Consolidation of a Molecular Signature of Healing in Cutaneous Leishmaniasis Is Achieved during the First 10 Days of Treatment.

The immune response is central to the pathogenesis of cutaneous leishmaniasis (CL). However, most of our current understanding of the immune response in human CL derives from the analysis of systemic responses, which only partially reflect what occurs in the skin. In this study, we characterized the transcriptional dynamics of skin lesions during the course of treatment of CL patients and identified gene signatures and pathways associated with healing and nonhealing responses. We performed a comparative transcriptome profiling of serial skin lesion biopsies obtained before, in the middle, and at the end of treatment of CL patients (eight who were cured and eight with treatment failure). Lesion transcriptomes from patients who healed revealed recovery of the stratum corneum, suppression of the T cell-mediated inflammatory response, and damping of neutrophil activation, as early as 10 d after initiation of treatment. These transcriptional programs of healing were consolidated before lesion re-epithelization. In stark contrast, downregulation of genes involved in keratinization was observed throughout treatment in patients who did not heal, indicating that in addition to uncontrolled inflammation, treatment failure of CL is mediated by impaired mechanisms of wound healing. This work provides insights into the factors that contribute to the effective resolution of skin lesions caused by Leishmania (Viannia) species, sheds light on the consolidation of transcriptional programs of healing and nonhealing responses before the clinically apparent resolution of skin lesions, and identifies inflammatory and wound healing targets for host-directed therapies for CL.

Cytokines signatures and susceptibility to cutaneous leishmaniasis in patients from Sistan and Baluchestan province of Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) is a complex, multifactorial disease that results from environmental factors such as parasite polymorphism, phlebotomine vectors, and host genetic factors. Some studies have identified specific genetic factors that may be associated with cutaneous leishmaniasis. The objective of this research was to resolve the association of 8 cytokine polymorphisms, including TNF-α -308 A/G (rs 1800629), TNF-α -238 A/G (rs 361525), TGF-ß1 -509 T/C (rs 1800469), TGF-ß1+ 915 G/C (rs 1800471), IFN-γ -874 T/A (rs 2430561), IFN-γ -179 G/A (rs 2069709), IL-10 -819 C/T (rs 1800871), and IL-10 -592 A/C (rs 1800872) with susceptibility to CL. METHODS: A total of 152 patients with designated CL and 100 healthy controls were selected from those referred to Sistan and Baluchestan hospitals. CL was diagnosed by microscopic examination of Giemsa-stained samples and culture. Leishmania species were identified using ITS2 gene PCR amplification with universal primers. Genetic polymorphism was determined by the ARMS PCR method on extracted genomic DNA of individuals. Eight SNPs cytokines were genotyped. RESULTS: Most of the Genotypic and allelic frequency comparisons between patients with CL and healthy subjects showed no difference, except 3. Individual SNP analysis showed highest association of TGF-ß1 -509 (rs1800469) -CC genotype (P = 0.03, OR = 7.05, 95 % CI = 3.3-15) with 5.7-fold increase, IFN-γ -874 (rs 2430561) -AA genotype (P = 0.04, OR = 4.72, 95 % CI = 1.6-14) with 4.2-fold increase, and IL10 -819 (rs1800871) -CC genotype (P = 0.05, OR = 3.63, 95 % CI = 2.5-5.3) with 1.9-fold increase, with CL. Odds ratios (ORs) and 95 % confidence intervals (CIs) were evaluated to assess the association power. CONCLUSION: Our results conclude that rs1800469 (TGF-ß1), rs2430561 (INF-γ), and rs1800872 (IL10) polymorphisms are associated with CL in southeastern Iranian people.

Genetic diversity and phylogeography of Phlebotomus argentipes (Diptera: Psychodidae, Phlebotominae), using COI and ND4 mitochondrial gene sequences.

