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RT-qPCR validation of some modulated genes in BALB/c and C57BL/6 BMDMs in response to L. amazonensis infection. Comparative analysis of the relative expression levels of selected genes determined by RNA-seq and validated by RT-qPCR. The bars represent the mean ± SD values of the fold changes in Il1b, Fcgr1, Ccr5, Smad6, Jun and Mapk14 expression determined with five independent biological replicates analyzed in duplicate. The fold changes were calculated through relative quantification using the ΔΔCt method. The data were normalized to Gapdh expression and the relative gene expression was set to 1 for the control (noninfected) samples. Statistical analysis was performed using the t-tests, and no significant differences were observed (p-value < 0.05) between the RT-qPCR and RNA-seq results for the BALB/c_La and C57BL/6_La groups. The bars for Amastin-like (LmxM.33.0960) show the mean after normalization to Gapdh in L. amazonensis infecting BALB/c and L. amazonensis infecting C57BL/6 macrophages. La, L. amazonensis.
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The fate of Leishmania infection can be strongly influenced by the host genetic background. In this work, we describe gene expression modulation of the immune system based on dual global transcriptome profiles of bone marrow-derived macrophages (BMDMs) from BALB/c and C57BL/6 mice infected with Leishmania amazonensis. A total of 12,641 host transcr...
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... validation assays were performed on some of the most modulated molecules from the RNA-seq data: Il1b, Fcgr1, Ccr5, Smad6, Jun and Mapk14. Comparative analyses showed concordance between the RNA-seq and RT-qPCR data with no statistically significant differences, thus validating the RNA-seq results (Fig. ...
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Citations
... The complete lists of genes are provided in S1 Table (24h) and S2 Table (48h). A direct comparison of deregulated genes at each timepoint revealed that the macrophage response to infection at 24 h was more pronounced compared to the response at 48 h, confirming previous findings in murine macrophage infection models, which evidenced a greater gene expression modulation during early infection and a progressive reduction at later timepoints until 72 h post-infection [4,6,28,29]. A fraction of these genes was consistently up-or downregulated both at 24 h and 48h, while another fraction (19% to 69%) was deregulated uniquely at 48h post-infection (Tables 2 and S3). ...
Background
In the Mediterranean basin, three Leishmania species have been identified: L . infantum , L . major and L . tropica , causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L . infantum , L . major , and L . tropica , as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L . infantum ) and parasites involved in CL (i.e., L . major , L . tropica ).
Methodology/Principal findings
U937 cells differentiated into macrophage-like cells were infected with L . infantum , L . major and L . tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L . infantum -infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization.
Conclusions
The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.
... Rab15 is involved in positive regulation of regulated secretory pathway-associated immunodeficiency [61]. The Fcgr1 gene encodes a protein that plays an important role in the immune response, acting as a high-affinity Fc-gamma receptor [62]. Upon examining the KEGG pathway using these genes, the cAMP signaling pathway, anti-inflammatory response, inflammatory mediator regulation of TRP channels, and FcγR-mediated phagocytosis pathways in cancer were included. ...
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... The CEBPB (ENSG00000172216) TF, a member of the bZIP family, is related to the main immune response processes. The expression of CEBPB gene up-regulated in response to Leishmania infection, as previously reported [61]. The CTCF (ENSG00000102974) (a C2H2 ZF TF) mediates the macrophage function of mice infected with Leishmania major [62]. ...
Background
Leishmaniasis is a parasitic disease caused by the Leishmania protozoan affecting millions of people worldwide, especially in tropical and subtropical regions. The immune response involves the activation of various cells to eliminate the infection. Understanding the complex interplay between Leishmania and the host immune system is crucial for developing effective treatments against this disease.
Methods
This study collected extensive transcriptomic data from macrophages, dendritic, and NK cells exposed to Leishmania spp. Our objective was to determine the Leishmania -responsive genes in immune system cells by applying meta-analysis and feature selection algorithms, followed by co-expression analysis.
