was obtained at enrolment and subsequently the following morning from all consenting individuals and used for smear preparation and acid-fast (Ziehl-Neelsen) staining. Smears were examined for presence of M. tuberculosis and bacilli were enumerated and scored according to the global guidelines for laboratory diagnosis of TB .
2.5. Haematological Enumerations
About 3.0 mL venous blood was collected from the study participants in ethylenediaminetetraacetic acid Vacutainer tubes (Becton Dickinson, Franklin Lakes, USA).
Complete blood count was performed using BC-3200 Mindray autohaematology analyser (Mindray Inc., Mahwah, USA).
The system was calibrated every morning before sample analysis, and hematologic analyses were performed within 10 minutes of blood collection.
2.6. Plasma Preparation
Plasma samples were prepared by centrifugation using bench-model centrifuge (Forma Scientific, Inc., Ohio, USA). Briefly, blood was centrifuged for 10 minutes at 1500 ×g, aliquoted into labelled cryovials, and frozen at −80°C until use for cytokine measurements.
2.7. Cytokine Measurements
Circulating levels of IFN-γ, IL-10, and adiponectin were determined in plasma samples using a sandwich enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s protocols (R&D Systems, Inc., Minneapolis, USA).
2.8. Statistical Analysis
Data analysis was conducted using GraphPad Prism v5 (GraphPad Inc., California, USA). Comparisons in age, anthropometric and hematologic measures, and cytokine levels between cases and controls were performed using Mann-Whitney test.
Fisher’s exact test was used for comparing gender distribution between the study groups.
Spearman’s rank correlation test was used to examine associations of cytokine levels and cytokine ratios with anthropometric and clinical measures in the TB cases.
2.9. Ethical Considerations
This study was approved by Kenyatta University Ethical Review Committee and was conducted according to Helsinki’s declarations. Written informed consent was obtained from all study participants prior to enrolment into the study. TB cases were treated according to Kenya national guidelines for TB treatment that is consisted of isoniazid .
3.1. Baseline Characteristics of the Study Participants
Baseline demographic and clinical information of the study participants are shown in Table 1. Age () and gender distribution () were similar between the study groups.
Anthropometric assessment showed significantly lower body weight (), body mass index (), hip circumference (), and middle upper arm circumference () in cases relative to controls. However, height (), waist girth (), and waist-to-hip ratio () were similar between the study groups.
Among the presenting clinical manifestations, axillary body temperature () was significantly elevated with lower systolic () and diastolic () pressures in the cases. However, pulse rate () was similar between the two groups. Chest examination indicated that 77.0% of the TB cases had congestion (30.8%) or crepitation (46.2%).
Hematologic analyses showed similar levels of total leucocyte (), neutrophil (), lymphocyte (), monocyte (), eosinophil (), and platelet () counts. Interestingly, TB cases presented with lower haemoglobin (), mean platelet volume (), and plateletcrit ().
Serum IFN-γ levels predict the therapeutic effect of mesenchymal stem cell transplantation in active rheumatoid arthritis