BACKGROUND: Phlebotomus argentipes complex is the primary vector for cutaneous leishmaniasis, a burgeoning health concern in contemporary Sri Lanka, where effective vector control is important for proper disease management. Understanding the genetic diversity of the P. argentipes population in Sri Lanka is vital before implementing a successful vector control program. Various studies have indicated that genetic divergence, caused by genetic drift or selection, can significantly influence the vector capacity of arthropod species. To devise innovative control strategies for P. argentipes, exploring genetic diversity and phylogeography can offer valuable insights into vector competence, key genetic trait transfer, and impact on disease epidemiology. The primary objective is to analyze the genetic diversity and phylogeography of the P. argentipes complex in Sri Lanka, based on two mitochondrial genomic regions in modern representatives of P. argentipes populations. METHODOLOGY: A total of 159 P. argentipes specimens were collected from five endemic areas of cutaneous leishmaniasis and identified morphologically. Two mitochondrial regions (Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4) were amplified using the total DNA and subsequently sequenced. Partial sequences of those mitochondrial genes were utilized to analyze genetic diversity indices and to explore phylogenetic and phylogeographic relationships. PRINCIPAL FINDINGS: Among five sampling locations, the highest genetic diversity for COI and ND4 was observed in Hambantota (Hd-0.749, π-0.00417) and Medirigiriya (Hd-0.977, π-0.01055), respectively. Phylogeographic analyses conducted using COI sequences and GenBank retrieved sequences demonstrated a significant divergence of P. argentipes haplotypes found in Sri Lanka. Results revealed that they have evolved from the Indian ancestral haplotype due to historical- geographical connections of the Indian subcontinent with Sri Lanka. CONCLUSIONS: Utilizing high-mutation-rate mitochondrial genes, such as ND4, can enhance the accuracy of genetic variability analysis in P. argentipes populations in Sri Lanka. The phylogeographical analysis of COI gene markers in this study provides insights into the historical geographical relationship between India and P. argentipes in Sri Lanka. Both COI and ND4 genes exhibited consistent genetic homogeneity in P. argentipes in Sri Lanka, suggesting minimal impact on gene flow. This homogeneity also implies the potential for horizontal gene transfer across populations, facilitating the transmission of genes associated with traits like insecticide resistance. This dynamic undermines disease control efforts reliant on vector control strategies.

Variants of CARD8 in Leishmania guyanensis-cutaneous leishmaniasis and influence of the variants genotypes on circulating plasma cytokines IL-1ß, TNFα and IL-8.

Nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein family (NLR) are intracellular pathogen recognition receptors mediating innate immunity, releasing proinflammatory cytokines IL-1ß and IL-18, and promoting pyroptotic cell death, upon sensing pathogenic or endogenous danger signals. In animal models, NLRP3 inflammasome has a dual role, pathogenic or protective in Leishmania-infection, depending on the Leishmania species and mice strain. Caspase recruitment containing domain 8 (CARD8) is a negative regulator of NLRP3 inflammasome and also an inhibitor of transcription factor NFĸB, a major transcription factor of proinflammatory cytokines. We investigated whether single nucleotide variants in CARD8 may partially explain why only a proportion of individuals coming from the same area of endemicity of leishmaniasis develop cutaneous leishmaniasis caused by Leishmania guyanensis. We genotyped four single nucleotide variants of the CARD8 gene by direct nucleotide sequencing in 1741 individuals from an endemic area of leishmaniasis, constituting 850 patients with CL and 891 healthy controls. The frequencies of the genotypes of the variants rs2288877 T>C, rs73944113 C>T, and rs2043211 A>T are similar among the patients with CL and HC, while the variant rs2288876 A>G) reveals an excess of the genotype AA among the patients with CL (44%) compared to 37% in the HC group. Allele A of the variant rs2288876 A>G) is associated with susceptibility to CL (OR = 1.2 [95%CI 1.03-1.4]; P = 0.01). Haplotype analysis showed that individuals harboring the haplotype CCAA have 280% odds of developing CL caused by L. guyanensis (OR = 3.8 [95% CI 2.0-7.7]; p = 0.00004). The variants rs2288877 T>C and rs2288876 A>G correlate with the plasma level of IL-8. Spearman correlation showed a significant positive correlation between the rs2288876 A>G allele A and the level of IL-8 (ρ = 0.22; p = 0.0002). CARD8 may partially contribute to the development of CL caused by L. guyanensis.

Vitamin D status, vitamin D receptor gene polymorphism, and haplotype in patients with cutaneous leishmaniasis: Correlation with susceptibility and parasite load index.