Results
As a result of meta-analysis, we discovered 703 differentially expressed genes (DEGs), primarily associated with the immune system and cellular metabolic processes. In addition, we have substantiated the significance of transcription factor families, such as bZIP and C2H2 ZF, in response to Leishmania infection. Furthermore, the feature selection techniques revealed the potential of two genes, namely G0S2 and CXCL8 , as biomarkers and therapeutic targets for Leishmania infection. Lastly, our co-expression analysis has unveiled seven hub genes, including PFKFB3 , DIAPH1 , BSG , BIRC3 , GOT2 , EIF3H , and ATF3 , chiefly related to signaling pathways.
Conclusions
These findings provide valuable insights into the molecular mechanisms underlying the response of immune system cells to Leishmania infection and offer novel potential targets for the therapeutic goals.
... The role of IL-1β and IL-18 in Leishmania infection has been the subject of numerous studies [39,46,48]. It was reported that IL-1β can modulate the immune responses, while IL-18 shifts the T-cell activation pathway towards Th2, however, both cytokines contribute to the progression of the disease [50,51]. Notably, IL-1β has been identified as a significant signaling factor for host resistance against infection, as this cytokine transmits signals through IL-1R and myeloid differentiation primary response protein (MyD) 88, leading to the induction of NOS2-mediated nitric oxide (NO) production. ...
Background
Cutaneous Leishmaniasis (CL) is a parasitic disease with diverse outcomes. Clinical diversity is influenced by various factors such as Leishmania species and host genetic background. The role of Leishmania RNA virus (LRV), as an endosymbiont, is suggested to not only affect the pathogenesis of Leishmania, but also impact host immune responses. This study aimed to investigate the influence of LRV2 on the expression of a number of virulence factors (VFs) of Leishmania and pro-inflammatory biomarkers.
Materials and methods
Sample were obtained from CL patients from Golestan province. Leishmania species were identified by PCR (LIN 4, 17), and the presence of LRV2 was checked using the semi-nested PCR (RdRp gene). Human monocyte cell line (THP-1) was treated with three isolates of L. major with LRV2 and one isolate of L. major without LRV2. The treatments with four isolates were administered for the time points: zero, 12, 24, 36, and 48 h after co-infection. The expression levels of Leishmania VFs genes including GP63, HSP83, and MPI, as well as pro-inflammatory biomarkers genes including NLRP3, IL18, and IL1β, were measured using quantitative real-time PCR.
Results
The expression of GP63, HSP83, and MPI revealed up-regulation in LRV2 + isolates compared to LRV2- isolates. The expression of the pro-inflammatory biomarkers including NLRP3, IL1β, and IL18 genes in LRV2- were higher than LRV2 + isolates.
Conclusion
This finding suggests that LRV2 + may have a probable effect on the Leishmania VFs and pro-inflammatory biomarkers in the human macrophage model.
... Balb/c mice have strong Th2 immune response, a common response in allergic reactions and infectious diseases, high production of IgG1 and IgA, high complement activity, and low interferon production. Additionally, Balb/c mice produce stronger humoral response against antigens in comparison with C57BL/6 strain (Bleul et al., 2021;Aoki et al., 2019;Zhang et al., 2023;Laurenti et al., 2004;Kuroda et al., 2002;Koo and Gan, 2006;Watanabe et al., 2004). ...
Inflammation is a general term for a wide variety of both physiological and pathophysiological processes in the body which primarily prevents the body from diseases and helps to remove dead tissues. It has a crucial part in the body immune system. Tissue damage can recruit inflammatory cells and cytokines and induce inflammation. Inflammation can be classified as acute, sub-acute, and chronic. If it remained unresolved and lasted for prolonged periods, it would be considered as chronic inflammation (CI), which consequently exacerbates tissue damage in different organs. CI is the main pathophysiological cause of many disorders such as obesity, diabetes, arthritis, myocardial infarction, and cancer. Thus, it is critical to investigate different mechanisms involved in CI to understand its processes and to find proper anti-inflammatory therapeutic approaches for it. Animal models are one of the most useful tools for study about different diseases and mechanisms in the body, and are important in pharmacological studies to find proper treatments. In this study, we discussed the various experimental animal models that have been used to recreate CI which can help us to enhance the understanding of CI mechanisms in human and contribute to the development of potent new therapies.
