Inhibition of Foxp3 expression in the placenta of mice infected intraperitoneally by toxoplasma gondii tachyzoites: insights into the PPARγ/miR-7b-5p/Sp1 signaling pathway.

BACKGROUND: Toxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii. METHODS: Quantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining. To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells. RESULTS: In T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site. CONCLUSIONS: T. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.

Association of polymorphism of IL-17A, IL-17F, and IL-6 with Toxoplasma gondii infection susceptibility in HIV/AIDS patients in Shiraz, southern Iran.

INTRODUCTION: Toxoplasma gondii infection is considered as one of the most important opportunistic infections and cause of death in HIV patients. METHODS: In this cross-sectional study, 334 HIV positive patients were included. The molecular test was performed by the restriction fragment length polymorphism-polymerase chain reaction method. Allelic frequency, haplotype analyses, and linkage disequilibrium were calculated. The odds ratio was calculated. The linear regression model was used to analysis of interleukin (IL)-17A, IL-17F, and IL-6 single-nucleotide polymorphism genotypes in HIV patients with and without toxoplasmosis. RESULTS: In total, 95 tested'patients (28.4%) were positive for toxoplasmosis. The risk of toxoplasma infection in the current study did not correlate with IL-17 and IL-6 polymorphism and the risk of contracting toxoplasma was also not significantly correlated in this study. There was no association between the frequency of alleles and the risk of toxoplasma infection in IL-17 haplotype analysis. CONCLUSION: The findings of this study revealed that there were significant differences in the serum levels of IL-6 and IL-17A, but not IL-17F, between the case and control groups in various genetic models. However, these polymorphisms did not show a significant relationship with toxoplasma infection in HIV-positive patients. This study represents the first investigation in Iran to explore the role of IL-6 and IL-17 polymorphisms in toxoplasma infection among HIV-positive patients.

Transcriptomic profiling of long non-coding RNAs and messenger RNAs in the liver of mice during Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan parasite that can infect a wide range of warm-blooded animals, including humans. It poses significant health risks, particularly in immunocompromised individuals and during pregnancy, leading to severe disease manifestations. The liver, being a crucial organ involved in immune response and metabolic regulation, plays a critical role in the host's defense against T. gondii infection. METHODS: In this study, we utilized RNA sequencing to investigate the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in the liver of mice infected with T. gondii. By employing this method, we obtained a comprehensive overview of the alterations in gene expression occurring in the liver during infection. RESULTS: By comparing the infected groups to the control groups, we identified numerous differentially expressed lncRNAs DElncRNAs and DEmRNAs at two stages of infection. Specifically, at the acute infection stage, we found 628 DElncRNAs, and 6346 DEmRNAs. At the chronic infection stage, we identified 385 DElncRNAs and 2513 DEmRNAs. Furthermore, we identified 1959 commonly expressed DEmRNAs, including IL27, Nos2, and Cxcr2, across two infection stages. Enrichment and co-location analyses revealed pathways linked to immune and inflammatory responses during T. gondii infection. Notably, through co-location analysis, our analysis revealed several DElncRNAs, including Gm29156, Gm29157, and Gm28644, which are potentially implicated in the progression of liver inflammation induced by T. gondii. Additionally, functional enrichment analysis disclosed stage-specific characteristics of liver inflammation and immune response, alongside changes in metabolic regulation and immunosuppression pathways. CONCLUSIONS: Our findings provide valuable insights into the expression patterns of lncRNAs and mRNAs in the liver at different stages of T. gondii infection. We identified potential regulatory factors and pathways implicated in liver inflammation, thereby enhancing our understanding of the molecular mechanisms underlying liver inflammation and immune responses during T. gondii infection. These findings could contribute to the development of targeted therapeutic strategies for liver inflammation in the context of T. gondii infection.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

Toxoplasma gondii infection regulates apoptosis of host cells via miR-185/ARAF axis.

