Mitochondrial DNA haplogroups and variants predispose to chagas disease cardiomyopathy

Cardiomyopathies are major causes of heart failure. Chagas disease (CD) is caused by the parasite Trypanosoma cruzi, and it is endemic in Central and South America. Thirty percent of cases evolve into chronic chagas cardiomyopathy (CCC), which has worse prognosis as compared with other cardiomyopathies. In vivo bioenergetic analysis and ex vivo proteomic analysis of myocardial tissues highlighted worse mitochondrial dysfunction in CCC, and previous studies identified nuclear-encoded mitochondrial gene variants segregating with CCC. Here, we assessed the role of the mitochondrial genome through mtDNA copy number variations and mtDNA haplotyping and sequencing from heart or blood tissues of severe, moderate CCC and asymptomatic/indeterminate Chagas disease as well as healthy controls as an attempt to help decipher mitochondrial-intrinsic genetic involvement in Chagas disease development. We have found that the mtDNA copy number was significantly lower in CCC than in heart tissue from healthy individuals, while blood mtDNA content was similar among asymptomatic Chagas disease, moderate, and severe CCC patients. An MtDNA haplogrouping study has indicated that African haplogroups were over represented in the Chagas subject groups in comparison with healthy Brazilian individuals. The European lineage is associated with protection against cardiomyopathy and the macro haplogroup H is associated with increased risk towards CCC. Using mitochondria DNA sequencing, 84 mtDNA-encoded protein sequence pathogenic variants were associated with CCC. Among them, two variants were associated to left ventricular non-compaction and two to hypertrophic cardiomyopathy. The finding that mitochondrial protein-coding SNPs and mitochondrial haplogroups associate with risk of evolving to CCC is consistent with a key role of mitochondrial DNA in the development of chronic chagas disease cardiomyopathy.

HLA-G Gene Variability Is Associated with Papillary Thyroid Carcinoma Morbidity and the HLA-G Protein Profile.

Human leukocyte antigen (HLA)-G is an immune checkpoint molecule that is highly expressed in papillary thyroid carcinoma (PTC). The HLA-G gene presents several functional polymorphisms distributed across the coding and regulatory regions (5'URR: 5' upstream regulatory region and 3'UTR: 3' untranslated region) and some of them may impact HLA-G expression and human malignancy. To understand the contribution of the HLA-G genetic background in PTC, we studied the HLA-G gene variability in PTC patients in association with tumor morbidity, HLA-G tissue expression, and plasma soluble (sHLA-G) levels. We evaluated 185 PTC patients and 154 healthy controls. Polymorphic sites defining coding, regulatory and extended haplotypes were characterized by sequencing analyses. HLA-G tissue expression and plasma soluble HLA-G levels were evaluated by immunohistochemistry and ELISA, respectively. Compared to the controls, the G0104a(5'URR)G*01:04:04(coding)UTR-03(3'UTR) extended haplotype was underrepresented in the PTC patients, while G0104a(5'URR)G*01:04:01(coding)UTR-03(3'UTR) was less frequent in patients with metastatic and multifocal tumors. Decreased HLA-G tissue expression and undetectable plasma sHLA-G were associated with the G010102a(5'URR)G*01:01:02:01(coding)UTR-02(3'UTR) extended haplotype. We concluded that the HLA-G variability was associated with PTC development and morbidity, as well as the magnitude of the encoded protein expression at local and systemic levels.

Epigenetic regulation of transcription factor binding motifs promotes Th1 response in Chagas disease cardiomyopathy.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.

Epigenetic regulation of transcription factor binding motifs promotes Th1 response in Chagas disease cardiomyopathy

Abstract: Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.

Plasma levels of soluble HLA-G and cytokines in papillary thyroid carcinoma before and after thyroidectomy.