BACKGROUND: CL endemicity was reported worldwide including in Saudi Arabia, imposing a major challenge on the health authorities. Vitamin D and its receptor (VDR) are key modulators of the immune response where the VDR is expressed. A remarkable lack of data exists in humans about the contribution of vitamin D and polymorphisms of the VDR gene in protozoan infections, especially cutaneous leishmaniasis (CL). OBJECTIVE: This is the first work conducted to assess the relationship between vitamin D status, polymorphisms of the VDR gene (BsmI, ApaI, TaqI, and FokI), and VDR haplotype with parasite tissue load and susceptibility to CL. METHODS: Fifty-two patients with confirmed CL (21 patients receiving vitamin D medication and 31 patients not receiving it) and 46 control subjects participated in this cross-sectional investigation. VDR genotyping was determined by restriction fragment length polymorphism analysis. Serum levels of 25-OH vitamin D were assessed using the ELISA method in all participants. The skin biopsy quantified the parasite load based on the Ridley parasitic index. RESULTS: The mean serum level of 25-OH vitamin D in CL patients who were not receiving vitamin D therapy was significantly lower compared to CL patients on vitamin D therapy and controls (p <0.001 for both) and CL patients with no history of vitamin D therapy had a significantly higher frequency of vitamin D deficiency compared to CL patients on vitamin D therapy and controls (p < 0.05). Compared to CL patients with no history of vitamin D therapy, CL patients receiving vitamin D therapy had a significantly lower mean size of the lesion and RPI (p = 0.02, .03 respectively). The frequency of genotype "aa" and its "a" allele in ApaI SNP of VDR was significantly lower in CL patients compared to controls (p = 0.006 and 0.03 respectively). However, patients with CL had a considerably greater frequency of the "A" allele than the controls (p = 0.03), suggesting its role in CL susceptibility. There was no statistically significant difference between the two groups in the genotype and allele frequency distributions of BsmI, TaqI, and FokI (p > 0.05). When compared to controls, CL cases had a considerably greater frequency of the "B-A-T-F" haplotype (p = 0.04), and a significantly lower frequency of the "B-a-T-F" haplotype (p = 0.01) suggesting that these haplotypes may have the potential susceptibility or protection against CL respectively. The "Aa" genotype in ApaI SNP of VDR had considerably lower levels of vitamin D with higher parasite load compared to the "AA" and: aa" genotypes (p = 0.02,0.02 respectively). A significant negative correlation was found between the parasite load and 25-OH vitamin D levels (r2 = -0.53, p< 0.001). CONCLUSIONS: According to these findings, vitamin D levels and "ApaI" VDR gene polymorphisms could affect the parasite load and susceptibility to infection, whereas BsmI, FokI, and TaqI polymorphisms did not. Correction of vitamin D levels may aid in CL management.

Identification of molecular mechanisms causing skin lesions of cutaneous leishmaniasis using weighted gene coexpression network analysis (WGCNA).

Leishmaniasis is an infectious disease, caused by a protozoan parasite. Its most common form is cutaneous leishmaniasis, which leaves scars on exposed body parts from bites by infected female phlebotomine sandflies. Approximately 50% of cases of cutaneous leishmaniasis fail to respond to standard treatments, creating slow-healing wounds which cause permanent scars on the skin. We performed a joint bioinformatics analysis to identify differentially expressed genes (DEGs) in healthy skin biopsies and Leishmania cutaneous wounds. DEGs and WGCNA modules were analyzed based on the Gene Ontology function, and the Cytoscape software. Among almost 16,600 genes that had significant expression changes on the skin surrounding Leishmania wounds, WGCNA determined that one of the modules, with 456 genes, has the strongest correlation with the size of the wounds. Functional enrichment analysis indicated that this module includes three gene groups with significant expression changes. These produce tissue-damaging cytokines or disrupt the production and activation of collagen, fibrin proteins, and the extracellular matrix, causing skin wounds or preventing them from healing. The hub genes of these groups are OAS1, SERPINH1, and FBLN1 respectively. This information can provide new ways to deal with unwanted and harmful effects of cutaneous leishmaniasis.

Low genetic heterogeneity of Leishmania major in different geographical regions of Iran.