BACKGROUND: Toxoplasmosis is a zoonosis with a worldwide presence that is caused by the intracellular parasite Toxoplasma gondii. Active regulation of apoptosis is an important immune mechanism by which host cells resist the growth of T. gondii or avoid excessive pathological damage induced by this parasite. Previous studies found that upregulated expression of microRNA-185 (miR-185) during T. gondii infection has a potential role in regulating the expression of the ARAF gene, which is reported to be associated with cell proliferation and apoptosis. METHODS: The expression levels of miR-185 and the ARAF gene were evaluated by qPCR and Western blot, respectively, in mice tissues, porcine kidney epithelial cells (PK-15) and porcine alveolar macrophages (3D4/21) following infection with the T. gondii ToxoDB#9 and RH strains. The dual luciferase reporter assay was then used to verify the relationship between miR-185 and ARAF targets in PK-15 cells. PK-15 and 3D4/21 cell lines with stable knockout of the ARAF gene were established by CRISPR, and then the apoptosis rates of the cells following T. gondii infection were detected using cell flow cytometry assays. Simultaneously, the activities of cleaved caspase-3, as a key apoptosis executive protein, were detected by Western blot to evaluate the apoptosis levels of cells. RESULTS: Infection with both the T. gondii ToxoDB#9 and RH strains induced an increased expression of miR-185 and a decreased expression of ARAF in mice tissues, PK-15 and 3D4/21 cells. MiR-185 mimic transfections showed a significantly negative correlation in expression levels between miR-185 and the ARAF gene. The dual luciferase reporter assay confirmed that ARAF was a target of miR-185. Functional investigation revealed that T. gondii infection induced the apoptosis of PK-15 and 3D4/21 cells, which could be inhibited by ARAF knockout or overexpression of miR-185. The expression levels of cleaved caspase-3 protein were significantly lower in cells with ARAF knockout than in normal cells, which were consistent with the results of the cell flow cytometry assays. CONCLUSIONS: Toxoplasma gondii infection could lead to the upregulation of miR-185 and the downregulation of ARAF, which was not related to the strain of T. gondii and the host cells. Toxoplasma gondii infection could regulate the apoptosis of host cells via the miR-185/ARAF axis, which represents an additional strategy used by T. gondii to counteract host-cell apoptosis in order to maintain survival and reproduce in the host cells.

LILRB4 regulates the function of decidual MDSCs via the SHP-2/STAT6 pathway during Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii infection can cause adverse pregnancy outcomes, such as recurrent abortion, fetal growth restriction and infants with malformations, among others. Decidual myeloid-derived suppressor cells (dMDSCs) are a novel immunosuppressive cell type at the fetal-maternal interface which play an important role in sustaining normal pregnancy that is related to their high expression of the inhibitory molecule leukocyte immunoglobulin-like receptor B4 (LILRB4). It has been reported that the expression of LILRB4 is downregulated on decidual macrophages after T. gondii infection, but it remains unknown whether T. gondii infection can induce dMDSC dysfunction resulting from the change in LILRB4 expression. METHODS: LILRB4-deficient (LILRB4-/-) pregnant mice infected with T. gondii with associated adverse pregnancy outcomes, and anti-LILRB4 neutralized antibodies-treated infected human dMDSCs were used in vivo and in vitro experiments, respectively. The aim was to investigate the effect of LILRB4 expression on dMDSC dysfunction induced by T. gondii infection. RESULTS: Toxoplasma gondii infection was observed to reduce STAT3 phosphorylation, resulting in decreased LILRB4 expression on dMDSCs. The levels of the main functional molecules (arginase-1 [Arg-1], interleukin-10 [IL-10]) and main signaling molecules (phosphorylated Src-homology 2 domain-containing protein tyrosine phosphatase [p-SHP2], phosphorylated signal transducer and activator of transcription 6 [p-STAT6]) in dMDSCs were all significantly reduced in human and mouse dMDSCs due to the decrease of LILRB4 expression induced by T. gondii infection. SHP-2 was found to directly bind to STAT6 and STAT6 to bind to the promoter of the Arg-1 and IL-10 genes during T. gondii infection. CONCLUSIONS: The downregulation of LILRB4 expression on dMDSCs induced by T. gondii infection could regulate the expression of Arg-1 and IL-10 via the SHP-2/STAT6 pathway, resulting in the dysfunction of dMDSCs, which might contribute to adverse outcomes during pregnancy by T. gondii infection.

TgMIF Promotes Hepatocyte Pyroptosis and Recruitment of Proinflammatory Macrophages During Severe Liver Injury in Acute Toxoplasmosis.