BACKGROUND: Increasing evidence shows that chronic inflammation plays an important role in thyroid tumorigenesis. Cytokines as central mediators in inflammatory microenvironment can present both pro-tumour and anti-tumour effects and cytokine release may be influenced by soluble HLA-G (sHLA-G), an immune checkpoint molecule whose expression can also be induced by certain cytokines. AIM: To understand the role of these soluble factors in papillary thyroid cancer (PTC). METHODS: We evaluated plasma levels of sHLA-G and of 13 cytokines using ELISA and flow cytometry, respectively, in PTC patients at two time points: pre- and post-thyroidectomy; and control subjects. RESULTS: Compared with controls, IL-6 levels were increased, while IL-1ß, IFN-α and TGF-ß1 levels were decreased in pre-thyroidectomy PTC patients. IFN-α and TGF-ß1 efficiently discriminated patients from controls and were associated with extrathyroidal extension and lymph node metastasis, respectively. In addition, TNF and IL-13 were associated with male gender, lymph node metastasis and Hashimoto thyroiditis, and sHLA-G with tumour invasion. Compared with pre-thyroidectomy, IL-4, IL-10, TNF, IFN-α and TGF-ß1 levels were increased in post-thyroidectomy. CONCLUSION: There are significant changes in the cytokine profile after surgical removal of the thyroid tumour, and IFN-α e TGF-ß1 showed to be promising cytokines for discriminating PTC patients from controls. We also found that different cytokines are associated with clinicohistopathological characteristics of PTC related to poor prognosis, suggesting that cytokines seem to play an important role in PTC development and management.

Liver HLA-E Expression Is Associated with Severity of Liver Disease in Chronic Hepatitis C.

Hepatitis C virus (HCV) can escape from innate and adaptive immunity, making the immune response ineffective. Human leukocyte antigen E (HLA-E) might regulate the antiviral function of immune response and contribute to the persistence of HCV and the severity of liver disease. This study aimed to evaluate the expression of HLA-E in the liver and its association with the severity of liver disease in HCV patients. We performed a retrospective analysis of liver biopsies from 125 HCV patients and from 20 control subjects without liver disease. Liver biopsies were reviewed and classified according to severity of fibrosis and inflammatory activity. The pathologist assessed the magnitude of HLA-E expression in a semiquantitative way, attributing scores from 0 to 3. Immunohistochemistry showed positive for HLA-E in hepatocyte and Kupffer cells. The rate of HLA-E positivity in hepatocytes and Kupffer cells was significantly higher in HCV patients compared to controls. The liver samples classified as severe fibrosis and necroinflammatory activity presented greater expression of HLA-E on Kupffer cells and hepatocytes, with a significant linear association. It indicates that HLA-E expression may have an immunomodulatory effect and a possible role in the severity of liver disease in chronic hepatitis C.

Canonical PI3Kγ signaling in myeloid cells restricts Trypanosoma cruzi infection and dampens chagasic myocarditis.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi (T. cruzi) and is an important cause of severe inflammatory heart disease. However, the mechanisms driving Chagas disease cardiomyopathy have not been completely elucidated. Here, we show that the canonical PI3Kγ pathway is upregulated in both human chagasic hearts and hearts of acutely infected mice. PI3Kγ-deficient mice and mutant mice carrying catalytically inactive PI3Kγ are more susceptible to T. cruzi infection. The canonical PI3Kγ signaling in myeloid cells is essential to restrict T. cruzi heart parasitism and ultimately to avoid myocarditis, heart damage, and death of mice. Furthermore, high PIK3CG expression correlates with low parasitism in human Chagas' hearts. In conclusion, these results indicate an essential role of the canonical PI3Kγ signaling pathway in the control of T. cruzi infection, providing further insight into the molecular mechanisms involved in the pathophysiology of chagasic heart disease.

The genetic diversity within the 1.4 kb HLA-G 5' upstream regulatory region moderately impacts on cellular microenvironment responses.

The HLA-G 5'URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5'URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5'URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5'URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5'URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-ß and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5'URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5'URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.

Human Leukocyte Antigen-G: A Promising Prognostic Marker of Disease Progression to Improve the Control of Human African Trypanosomiasis.

BACKGROUND: Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense can be diagnosed in the early hemolymphatic stage (stage 1 [S1]) or meningoencephalitic stage (stage 2 [S2]). Importantly, individuals harbouring high and specific antibody responses to Tbg antigens but negative parasitology are also diagnosed in the field (seropositive [SERO]). Whereas some develop the disease in the months following their initial diagnosis (SERO/HAT), others remain parasitologically negative for long periods (SERO) and are apparently able to control infection. Human leucocyte antigen (HLA)-G, an immunosuppressive molecule, could play a critical role in this variability of progression between infection and disease. METHODS: Soluble HLA-G (sHLA-G) was measured in plasma for patients in the SERO (n = 65), SERO/HAT (n = 14), or HAT (n = 268) group and in cerebrospinal fluid for patients in S1 (n = 55), early S2 (n = 93), or late S2 (n = 110). Associations between these different statuses and the soluble level or genetic polymorphisms of HLA-G were explored. RESULTS: Plasma sHLA-G levels were significantly higher in HAT (P = 6 × 10-7) and SERO/HAT (P = .007) than SERO patients. No difference was observed between the SERO/HAT and HAT groups. Within the HAT group, specific haplotypes (HG010102 and HG0103) displayed increased frequencies in S1 (P = .013) and late S2 (P = .036), respectively. CONCLUSIONS: These results strongly suggest the involvement of HLA-G in HAT disease progression. Importantly, high plasma sHLA-G levels in SERO patients could be predictive of subsequent disease development and could represent a serological marker to help guide therapeutic decision making. Further studies are necessary to assess the predictive nature of HLA-G and to estimate both sensitivity and specificity.