... Intriguingly, the functional enrichment analysis carried out using DAVID revealed that many pathways related to parasitic diseases such as amoebiasis, trypanosomiasis, toxoplasmosis, and leishmaniasis were also enriched in the up-regulated dataset of the infected animals (O'Gorman et al., 2006;Aoki et al., 2019;Oliveira et al., 2020;Kalavi et al., 2021;Salloum et al., 2021;Sun et al., 2021;Wang et al., 2022). The constituent genes associated with these infections and their expression statistics observed in our study are shown in Table 2. ...
Bovine anaplasmosis caused by Anaplasma marginale is a tick-borne disease of livestock with widespread prevalence and huge economic implications. In order to get new insights into modulation of host gene expression in response to natural infections of anaplasmosis, this study is the first attempt that compared the transcriptome profiles of peripheral blood mononuclear cells (PBMCs) of A. marginale infected and healthy crossbred cattle. Transcriptome analysis identified shared as well as unique functional pathways in the two groups. Translation and structural constituent of ribosome were the important terms for the genes abundantly expressed in the infected as well as healthy animals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes revealed that immunity and signal transduction related terms were enriched for the up-regulated genes in the infected animals. The over-represented pathways were cytokine-cytokine receptor interaction and signaling pathways involving chemokines, Interleukin 17 (IL17), Tumour Necrosis Factor (TNF), Nuclear Factor Kappa B (NFKB) etc. Interestingly, many genes previously associated with parasite-borne diseases such as amoebiasis, trypanosomiasis, toxoplasmosis, and leishmaniasis were profusely expressed in the dataset of the diseased animals. High expression was also evident for the genes for acute phase response proteins, anti-microbial peptides and many inflammatory cytokines. Role of cytokines in mediating communication between immune cells was the most conspicuous gene network identified through the Ingenuity Pathway Analysis. This study provides comprehensive information about the crosstalk of genes involved in host defense as well as parasite persistence in the host upon infection with A. marginale.
... Previous studies have compared the differential responses of macrophages from resistant and susceptible mice induced by Leishmania (6,7). However, to the best of our knowledge, no study has focused on the impact of the basal transcriptomic profile of macrophages, from different host backgrounds, on their response to Leishmania infection. ...
... Global transcriptomic events associated with M-CSFdependent monocyte-to-macrophage differentiation have been analyzed and associated with major changes in the global transcriptomic profile (17). Moreover, differential basal backgrounds in non-infected BALB/c vs. non-infected C57BL/6 L929 differentiated macrophages have also been previously reported (6). Among the DEGs between the two mouse strains, our analysis showed that Cathepsin E (Supplementary File 1) is one of the most strongly modulated genes in the BALB/c derived macrophages. ...
... In particular, these profiles could account for the limited amplitude of the inflammatory response observed in Leishmaniainfected BALB/c BMdMs, while the conditions that prevail in uninfected C57BL/6 BMdMs could explain the greater magnitude of the immune response induced by Leishmania infection in resistant BMDMs (Figure 2). Similarly, L. amazoniensis has also been reported to induce a stronger immune response activation in BMDMs from C57BL/6 mice as compared to BALB/c mice (6). In vivo, these different amounts of cytokines/chemokines produced by the macrophages in different backgrounds could affect leukocyte recruitment, especially at the site of infection. ...
Leishmaniases are a group of diseases with different clinical manifestations. Macrophage-Leishmania interactions are central to the course of the infection. The outcome of the disease depends not only on the pathogenicity and virulence of the parasite, but also on the activation state, the genetic background, and the underlying complex interaction networks operative in the host macrophages. Mouse models, with mice strains having contrasting behavior in response to parasite infection, have been very helpful in exploring the mechanisms underlying differences in disease progression. We here analyzed previously generated dynamic transcriptome data obtained from Leishmania major (L. major) infected bone marrow derived macrophages (BMdMs) from resistant and susceptible mouse. We first identified differentially expressed genes (DEGs) between the M-CSF differentiated macrophages derived from the two hosts, and found a differential basal transcriptome profile independent of Leishmania infection. These host signatures, in which 75% of the genes are directly or indirectly related to the immune system, may account for the differences in the immune response to infection between the two strains. To gain further insights into the underlying biological processes induced by L. major infection driven by the M-CSF DEGs, we mapped the time-resolved expression profiles onto a large protein-protein interaction (PPI) network and performed network propagation to identify modules of interacting proteins that agglomerate infection response signals for each strain. This analysis revealed profound differences in the resulting responses networks related to immune signaling and metabolism that were validated by qRT-PCR time series experiments leading to plausible and provable hypotheses for the differences in disease pathophysiology. In summary, we demonstrate that the host’s gene expression background determines to a large degree its response to L. major infection, and that the gene expression analysis combined with network propagation is an effective approach to help identifying dynamically altered mouse strain-specific networks that hold mechanistic information about these contrasting responses to infection.
... Spermidine reduces the secretion of TNF-α and IL-1β in LPS-stimulated RAW 264.7 macrophages [67] and MCP-1 secretion in THP-1-macrophages treated with IFN-γ [90]. IL-1β induces NOS2 and NO production and resistance to infection in C57BL/6 BMDM infected with L. amazonensis [91], and transcriptome data showed downregulation of Il1b in L. amazonensis infected BALB/c-BMDM [92]. In L. amazonensis skin lesions on C57BL/6 mice, the lack of CCR2 (receptor for MCP-1) CD11b +cells showed lower ARG1 and NOS2 and a reduction in parasite load [70]. ...
Leishmania is a protozoan that causes leishmaniasis, a neglected tropical disease with clinical manifestations classified as cutaneous, mucocutaneous, and visceral leishmaniasis. In the infection context, the parasite can modulate macrophage gene expression affecting the microbicidal activity and immune response. The metabolism of L-arginine into polyamines putrescine, spermidine, and spermine reduces nitric oxide (NO) production, favoring Leish-mania survival. Here, we investigate the effect of supplementation with L-arginine and poly-amines in infection of murine BALB/c macrophages by L. amazonensis and in the transcriptional regulation of genes involved in arginine metabolism and proinflammatory response. We showed a reduction in the percentage of infected macrophages upon putres-cine supplementation compared to L-arginine, spermidine, and spermine supplementation. Unexpectedly, deprivation of L-arginine increased nitric oxide synthase (Nos2) gene expression without changes in NO production. Putrescine supplementation increased transcript levels of polyamine metabolism-related genes Arg2, ornithine decarboxylase (Odc1), Sper-midine synthase (SpdS), and Spermine synthase (SpmS), but reduced Arg1 in L. amazo-nensis infected macrophages, while spermidine and spermine promoted opposite effects. Putrescine increased Nos2 expression without leading to NO production, while L-arginine plus spermine led to NO production in uninfected macrophages, suggesting that polyamines can induce NO production. Besides, L-arginine supplementation reduced Il-1b during infection , and L-arginine or L-arginine plus putrescine increased Mcp1 at 24h of infection, suggesting that polyamines availability can interfere with cytokine/chemokine production. Our data showed that putrescine shifts L-arginine-metabolism related-genes on BALB/c macro-phages and affects infection by L. amazonensis.
... Previous studies determined the transcriptome of Li infection in THP-1 derived macrophages (Gatto et al., 2020), La and L. major infection in primary human macrophages (hMDM) (Fernandes et al., 2016) and Lb infection in patient's lesions (Maretti-Mira et al., 2012). In murine macrophages, the transcriptome of BALB/c and C57BL/6 macrophages infected with La indicated an inflammatory response different from the spectrum extremes M1 and M2 polarized macrophages (Osorio y Fortéa et al., 2009;Aoki et al., 2019) observed in L. major (Sacks & Noben-Trauth, 2002). All of the above-mentioned results of transcriptome-wide experiments exhibit some contrasting results with the literature because macrophage response to Leishmania is highly dependent on the parasite species and strain and host cell type, thus the importance of investigating different models (Salloum et al., 2021). ...
It is well established that infection with Leishmania alters the host cell’s transcriptome. Since mammalian cells have multiple mechanisms to control gene expression, different molecules, such as noncoding RNAs, can be involved in this process. MicroRNAs have been extensively studied upon Leishmania infection, but whether long noncoding RNAs (lncRNAs) are also altered in macrophages is still unexplored. We performed RNA-seq from THP-1-derived macrophages infected with Leishmania amazonensis (La), L. braziliensis (Lb), and L. infantum (Li), investigating a previously unappreciated fraction of macrophage transcriptome. We found that more than 24% of the total annotated transcripts and 30% of differentially expressed (DE) RNAs in Leishmania-infected macrophage correspond to lncRNAs. LncRNAs and protein coding RNAs with altered expression are similar among macrophages infected with the Leishmania species. Still, some species-specific alterations could occur due to distinct pathophysiology in which Li infection led to a more significant number of exclusively DE RNAs. The most represented classes among DE lncRNAs were intergenic and antisense lncRNAs. We also found enrichment for immune response-related pathways in the DE protein coding RNAs, as well as putative targets of the lncRNAs. We performed a coexpression analysis to explore potential cis regulation of coding and antisense noncoding transcripts. We identified that antisense lncRNAs are similarly regulated as its neighbor protein coding genes, such as the BAALC/BAALC-AS1, BAALC/BAALC-AS2, HIF1A/HIF1A-AS1, HIF1A/HIF1A-AS3 and IRF1/IRF1-AS1 pairs, which can occur as a species-specific modulation. These findings are a novelty in the field because, to date, no study has focused on analyzing lncRNAs in Leishmania-infected macrophage. Our results suggest that lncRNAs may account for a novel mechanism by which Leishmania can control macrophage function. Further research must validate putative lncRNA targets and provide additional prospects in lncRNA function during Leishmania infection.
... The advent of transcriptomics has contributed to the global comprehension of how macrophages respond toward infection, including infection by Leishmania. A large number of genes modulated by Leishmania were related to the immune response (pro-and anti-inflammatory), glycolysis, lipid metabolism, biogenesis, and phagocytosis (Fernandes et al., 2016;Aoki et al., 2017;Aoki et al., 2019;Shadab et al., 2019;Restrepo et al., 2021;Reverte et al., 2021;Salloum et al., 2021;Chaparro et al., 2022). While these studies have shed light on a plethora of mechanisms potentially perturbed by Leishmania, they have not explored the impact that coexposure to additional agents may have on the immune response mounted toward Leishmania. ...
Leishmania RNA virus 1 (LRV1) is a double-stranded RNA virus found in some strains of the human protozoan parasite Leishmania, the causative agent of leishmaniasis, a neglected tropical disease. Interestingly, the presence of LRV1 inside Leishmania constitutes an important virulence factor that worsens the leishmaniasis outcome in a type I interferon (IFN)–dependent manner and contributes to treatment failure. Understanding how macrophages respond toward Leishmania alone or in combination with LRV1 as well as the role that type I IFNs may play during infection is fundamental to oversee new therapeutic strategies. To dissect the macrophage response toward infection, RNA sequencing was performed on murine wild-type and Ifnar-deficient bone marrow–derived macrophages infected with Leishmania guyanensis (Lgy) devoid or not of LRV1. Additionally, macrophages were treated with poly I:C (mimetic virus) or with type I IFNs. By implementing a weighted gene correlation network analysis, the groups of genes (modules) with similar expression patterns, for example, functionally related, coregulated, or the members of the same functional pathway, were identified. These modules followed patterns dependent on Leishmania, LRV1, or Leishmania exacerbated by the presence of LRV1. Not only the visualization of how individual genes were embedded to form modules but also how different modules were related to each other were observed. Thus, in the context of the observed hyperinflammatory phenotype associated to the presence of LRV1, it was noted that the biomarkers tumor-necrosis factor α (TNF-α) and the interleukin 6 (IL-6) belonged to different modules and that their regulating specific Src-family kinases were segregated oppositely. In addition, this network approach revealed the strong and sustained effect of LRV1 on the macrophage response and genes that had an early, late, or sustained impact during infection, uncovering the dynamics of the IFN response. Overall, this study contributed to shed light and dissect the intricate macrophage response toward infection by the Leishmania-LRV1 duo and revealed the crosstalk between modules made of coregulated genes and provided a new resource that can be further explored to study the impact of Leishmania on the macrophage response.