Liver injury is a common complication during infection of Toxoplasma gondii. However, the Toxoplasma effector proteins involved remain unknown. Herein, we identified that T. gondii macrophage migration inhibitory factor (TgMIF) is a critical pathogenic factor of liver injury in acute toxoplasmosis mouse model induced by a less virulent strain, which is widely prevalent in humans. We show that TgMIF is a novel activator of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in hepatocytes, resulting in subsequent pyroptosis. Furthermore, T. gondii promotes the TgMIF-dependent infiltration of Ly6Chi proinflammatory macrophages to release cytokines, leading to hepatocyte apoptosis. Although the intense inflammation induced by TgMIF inhibits the proliferation of intracellular parasites, it results in fatal liver damage. In contrast, parasites with TgMIF gene deletion significantly alleviate liver injury and prolong mice survival. The discovery of novel Toxoplasma virulence factor may expedite the development of human toxoplasmosis control strategies.

Comprehensive analysis of mRNA-lncRNA co-expression profiles in mouse brain during infection with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite which seriously threatens the health of domestic animals and humans. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts greater than 200 nucleotides, which are widely involved in transcriptional and epigenetic regulations. However, little is known about the roles of host lncRNAs in the response to T. gondii infections. In this study, using Illumina sequencing technology, we analyzed the expression profiles of mRNAs and lncRNAs in BALB/c mouse brain following infection by T. gondii PRU strain (type II genotype) cysts. The identified differentially expressed (DE) RNAs were subjected to bioinformatics analysis. A total of 2,090 annotated lncRNAs along with 3,577 novel lncRNAs were identified. In the acutely infected mouse brain, a total of 330 mRNAs and 19 lncRNAs were dys-regulated, whereas 136 DE mRNAs and 9 DE lncRNAs were identified in chronically infected mouse brain. GO analysis revealed that these DE mRNAs identified at acute infection stage were involved in immune response, whereas DE mRNAs found at chronic infection stage were mostly enriched in response to protozoan. KEGG analysis showed that DE mRNAs were significantly enriched in disease related pathways. In addition, the putative mRNA-lncRNA co-expression network was constructed, and several hub regulatory RNAs were identified based on the transcriptome data. This study firstly characterized the co-expression profile of mRNAs and lncRNAs in mouse brain infected with T. gondii and provided a framework for further studies of the roles of lncRNAs in host neuropathology during toxoplasmosis progression.

CircRNA and miRNA expression analysis in livers of mice with Toxoplasma gondii infection.

Toxoplasmosis is an important zoonotic parasitic disease caused by Toxoplasma gondii (T. gondii). However, the functions of circRNAs and miRNAs in response to T. gondii infection in the livers of mice at acute and chronic stages remain unknown. Here, high-throughput RNA sequencing was performed for detecting the expression of circRNAs and miRNAs in livers of mice infected with 20 T. gondii cysts at the acute and chronic stages, in order to understand the potential molecular mechanisms underlying hepatic toxoplasmosis. Overall, 265 and 97 differentially expressed (DE) circRNAs were found in livers at the acute and chronic infection stages in comparison with controls, respectively. In addition, 171 and 77 DEmiRNAs were found in livers at the acute and chronic infection stages, respectively. Functional annotation showed that some immunity-related Gene ontology terms, such as "positive regulation of cytokine production", "regulation of T cell activation", and "immune receptor activity", were enriched at the two infection stages. Moreover, the pathways "Valine, leucine, and isoleucine degradation", "Fatty acid metabolism", and "Glycine, serine, and threonine metabolism" were involved in liver disease. Remarkably, DEcircRNA 6:124519352|124575359 was significantly correlated with DEmiRNAs mmu-miR-146a-5p and mmu-miR-150-5p in the network that was associated with liver immunity and pathogenesis of disease. This study revealed that the expression profiling of circRNAs in the livers was changed after T. gondii infection, and improved our understanding of the transcriptomic landscape of hepatic toxoplasmosis in mice.

Genomic insights into host and parasite interactions during intracellular infection by Toxoplasma gondii.

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii. We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T. gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite's genome, including more accurate mapping of known genes and the identification of new genes and cis-regulatory elements. Motif analysis reveals not only the known T. gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T. gondii's perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T. gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

Toxoplasma gondii Seropositivity Interacts with Catechol-O-methyltransferase Val105/158Met Variation Increasing the Risk of Schizophrenia.