HLA-E coding and 3' untranslated region variability determined by next-generation sequencing in two West-African population samples.

HLA-E is a non-classical Human Leucocyte Antigen class I gene with immunomodulatory properties. Whereas HLA-E expression usually occurs at low levels, it is widely distributed amongst human tissues, has the ability to bind self and non-self antigens and to interact with NK cells and T lymphocytes, being important for immunosurveillance and also for fighting against infections. HLA-E is usually the most conserved locus among all class I genes. However, most of the previous studies evaluating HLA-E variability sequenced only a few exons or genotyped known polymorphisms. Here we report a strategy to evaluate HLA-E variability by next-generation sequencing (NGS) that might be used to other HLA loci and present the HLA-E haplotype diversity considering the segment encoding the entire HLA-E mRNA (including 5'UTR, introns and the 3'UTR) in two African population samples, Susu from Guinea-Conakry and Lobi from Burkina Faso. Our results indicate that (a) the HLA-E gene is indeed conserved, encoding mainly two different protein molecules; (b) Africans do present several unknown HLA-E alleles presenting synonymous mutations; (c) the HLA-E 3'UTR is quite polymorphic and (d) haplotypes in the HLA-E 3'UTR are in close association with HLA-E coding alleles. NGS has proved to be an important tool on data generation for future studies evaluating variability in non-classical MHC genes.

Human leucocyte antigen-G (HLA-G) and its murine functional homolog Qa2 in the Trypanosoma cruzi Infection.

Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3' untranslated region (3'UTR) polymorphic sites (associated with mRNA stability and target for microRNA binding) and HLA-G tissue expression (heart, colon, and esophagus) in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3'UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate). HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, and NOS-2) genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection.

The Role of HLA-G Molecule and HLA-G Gene Polymorphisms in Tumors, Viral Hepatitis, and Parasitic Diseases.

Considering that the non-classical HLA-G molecule has well-recognized tolerogenic properties, HLA-G expression is expected to be deleterious when present in tumor cells and in cells chronically infected by viruses, whereas HLA-G expression is expected to be advantageous in autoimmune disorders. The expression of HLA-G on tissue or peripheral blood cells, the levels of soluble HLA-G and polymorphic sites along the gene have been studied in several disorders. In this study, we revised the role of the molecule and polymorphic sites along the HLA-G gene in tumors, viral hepatitis, and parasitic disorders. Overall, several lines of evidence clearly show that the induction of HLA-G expression in tumors has been associated with worse disease outcome and disease spread. In addition, the few studies conducted on hepatitis and parasitic disorders indicate that HLA-G may contribute to disease pathogenesis. Few isolated polymorphic sites, primarily located at the coding or 3' untranslated HLA-G region, have been evaluated in these disorders, and a complete HLA-G typing together with the study of gene regulatory elements may further help on the understanding of the influence of the genetic background on disease susceptibility.

Polymorphic sites at the immunoregulatory CTLA-4 gene are associated with chronic chagas disease and its clinical manifestations.