To examine the genetic diversity of Leishmania major, 100 Giemsa-stained positive slides were collected from endemic foci of Iran (Northeast, Central, and Southwest provinces) over two consecutive years during 2019-2021. The Leishmania ITS-rDNA gene was amplified and Leishmania sp. was recognized by PCR-RFLP and sequencing. In addition, 178 registered ITS-rDNA sequences from other geographical regions of Iran were retrieved from GenBank, including different host species (human, sandfly and rodent). A total of 40 new haplotypes were discovered using the ITS-rDNA sequence analysis. IR29 (20.6%) and IR34 (61%) were the two most common haplotypes, represented by a star-like feature in the overall population. Analysis of the molecular variance test revealed low genetic diversity of L. major in human cases (Haplotype diversity; 0.341), rodent (Hd; 0.387) and sandfly (Hd; 0.390) sequences. The lowest genetic diversity of L. major was observed in Southwest/Southeast Iran (Hd: 0.104-0.286). The statistically Fst value indicated that L. major is not genetically differentiated between geographic regions of Iran, except for the Northeast-Southwest (Fst: 0.29055) and Central-Southwest (Fst: 0.30294) population pairs. The current study as the first investigation discloses new perspectives for further evaluation in the identification local transmission paradigms and initiating effective prevention strategies.

Variants of NOD2 in Leishmania guyanensis-infected patients with cutaneous leishmaniasis and correlations with plasma circulating pro-inflammatory cytokines.

Leishmaniases, a group of vector-borne diseases, are caused by the protozoan intracellular parasite Leishmania (L.) and are transmitted by the phlebotomine sandflies. A wide range of clinical manifestations in L- infection is observed. The clinical outcome ranges from asymptomatic, cutaneous leishmaniasis (CL) to severe mucosal leishmaniasis (ML) or visceral leishmaniasis (VL), depending on the L. species. Interestingly, only a fraction of L.-infected individuals progress to disease development, suggesting a key role of host genetics in the clinical outcome. NOD2 plays a critical role in the control of host defense and inflammation. The NOD2-RIK2 pathway is involved in developing a Th1- type response in patients with VL and C57BL/6 mice infected with L. infantum. We investigated whether variants in the NOD2 gene (R702W rs2066844, G908R rs2066845, and L1007fsinsC rs2066847) are associated with susceptibility to CL caused by L. guyanensis (Lg) in 837 patients with Lg-Cl and 797 healthy controls (HC) with no history of leishmaniasis. Both patients and HC are from the same endemic area of the Amazonas state of Brazil. The variants R702W and G908R were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and L1007fsinsC was by direct nucleotide sequencing. The minor allele frequency (MAF) of L1007fsinsC was 0.5% among the patients with Lg-CL and 0.6% in the healthy controls group. R702W genotypes frequencies were similar in both groups. Only 1% and 1.6% were heterozygous for G908R among the patients with Lg-CL and HC, respectively. None of the variants revealed any association with susceptibility to the development of Lg-CL. Correlations of genotypes with the level of plasma cytokines revealed that individuals with the mutant alleles of R702W tend to have low levels of IFN-γ. G908R heterozygotes also tend to have low IFN-γ, TNF-α, IL-17, and IL-8. Variants of NOD2 are not involved in the pathogenesis of Lg-CL.

Effect of Leishmania RNA virus 2 on virulence factors and cytokines gene expression in a human macrophage infected with Leishmania major: A preliminary study.

Cutaneous leishmaniasis (CL) is one of the most important infectious parasitic diseases in the world caused by the Leishmania parasite. In recent decades, the presence of a virus from the Totiviridae family has been proven in some Leishmania species. Although the existence of LRV2 in the Old world Leishmania species has been confirmed, almost no studies have been done to determine the potential impact of LRV2 on the immunopathogenicity of the Leishmania parasite. In this preliminary study, we measured the expression of target genes, including Glycoprotein 63 (gp63), Heat Shock Protein 70 (hsp70), Cysteine Protease b (cpb), Interleukin 1 beta (IL-1ß), IL8 and IL-12 in LRV2 positive Leishmania major strain (LRV2+L. major) and LRV2 negative L. major strain (LRV2-L. major). We exposed THP-1, a human leukemia monocytic cell line, to promastigotes of both strains. After the initial infection, RNA was extracted at different time points, and the relative gene expression was determined using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Findings showed that the presence of LRV2 in L. major was able to increase the expression of gp63, hsp70, and cpb genes; also, we observed lower levels of expression in cytokine genes of IL-1ß, IL-8, IL-12 in the presence of LRV2+, which are critical factors in the host's immune response against leishmaniasis. These changes could suggest that the presence of LRV2 in L. major parasite may change the outcome of the disease and increase the probability of Leishmania survival; nevertheless, further studies are needed to confirm our results.

Genetic Iron Overload Hampers Development of Cutaneous Leishmaniasis in Mice.

The survival, growth, and virulence of Leishmania spp., a group of protozoan parasites, depends on the proper access and regulation of iron. Macrophages, Leishmania's host cell, may divert iron traffic by reducing uptake or by increasing the efflux of iron via the exporter ferroportin. This parasite has adapted by inhibiting the synthesis and inducing the degradation of ferroportin. To study the role of iron in leishmaniasis, we employed Hjv-/- mice, a model of hemochromatosis. The disruption of hemojuvelin (Hjv) abrogates the expression of the iron hormone hepcidin. This allows unrestricted iron entry into the plasma from ferroportin-expressing intestinal epithelial cells and tissue macrophages, resulting in systemic iron overload. Mice were injected with Leishmania major in hind footpads or intraperitoneally. Compared with wild-type controls, Hjv-/- mice displayed transient delayed growth of L. major in hind footpads, with a significant difference in parasite burden 4 weeks post-infection. Following acute intraperitoneal exposure to L. major, Hjv-/- peritoneal cells manifested increased expression of inflammatory cytokines and chemokines (Il1b, Tnfa, Cxcl2, and Ccl2). In response to infection with L. infantum, the causative agent of visceral leishmaniasis, Hjv-/- and control mice developed similar liver and splenic parasite burden despite vastly different tissue iron content and ferroportin expression. Thus, genetic iron overload due to hemojuvelin deficiency appears to mitigate the early development of only cutaneous leishmaniasis.

Cutaneous leishmaniasis: multiomics approaches to unravel the role of immune cells checkpoints.

INTRODUCTION: Cutaneous leishmaniasis (CL) is the most frequent form of leishmaniases, associated with skin inflammation and ulceration. Understanding the interaction of different phagocytic cells in the recognition and uptake of different Leishmania species is critical for controlling the infection. Phagocytic cells have a pivotal role as professional antigen-presenting cells that bridge the innate and adaptive immunity and shape the outcome of the disease. AREAS COVERED: Here we reviewed new technologies with high-throughput data collection capabilities along with systems biology approaches which are recently being used to decode the paradox of CL immunology. EXPERT OPINION: We emphasized on the crosstalk between DC and T-cells while focusing on the immune checkpoints interactions between the human immune system and the Leishmania species. Further, we discussed omics technologies including bulk RNA sequencing, reverse transcriptase-multiplex ligation dependent probe amplification (RT-MLPA), and proximity extension assay (PEA) in studies on human blood or tissue-driven samples from CL patients in which we have so far been involved.

Screening of potential hub genes involved in Cutaneous Leishmaniasis infection via bioinformatics analysis.

BACKGROUND: Cutaneous Leishmaniasis (CL) is the most common clinical form of leishmaniasis. Despite its low mortality, CL deserves further attention because its pathogenesis is currently no well-known or well-researched. METHODS: We downloaded the gene expression datasets of GSE55664 and GSE63931 with respect to leishmaniasis from the Gene Expression Synthesis (GEO) database. Additionally, the differentially expressed genes (DEGs) in the infection and control groups were identified by packages of R software. The Gene Ontology (GO) function, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) pathway were utilized for the biological functional analysis. Subsequently, we identified the top ten hub genes from protein-protein interaction (PPI) networks based on STRING and Cytoscape software. The hub genes were validated in GraphPad Prism 8.0 using the GSE162760 dataset. Further, CIBERSORT was used to evaluate the immune cell infiltration proportions between the CL infection samples and the control samples based on the GSE43880 and GSE55664 datasets. RESULTS: The enrichment analysis revealed that DEGs were significantly involved in cell-mediated immune responses, such as leukocyte cell-cell adhesion and T-cell activation. STAT1, CCR7, CCR2, and CXCL10 were identified as hub genes with statistical significance. These hub genes showed close correlations with various immune cells, such as M1 cells and CD4-activated memory T-cells. CONCLUSIONS: In our research, we used bioinformatics analysis to identify some molecular biomarkers and significant pathways in CL infection. These hub genes may provide new options for future diagnosis and treatment.

DNA methylation alterations caused by Leishmania infection may generate a microenvironment prone to tumour development.

DNA methylation is an epigenetic signature consisting of a methyl group at the 5' cytosine of CpG dinucleotides. Modifications in DNA methylation pattern have been detected in cancer and infectious diseases and may be associated with gene expression changes. In cancer development DNA methylation aberrations are early events whereas in infectious diseases these epigenetic changes may be due to host/pathogen interaction. In particular, in leishmaniasis, a parasitic disease caused by the protozoan Leishmania, DNA methylation alterations have been detected in macrophages upon infection with Leishmania donovani and in skin lesions from patients with cutaneous leishmaniasis. Interestingly, different types of cancers, such as cutaneous malignant lesions, lymphoma and hepatocellular carcinoma, have been diagnosed in patients with a history of leishmaniasis. In fact, it is known that there exists an association between cancer and infectious diseases. Leishmania infection may increase susceptibility to develop cancer, but the mechanisms involved are not entirely clear. Considering these aspects, in this review we discuss the hypothesis that DNA methylation alterations induced by Leishmania may trigger tumorigenesis in long term infection since these epigenetic modifications may enhance and accumulate during chronic leishmaniasis.

Blood transcriptional profiles distinguish different clinical stages of cutaneous leishmaniasis in humans.

Cutaneous leishmaniasis (CL) is a neglected tropical disease with severe morbidity and socioeconomic sequelae. A better understanding of underlying immune mechanisms that lead to different clinical outcomes of CL could inform the rational design of intervention measures. While transcriptomic analyses of CL lesions were recently reported by us and others, there is a dearth of information on the expression of immune-related genes in the blood of CL patients. Herein, we investigated immune-related gene expression in whole blood samples collected from individuals with different clinical stages of CL along with healthy volunteers in an endemic CL region where Leishmania (L.) tropica is prevalent. Study participants were categorized into asymptomatic (LST+) and healthy uninfected (LST-) groups based on their leishmanin skin test (LST). Whole blood PAXgene samples were collected from volunteers, who had healed CL lesions, and patients with active L. tropica cutaneous lesions. Quality RNA extracted from 57 blood samples were subjected to Dual-color reverse-transcription multiplex ligation-dependent probe amplification (dcRT-MLPA) assay for profiling 144 immune-related genes. Results show significant changes in the expression of genes involved in interferon signaling pathway in the blood of active CL patients, asymptomatics and healed individuals. Nonetheless, distinct profiles for several immune-related genes were identified in the healed, the asymptomatic, and the CL patients compared to the healthy controls. Among others, IFI16 and CCL11 were found as immune transcript signatures for the healed and the asymptomatic individuals, respectively. These results warrant further exploration to pinpoint novel blood biomarkers for different clinical stages of CL.

Human genetic polymorphism and Leishmaniasis.

Leishmaniasis is a disease of the subtropical and tropical spheres of the earth and has various clinical manifestations. The different form of leishmaniasis includes cutaneous leishmaniasis, mucocutaneous leishmaniasis, most lethal visceral leishmaniasis and PKDL form. These different forms depend on many factors such as parasite and vector species, geographical, environmental conditions and population ethnicity. Host genetic factors have been widely investigated for their role in developing the disease in various infections. There are several reports on associations or resistance between candidate gene polymorphisms and the risk and outcome of Leishmania infection. Polymorphism in genes involved in both innate and adaptive immune systems, as well as genes of metabolic processes contributes to disease manifestation. The wide availability and advancement of molecular techniques permits to exploration of hereditary factors related to leishmaniasis. Many candidate gene studies were conducted on family-based and population to identify novel biomarkers for understanding disease pathogenesis pathways and possible drug targets. This comprehensive review presents an update on various human genes polymorphism that influence the outcome of different forms of Leishmania infection in endemic regions of the world. Various electronic databases were searched systematically for relevant publications and thoroughly analyzed. Most of the candidate gene studies were found with discrepancies in findings. Genetic and functional studies with adequate power are needed to validate the contribution of host genes in susceptibility or resistance towards Leishmania infection and understanding pathogenesis.

Mitogen-Activated Protein Kinase and Aquaglyceroporin Gene Expression in Treatment Failure Leishmania major.

PURPOSE: Leishmaniasis comprises various clinical forms mainly including cutaneous, muco-cutaneous, and visceral leishmaniasis; caused by Leishmania species. Antimoniate is the first-line treatment but some cases showed no response to treatment in the worldwide. In this study, mitogen-activated protein kinase (MAPK) and aquaglyceroporin 1 (AQP1) gene expressions were assessed in treatment failure clinical isolates of Leishmania major. Also, molecular and phylogenic analyses of the mentioned isolates were performed. METHODS: Samples were obtained from the patients with suspicious CL referred to the laboratory of Diagnosis Center, Gorgan Province, Iran, from October 2016 to December 2019. Detection and identification of the parasite was performed. The genes expressions of MAPK1 and AQP1 were done using SYBR Green real-time PCR. The AQP1 gene from the isolates with treatment failure was sequenced and analyzed using BLAST and multiple alignments. The phylogenic analysis was done using MEGA7. The statistical analysis was done using SPSS 16.0 by non-parametric Mann-Whitney U test. RESULTS: All clinical isolates were detected L. major. The mean AQP1 and MAPK1 gene expressions in treatment failure isolates were 58.71 and 6.139 fold less than the ones in treatment response isolates, respectively. Based on the AQP1 gene sequence, a nucleotide change of aspartic acid with asparagine at the site 234 was observed. Phylogenic tree analysis showed three groups with the minimum dissimilarity of 0.008 between TF isolates with the standard L. major strains. CONCLUSION: We showed that MAPK1 and AQP1 may have critical roles in response to antimoniate in clinical isolates L. major in this study.

IL-23R Variant rs11805303 Is Associated With Susceptibility to the Development of Cutaneous Leishmaniasis in Leishmania guyanensis-Infected Individuals.

BACKGROUND: Emerging evidence suggests that the interleukin (IL) 17/ IL-23 axis may play a role in the pathogenesis of leishmaniasis. Our aim was to investigate whether the IL-23R variant rs11805303 is a risk factor for the development of cutaneous leishmaniasis (CL) in Leishmania guyanensis-infected individuals. METHODS: We genotyped by polymerase chain reaction-restriction fragment length polymorphism the rs11805303 C/T in 828 patients with CL and 806 healthy individuals. Plasma tumor necrosis factor-α, IL-6, interferon-γ, IL-1ß, and IL-17 were measured with the Bioplex assay. RESULTS: The distribution of the genotypes differed between patients with CL and healthy controls with a common odds ratio of 1.78 (P = 2.2 × 10-11) for the disease-associated T allele. Leishmania guyanensis-infected individuals homozygous for the T allele show a 200% increased risk of progressing to disease development, with a 95% confidence interval ranging from 81% to 400% (P = 9.9 × 10-6) in comparison to individuals homozygous for the C allele. Males homozygous for the T allele have higher plasma levels of IL-17 compared with heterozygous or homozygous CC individuals. CONCLUSIONS: The present association of the IL-23R variant rs11805303 with the development of CL suggests that the IL-17/IL-23 axis may play an important role in the pathogenesis of CL.

Resistance Against Leishmania major Infection Depends on Microbiota-Guided Macrophage Activation.

Innate immune cells present a dual role during leishmaniasis: they constitute the first line of host defense but are also the main host cells for the parasite. Response against the infection that results in the control of parasite growth and lesion healing depends on activation of macrophages into a classical activated phenotype. We report an essential role for the microbiota in driving macrophage and monocyte-derived macrophage activation towards a resistance phenotype against Leishmania major infection in mice. Both germ-free and dysbiotic mice showed a higher number of myeloid innate cells in lesions and increased number of infected cells, mainly dermal resident and inflammatory macrophages. Despite developing a Th1 immune response characterized by the same levels of IFN-γ production as the conventional mice, germ-free mice presented reduced numbers of iNOS+ macrophages at the peak of infection. Absence or disturbance of host microbiota impaired the capacity of bone marrow-derived macrophage to be activated for Leishmania killing in vitro, even when stimulated by Th1 cytokines. These cells presented reduced expression of inos mRNA, and diminished production of microbicidal molecules, such as ROS, while presenting a permissive activation status, characterized by increased expression of arginase I and il-10 mRNA and higher arginase activity. Colonization of germ-free mice with complete microbiota from conventional mice rescued their ability to control the infection. This study demonstrates the essential role of host microbiota on innate immune response against L. major infection, driving host macrophages to a resistance phenotype.

Variants of MIRNA146A rs2910164 and MIRNA499 rs3746444 are associated with the development of cutaneous leishmaniasis caused by Leishmania guyanensis and with plasma chemokine IL-8.

Leishmania are intracellular protozoan parasites that cause a wide spectrum of clinical manifestations in genetically susceptible individuals with an insufficient or balanced Th1 immune response to eliminate the parasite. MiRNAs play important regulatory role in numerous biological processes including essential cellular functions. miR146-a acts as an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) present in the toll-like receptors pathway while miR499a modulates TGF-ß and TNF signalling pathways. Here, we investigated whether MIRNA146A rs2910164 and MIRNA499 rs3746444 variants are associated with the development of L. guyanensis (Lg)-cutaneous leishmaniasis (CL). The variants MIR146A rs2910164 and MIR499A rs3746444 were assessed in 850 patients with Lg-CL and 891 healthy controls by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma cytokines were measured using the BioPlex assay. Carriers of rs2910164 CC genotype have 30% higher odds of developing CL (ORadjage/sex = 1.3 [95%CI 0.9-1.8]; Padjage/sex 0.14) compared to individuals with the genotype GG (ORadjage/sex = 0.77 [95%CI 0.56-1.0]; Padjage/sex 0.14) if exposed to Lg-infection. Heterozygous GC individuals also showed lower odds of developing CL (ORadjage/sex = 0.77 [95%CI 0.5-1.1]; Padjage/sex 0.09). Homozygosity for the allele C is suggestive of an association with the development of Lg-CL among exposed individuals to Lg-infection. However, the odds of developing CL associated with the CC genotype was evident only in male individuals (ORadjage = 1.3 [95% CI = 0.9-2.0]; Padjage = 0.06). Individuals homozygous for the G allele tend to have higher plasma IL-8 and CCL5. Similarly, for the MIR499A rs3746444, an association with the G allele was only observed among male individuals (OR = 1.4 [1.0-1.9]; P = 0.009). In a dominant model, individuals with the G allele (GG-GA) when compared to the AA genotype reveals that carriers of the G allele have 40% elevated odds of developing Lg-CL (ORadjage = 1.4 [1.1-1.9]). Individuals with the GG genotype have higher odds of developing Lg-CL (ORadjage/sex = 2.0 [95%CI 0.83-5.0]; Padjage = 0.01. Individuals homozygous for the G allele have higher plasma IL-8. Genetic combinations of both variants revealed that male individuals exposed to Lg bearing three or four susceptible alleles have higher odds of developing Lg-CL (OR = 2.3 [95% CI 1.0-4.7]; p = 0.017). Both MIR146A rs2910164 and MIR499A rs3746444 are associated with the development of Lg-CL and this association is prevalent in male individuals.

Novel IL-12Rß1 deficiency-mediates recurrent cutaneous leishmaniasis.

BACKGROUND: The IL-12/IFN-γ axis plays a vital role in the control of intramacrophagic pathogens including Leishmania infections. OBJECTIVE: The aim of this study was to investigate genetic defects in the IL-12/IFN-γ axis in cutaneous leishmaniasis patients, using immunological and genetic evaluation. METHODS: Enzyme-linked immunosorbent assay was used to quantify IFN-γ , while flow cytometry was performed to analyze surface IL-12Rß1/IL-12Rß2 expression and phosphorylation of signal transducers as well as the activator of transcription 4 (pSTAT4). Sequencing was carried out for genetic analysis. RESULTS: The peripheral blood mononuclear cells from the two patients (P1 and P2) demonstrated impaired production of IFN-γ. Furthermore, abolishment of the surface expression of Il-12Rß1 was observed in lymphocytes, with consequent impairment of STAT4 phosphorylation in the lymphocytes of P1 and P2. IL-12Rß1 deficiency was identified, which was caused by a novel homozygous missense mutation (c.485>T/p.P162L) and a novel homozygous nonsense mutation (c.805G>T/P.E269*) in the IL-12Rß2 gene of P1 and P2, respectively. In silico analyses predicted these novel mutations as being pathogenic, causing truncated proteins, with consequent inactivation. CONCLUSION: Our data have expanded the phenotype and mutation spectra associated with IL-12Rß1 deficiency, and suggest that patients with CL should be screened for mutations in genes of the IL-12/IFN-γ axis.