Schizophrenia is a heterogeneous and severe psychotic disorder. Epidemiological findings have suggested that the exposure to infectious agents such as Toxoplasma gondii (T. gondii) is associated with an increased risk for schizophrenia. On the other hand, there is evidence involving the catechol-O-methyltransferase (COMT) Val105/158Met polymorphism in the aetiology of schizophrenia since it alters the dopamine metabolism. A case−control study of 141 patients and 142 controls was conducted to analyse the polymorphism, the prevalence of anti-T. gondii IgG, and their interaction on the risk for schizophrenia. IgG were detected by ELISA, and genotyping was performed with TaqMan Real-Time PCR. Although no association was found between any COMT genotype and schizophrenia, we found a significant association between T. gondii seropositivity and the disorder (χ2 = 11.71; p-value < 0.001). Furthermore, the risk for schizophrenia conferred by T. gondii was modified by the COMT genotype, with those who had been exposed to the infection showing a different risk compared to that of nonexposed ones depending on the COMT genotype (χ2 for the interaction = 7.28, p-value = 0.007). This study provides evidence that the COMT genotype modifies the risk for schizophrenia conferred by T. gondii infection, with it being higher in those individuals with the Met/Met phenotype, intermediate in heterozygous, and lower in those with the Val/Val phenotype.

The Core Human MicroRNAs Regulated by Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii (T. gondii) is an intracellular zoonotic protozoan parasite known to effectively modulate the host system for its survival. A large number of microRNAs (miRNAs) regulated by different strains of T. gondii in diverse types of host cells/tissues/organs have been reported across multiple studies. OBJECTIVE: We aimed to decipher the complexity of T. gondii regulated spectrum of miRNAs to derive a set of core miRNAs central to different strains of T. gondii infection in diverse human cell lines. METHODS: We first assembled miRNAs hat are regulated by T. gondii altered across the various assortment of infections and time points of T. gondii infection in multiple cell types. For these assembled datasets, we employed specific criteria to filter the core miRNAs regulated by T. gondii. Subsequently, accounting for the spectrum of miRNA-mRNA target combinations, we applied a novel confidence criterion to extract their core experimentally-validated mRNA targets in human cell systems. RESULTS: This analysis resulted in the extraction of 74 core differentially regulated miRNAs and their 319 high-confidence mRNA targets. Based on these core miRNA-mRNA pairs, we derived the central biological processes perturbed by T. gondii in diverse human cell systems. Further, our analysis also resulted in the identification of novel autocrine/paracrine signalling factors that could be associated with host response modulated by T. gondii. CONCLUSION: The current analysis derived a set of core miRNAs, their targets, and associated biological processes fine-tuned by T. gondii for its survival within the invaded cells.

Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis.

Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)|-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.

Toxoplasma gondii promotes microRNA-34a to inhibit Foxp3 expression in adverse outcomes of pregnancy in mice.

Infection with Toxoplasma gondii (T. gondii) during the pregnant period causes microcephaly, mental and psychomotor retardation or death. Miserable outcomes are mainly linked with regulatory T cells (Tregs) dysfunction. Forkhead box P3 (Foxp3), a vital regulator of establishment and maintenance of Tregs, can be modulated by microRNAs (miRNAs). Previously, our study revealed that T. gondii infection in pregnant mice induced Tregs dysfunction, accompanied with reduced Foxp3 expression. The role of miRNAs in the inhibition of Foxp3 triggered by T. gondii remains unclear. Herein, T. gondii infection promotes miR-34a expression in the placenta of mice. miR-34a mimic inhibits Foxp3 expression via targeting 3' untranslated region (3'UTR) whereas its inhibitor promotes Foxp3 expression in vitro. T. gondii antigens could enhance the activity of miR-34a promoter via a Smad4-dependent mechanism. Collectively, our data reveal a new avenue through which T. gondii inhibits Foxp3 expression necessary to drive adverse outcomes of pregnancy in mice.