BACKGROUND: Chagas disease affects approximately 10 million people mainly in Latin America. The immune regulation by the host seems to be an essential factor for disease evolution, and immune system inhibitory molecules such as CTLA-4 and PD-1 favor the maintenance of peripheral tolerance. Considering that polymorphisms at the immunoregulatory CTLA-4 and PDCD1 genes may alter their inhibitory function, we investigated the association of alleles, genotypes and haplotypes of polymorphic sites observed at the CTLA-4 and PDCD1 genes with different clinical manifestations of chronic Chagas disease (indeterminate, cardiac, digestive and mixed). METHODS: The polymorphisms at the CTLA-4 (-1722T/C, -318C/T and +49A/G) and PDCD1 (PD-1.3G/A) genes were typed using TaqMan methodology in 277 chronic Chagas disease patients classified into four groups, according to clinical characteristics, and 326 non-infected controls. RESULTS: Our results showed that CTLA-4 -1722CC genotype (22%), -1722C allele (27%) and CTLA-4 TCG (8.6%), TCA (26%) and CCA (15%) haplotypes were strongly associated with the indeterminate form, while the CTLA-4-318CT genotype (82%) and CTLA-4-318T allele (47%) were found mainly in patients with the mixed form of the disease. The CTLA-4 TCG haplotype (10.2%) was associated with the digestive form. On the other hand, the PD-1.3G/A polymorphism was not associated with chronic Chagas disease and its clinical manifestations. CONCLUSIONS: Here, we showed that alleles, genotypes and haplotypes reported to increase the expression of the regulatory molecule CTLA-4 were associated with the indeterminate form of the disease. Taken together, our data support the idea that polymorphic sites at immunoregulatory genes may influence the development of Chagas disease variants.

Galectin-3 negatively regulates the frequency and function of CD4(+) CD25(+) Foxp3(+) regulatory T cells and influences the course of Leishmania major infection.

Galectin-3, an endogenous glycan-binding protein, plays essential roles during microbial infection by modulating innate and adaptive immunity. However, the role of galectin-3 within the CD4(+) CD25(+) Foxp3(+) T regulatory (TREG ) cell compartment has not yet been explored. Here, we found, in a model of Leishmania major infection, that galectin-3 deficiency increases the frequency of peripheral TREG cells both in draining lymph nodes (LNs) and sites of infection. These observations correlated with an increased severity of the disease, as shown by increased footpad swelling and parasite burden. Galectin-3-deficient (Lgals3(-/-) ) TREG cells displayed higher CD103 expression, showed greater suppressive capacity, and synthesized higher amounts of IL-10 compared with their wild-type (WT) counterpart. Furthermore, both TREG cells and T effector (TEFF ) cells from Lgals3(-/-) mice showed higher expression of Notch1 and the Notch target gene Hes-1. Interestingly, Notch signaling components were also altered in both TREG and TEFF cells from uninfected Lgals3(-/-) mice. Thus, endogenous galectin-3 regulates the frequency and function of CD4(+) CD25(+) Foxp3(+) TREG cells and alters the course of L. major infection.

Genome survey sequence analysis and identification of homologs of major surface protease (gp63) genes in Trypanosoma rangeli.

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

Organization of H locus conserved repeats in Leishmania (Viannia) braziliensis correlates with lack of gene amplification and drug resistance.

Resistance to antimonials is a major problem when treating visceral leishmaniasis in India and has already been described for New World parasites. Clinical response to meglumine antimoniate in patients infected with parasites of the Viannia sub-genus can be widely variable, suggesting the presence of mechanisms of drug resistance. In this work, we have compared L. major and L. braziliensis mutants selected in different drugs. The cross-resistance profiles of some cell lines resembled those of mutants bearing H locus amplicons. However, amplified episomal molecules were exclusively detected in L. major mutants. The analysis of the L. braziliensis H region revealed a strong conservation of gene synteny. The typical intergenic repeats that are believed to mediate the amplification of the H locus in species of the Leishmania sub-genus are partially conserved in the Viannia species. The conservation of these non-coding elements in equivalent positions in both species is indicative of their relevance within this locus. The absence of amplicons in L. braziliensis suggests that this species may not favour extra-chromosomal gene amplification as a source of phenotypic heterogeneity and fitness maintenance in changing environments.

Specificity of modified Drosophila mariner transposons in the identification of Leishmania genes.

Genetic manipulation of the protozoan Leishmania has led to a better understanding of the survival and development of these pathogens within their hosts. The association of the Leishmania genome sequencing information with the ability of transposons to introduce or destroy phenotypes allows a global perspective on the role and importance of genes in cellular pathways. Herein we report the construction and testing of mariner transposable elements carrying the neomycin phosphotransferase, green fluorescent protein, or beta-glucuronidase genes as reporters for translational fusion events. We demonstrate that the expression of the reporter genes will occur only when the genes are inserted in-frame within predicted genes. Our results not only add to the mariner toolkit for gene manipulation but also strengthen the evidence that the mariner system is a reliable means for the study of gene expression in Leishmania.