Cluster analysis of splenocyte microRNAs in the pig reveals key signal regulators of immunomodulation in the host during acute and chronic Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that can cause a geographically widespread zoonosis. Our previous splenocyte microRNA profile analyses of pig infected with T. gondii revealed that the coordination of a large number of miRNAs regulates the host immune response during infection. However, the functions of other miRNAs involved in the immune regulation during T. gondii infection are not yet known. METHODS: Clustering analysis was performed by K-means, self-organizing map (SOM), and hierarchical clustering to obtain miRNA groups with the similar expression patterns. Then, the target genes of the miRNA group in each subcluster were further analyzed for functional enrichment by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway to recognize the key signaling molecules and the regulatory signatures of the innate and adaptive immune responses of the host during T. gondii infection. RESULTS: A total of 252 miRNAs were successfully divided into 22 subclusters by K-means clustering (designated as K1-K22), 29 subclusters by SOM clustering (designated as SOM1-SOM29), and six subclusters by hierarchical clustering (designated as H1-H6) based on their dynamic expression levels in the different infection stages. A total of 634, 660, and 477 GO terms, 15, 26, and 14 KEGG pathways, and 16, 15, and 7 Reactome pathways were significantly enriched by K-means, SOM, and hierarchical clustering, respectively. Of note, up to 22 miRNAs mainly showing downregulated expression at 50 days post-infection (dpi) were grouped into one subcluster (namely subcluster H3-K17-SOM1) through the three algorithms. Functional analysis revealed that a large group of immunomodulatory signaling molecules were controlled by the different miRNA groups to regulate multiple immune processes, for instance, IL-1-mediated cellular response and Th1/Th2 cell differentiation partly depending on Notch signaling transduction for subclusters K1 and K2, innate immune response involved in neutrophil degranulation and TLR4 cascade signaling for subcluster K15, B cell activation for subclusters SOM17, SOM1, and SOM25, leukocyte migration, and chemokine activity for subcluster SOM9, cytokine-cytokine receptor interaction for subcluster H2, and interleukin production, chemotaxis of immune cells, chemokine signaling pathway, and C-type lectin receptor signaling pathway for subcluster H3-K17-SOM1. CONCLUSIONS: Cluster analysis of splenocyte microRNAs in the pig revealed key regulatory properties of subcluster miRNA molecules and important features in the immune regulation induced by acute and chronic T. gondii infection. These results contribute new insight into the identification of physiological immune responses and maintenance of tolerance in pig spleen tissues during T. gondii infection.

The association between serum level and genetic variation of IL-3 (rs40401) in recurrent abortion women infected with toxoplasmosis in Iraq.

Toxoplasmosis is one of the most infections during pregnancy transmitted from mother to child via the placenta and causes morbidity and mortality prenatally. IL-3 is a hematopoiesis enhancing factor and assist in the implantation embryo and placental growth. The objective of this study is to provide valuable information about the risks of IL-3 elevated levels in aborted women with toxoplasmosis by determining the risk, or the protective role of alleles or genotypes for single nucleotide polymorphism (SNP) of IL-3 (rs40401) which might have a relationship with the susceptibility to toxoplasmosis. The levels of IL-3 in blood samples of patients and controls were detected by ELISA while the allelic discrimination method was used for SNP IL-3 (rs40401). The results suggested the IL-3 serum concentration of healthy pregnant women(HP), recurrent abortion women without toxoplasmosis (RAWOT), and recurrent abortion women with toxoplasmosis(RAWT) was declined versus healthy non pregnant women (HNP) with a statistically significant difference (p < 0.05). Moreover, the results SNP of IL-3 showed no significant association between patients and controls. Considering the distribution of serum levels for IL-3 by SNPs, the results have shown the serum level of IL-3 for the genotype CC, CT, TT in RAWOT and RAWT declined comparing to HNP women with significant differences (p < 0.05). In conclusion, SNP of IL-3 has not represented as a risk factor in recurrent abortion women with toxoplasmosis. Although the serum levels of IL-3 differed in patients according to genotype with significant differences compared to control.

Expression of Interleukin-33 Gene in Hemodialysis Patients with Chronic Toxoplasmosis in Baghdad, Iraq.

Toxoplasmosis is a protozoan parasite with high distribution, leading to different abnormalities in hosts. The present study aimed to determine the distribution of toxoplasmosis in hemodialysis patients and the expression of the Interleukin (IL)-33 gene in chronic Toxoplasmosis. The present study evaluated 120 subjects, including 60 patients who were undergoing dialysis and 60 healthy samples as the control group, from the 1st February to 1st November 2021. Anti-Toxoplasma gondii IgG was detected by using the enzyme-linked immunosorbent assay (ELISA) technique and the real-time polymerase-chain-reaction (PCR) was used to perform IL-33. The results demonstrated that the highest anti-toxoplasmosis IgG antibody rate was observed in the age group 51-70 years who were undergoing dialysis, in comparison with that in the control group (P<0.05). The male patients who had anti-toxoplasmosis IgG antibodies outnumbered the healthy people (P<0.05), while the female patients did not significantly differ from the healthy group. Chronic toxoplasmosis showed a higher number according to residency (the urban and rural patients), compared to healthy people. The frequency of dialysis times per week in chronic Toxoplasmosis patients was significantly higher among the infected patients. The findings were displayed to be positive in dialysis at 2 weeks (P<0.05). The expression of the IL-33 gene was investigated in patients who were undergoing hemodialysis and in healthy controls by using real-time PCR. The findings demonstrated that there was a high Ct value for patients and controls with a high Ct value of templates, preoperational to the gene concentration. The high prevalence of toxoplasmosis in dialysis patients and the role of IL-33 in cellular immunity in these patients highlight the need to investigate the mechanisms limiting infection with intracellular protozoa.

Association between genetic polymorphism of IL-27 (rs153109) and toxoplasmosis in Iraqi women with recurrent abortion.

Single nucleotide polymorphisms (SNPs) are predictive markers for diseases, also cytokines are undergoing genetic controls and their genetic polymorphisms have a functional role in regulating the levels of cytokine gene expression. This study aims to reveal the association of toxoplasmosis with serum levels and SNP of IL-27 in aborted women. Total, 200 blood samples of patients and controls were collected from Al-Alawiya Maternity Teaching Hospital/Baghdad/Iraq from 2019­2020 for detecting the level of IL-27 by ELISA while the allelic discrimination method was used for SNP IL-27 (rs153109). The results indicated the IL-27 serum concentration elevated with significant differences in recurrent abortion with toxoplasmosis group compared to healthy women, pregnant women, and recurrent abortion. Also, recurrent abortion had significant differences compared to healthy women and pregnant women (P<0.05). Moreover, SNP results of IL-27 showed no significant association between patients and controls. Considering the distribution of serum levels for IL-27 by SNP, it was observed that IL-27 serum levels for TT, TC, and CC genotypes elevated in the patient group versus the control group. In addition, it was observed elevation serum level of IL-27 for the genotypes TT, TC, and CC in recurrent abortion with toxoplasmosis in contrast to healthy women, pregnant women, and recurrent abortion (P<0.05). Also, in recurrent abortion, the level of IL-27 for TC, and CC genotype showed significant differences comparing to healthy and pregnant women (P<0.05). In conclusion, the level of IL-27 in recurrent abortion women with toxoplasmosis was higher than the recurrent abortion women, which may be due to the inflammatory response to toxoplasmosis. SNP of IL-27 has not represented as a risk factor in recurrent abortion women with toxoplasmosis.

Genetic Analyses of Common Infections in the Avon Longitudinal Study of Parents and Children Cohort.

The burden of infections on an individual and public health is profound. Many observational studies have shown a link between infections and the pathogenesis of disease; however a greater understanding of the role of host genetics is essential. Children from the longitudinal birth cohort, the Avon Longitudinal Study of Parents and Children, had 14 antibodies measured in plasma at age 7: Alpha-casein protein, beta-casein protein, cytomegalovirus, Epstein-Barr virus, feline herpes virus, Helicobacter pylori, herpes simplex virus 1, influenza virus subtype H1N1, influenza virus subtype H3N2, measles virus, Saccharomyces cerevisiae, Theiler's virus, Toxoplasma gondii, and SAG1 protein domain, a surface antigen of Toxoplasma gondii measured for greater precision. We performed genome-wide association analyses of antibody levels against these 14 infections (N = 357 - 5010) and identified three genome-wide signals (P < 5×10-8), two associated with measles virus antibodies and one with Toxoplasma gondii antibodies. In an association analysis focused on the human leukocyte antigen (HLA) region of the genome, we further detected 15 HLA alleles at a two-digit resolution and 23 HLA alleles at a four-digit resolution associated with five antibodies, with eight HLA alleles associated with Epstein-Barr virus antibodies showing strong evidence of replication in UK Biobank. We discuss how our findings from antibody levels complement other studies using self-reported phenotypes in understanding the architecture of host genetics related to infections.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention.