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Background: The aim of this study is to investigate tubular and glomerular function after visceral leishmaniasis (VL) treatment with pentavalent antimonials. Methods: This is a prospective study including 14 patients with VL diagnosis treated with pentavalent antimonials. Urine acidification and concentration tests were performed. Estimated glomerular filtration rate (eGFR), fractional excretion of sodium (FE Na ) and potassium (FE K ) and free water clearance (C H2O ) were measured to assess glomerular and tubular function. Results: The VL group had a significantly lower FE K , serum sodium and plasma osmolality (P osm ). No significant differences were found regarding proteinuria, eGFR, FE Na or C H2O . Patients in the VL group had lower urinary osmolality (U osm ) before DDAVP use when compared to the control group, as well as a lower U/P osm . The urinary pH before and after CaCl 2 load was higher in the VL group. Conclusion: This study shows evidence of reversal of some tubular dysfunction in VL, but other dysfunctions may persist, especially urinary acidification capacity.

Despite the withdrawal of pentavalent-antimonials in treating Visceral leishmaniasis from India, recent clinical isolates of Leishmania donovani (LD) exhibit unresponsiveness towards pentavalent-antimony (LD-R). This antimony-unresponsiveness points towards a genetic adaptation that underpins LD-R’s evolutionary persistence and dominance over sensitive counterparts (LD-S). This study highlights how LD evolutionarily tackled antimony exposure and gained increased potential of scavenging host-iron within its parasitophorous vacuoles (PV) to support its aggressive proliferation. Even though anti-leishmanial activity of pentavalent antimonials relies on triggering oxidative outburst, LD-R exhibits a surprising strategy of promoting reactive oxygen species (ROS) generation in infected macrophages. An inherent metabolic shift from glycolysis to Pentose Phosphate shunt allows LD-R to withstand elevated ROS by sustaining heightened levels of NADPH. Elevated ROS levels on the other hand trigger excess iron production, and LD-R capitalizes on this surplus iron by selectively reshuffling macrophage-surface iron exporter, Ferroportin, around its PV thereby gaining a survival edge as a heme-auxotroph. Higher iron utilization by LD-R leads to subsequent iron insufficiency, compensated by increased erythrophagocytosis through the breakdown of SIRPα-CD47 surveillance, orchestrated by a complex interplay of two proteases, Furin and ADAM10. Understanding these mechanisms is crucial for managing LD-R-infections and their associated complications like severe anemia, and may also provide valuable mechanistic insights into understanding drug unresponsiveness developed in other intracellular pathogens that rely on host iron.

Intracellular phagosomal pathogens represent a formidable challenge for innate immune cells, as, paradoxically, these phagocytic cells can act as both host cells that support pathogen replication and, when properly activated, are the critical cells that mediate pathogen elimination. Infection by parasites of the Leishmania genus provides an excellent model organism to investigate this complex host-pathogen interaction. In this review we focus on the dynamics of Leishmania amazonensis infection and the host innate immune response, including the impact of the adaptive immune response on phagocytic host cell recruitment and activation. L. amazonensis infection represents an important public health problem in South America where, distinct from other Leishmania parasites, it has been associated with all three clinical forms of leishmaniasis in humans: cutaneous, muco-cutaneous and visceral. Experimental observations demonstrate that most experimental mouse strains are susceptible to L. amazonensis infection, including the C57BL/6 mouse, which is resistant to other species such as Leishmania major , Leishmania braziliensis and Leishmania infantum . In general, the CD4 + T helper (Th)1/Th2 paradigm does not sufficiently explain the progressive chronic disease established by L. amazonensis , as strong cell-mediated Th1 immunity, or a lack of Th2 immunity, does not provide protection as would be predicted. Recent findings in which the balance between Th1/Th2 immunity was found to influence permissive host cell availability via recruitment of inflammatory monocytes has also added to the complexity of the Th1/Th2 paradigm. In this review we discuss the roles played by innate cells starting from parasite recognition through to priming of the adaptive immune response. We highlight the relative importance of neutrophils, monocytes, dendritic cells and resident macrophages for the establishment and progressive nature of disease following L. amazonensis infection.

Leishmania is an intracellular protozoan parasite that causes leishmaniasis, a disease prevalent in 97 countries. Co-infection with HIV increases susceptibility to visceral leishmaniasis (VL), accelerating HIV’s progression to AIDS. Managing VL in HIV-infected individuals is challenging due to atypical presentations and limited therapeutic responses, highlighting the need to develop new disease management strategies. Extracellular vesicles (EVs) hold great promise for this goal as they can be used for a higher understanding of biological processes and biomarker discovery. In this context, a proteomic analysis was carried out from plasma-EVs of an HIV/VL patient over two years and compared to HIV and healthy controls. The analysis confirmed classical EV markers but showed limited detection of Leishmania proteins. However, variations in human protein abundance related to relevant immunological processes were observed. Notably, the macrophage receptor with a collagenous structure (MARCO) was consistently detected only in the patient and not in the control groups. Significantly, the relevance of MARCO as a possible VL biomarker was confirmed using a validation cohort with five VL patients and its detection by Western Blot was possible. Although MARCO warrants further investigation as a VL related biomarker, the study of EVs confirmed their promise of being a privileged window into this disease. Future studies are needed to broaden data on EVs in infections to improve clinical management.

Leishmania donovani (Ld) is the causative agent of visceral leishmaniasis, which results in death if not treated. In mammalian cells, Ld live in vacuolar compartments called Leishmania parasitophorous vacuoles (LdLPVs) that enigmatically divide following parasite replication. We evaluated the role of the endosomal sorting complex required for transport (ESCRT) machinery in the scission of LdLPVs. We found that ESCRT components are constitutively recruited to LdLPVs. We propose that this recruitment depends on the expression of PI(3,4)P2 on LdLPVs. The knockdown (KD) of upstream components of the ESCRT machinery revealed that ALIX, but not TSG101 or VPS28, led to a significant reduction in the parasite burden in infected cultures. Interestingly, LdLPVs in ALIXKDs were more distended and harbored more than 2 parasites. Incorporation of BrdU into Leishmania in THP-1 macrophages revealed that parasite replication was inhibited in ALIXKD due to defective LdLPV scission. These findings establish that non-canonical activation of the ESCRT machinery is required for Leishmania to replicate within macrophages.

Lipid metabolism plays a decisive role in host-pathogen interactions and immune regulation, with apolipoproteins (Apo) being central to this process. However, their role in leishmaniasis remains unexplored. Herein, we deliberate the immunoregulatory function of ApoA1 during Leishmania donovani infection using THP-1-derived macrophages alone and in combination with T lymphocytes derived from human PBMC. We found low serum ApoA1 levels in active VL and PKDL than in healthy controls. It was shown that direct interaction of ApoA1 with ABCA1 (ATP-binding cassette transporter A1) on macrophages promotes cholesterol efflux, reflected by increased HDL levels and reduced total cellular cholesterol. This phenomenon was associated with reduced Leishmania infectivity and its downstream signaling in macrophages, i.e., downregulation of PPAR-γ and the endoplasmic reticulum-stress marker CHOP. Additionally, ApoA1 in the presence of extracellular HDL slightly promoted macrophage polarization towards M1, as indicated by increased expression of IL-12 and iNOS2 or nitric oxide production, alongside reduced expression of M2 phenotype-associated markers, including IL-10 and arginase. In co-culture with PBMC-derived T-cells, ApoA1-primed macrophages facilitated Th1 polarization, as demonstrated by increased IFN-γ and STAT1, and indirectly by reduced expression of Th2-specific markers (GATA-3 and IL-4). Overall, these results implicate ApoA1 as a vital immunomodulatory factor and potential therapeutic target in leishmaniasis.

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, remains a significant public health concern that cannot be overlooked in underdeveloped regions. Studies suggest that lipids play a crucial role in the survival of Leishmania parasites in mammalian hosts. However, a comprehensive understanding of the characteristics and underlying mechanisms of lipid metabolism in VL hosts is lacking. In this study, we conducted lipidomic and transcriptomic analyses of liver tissues from VL golden hamsters at 12 weeks post-infection (WPI) and performed integrated analysis. Simultaneously, qPCR validation of several key regulatory enzymes was performed at the tissue level. The results revealed a decreased abundance of phospholipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and an increased abundance of their metabolites, including lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and platelet-activating factor (PAF). Conjoint pathway analysis revealed that glycerophospholipid (GPL) metabolism, arachidonic acid (AA) metabolism, glycerolipid metabolism, and linolenic acid metabolism were the pathways with relatively high proportions of common enrichment. In the GPL metabolism and AA metabolism pathways, the transcription levels of genes such as phospholipase A2 (PLA2) family enzymes, cyclooxygenase-2 (Cox-2), arachidonate 5-lipoxygenase (Alox5), and hematopoietic prostaglandin D synthase (Hpgds), all of which regulate phospholipid hydrolysis and lipid mediator production, were significantly increased. Additionally, we found that the expression of lysophosphatidylcholine acyltransferase 1/2 (Lpcat1/2), the enzyme regulating PC remodeling, was upregulated and that the levels of saturated PCs (PC30:0, PC32:0, and PC34:0) were simultaneously significantly increased simultaneously. These findings suggest that Leishmania infection may regulate PC remodeling in the host liver and increase membrane phospholipid metabolism, resulting in the production of a series of lipid mediators that participate in immune regulation; this could have a significant impact on the survival of Leishmania in the host and on the progression of the disease.

Visceral leishmaniasis (VL) occurs more frequently in immunosuppressed individuals, especially those undergoing immunosuppressive drug therapy for an autoimmune disease. In those receiving TNF antagonist therapy (anti-TNF), the course of VL is more severe and the response to traditional leishmanicidal treatments, such as antimonials (Sb), is often reduced. This effect of anti-TNF treatment is observed in our immunosuppressed-mouse model of VL. In this model, we compared anti-TNF immunosuppression with no immunosuppression before and after VL treatment with Sb. Serum-derived extracellular vesicles (EVs) were analyzed through label-free quantitative proteomics to identify proteins involved in both VL severity and the impact of anti-TNF immunosuppression on treatment outcome. In total, 223 dysregulated proteins were found in the pre-treatment groups, the majority of which, such as vitronectin, haemopexin or caveolin-1, were downregulated in the anti-TNF samples. In contrast, 173 proteins were identified in the Sb-treatment groups, most of which were found enriched in the anti-TNF plus treatment samples (anti-TNF+Sb) including fibronectin, transferrin, vitronectin and dipeptidyl peptidase-4. These differentially-expressed proteins were associated with pathways related to the immune system, liver regeneration, and ion transport. Our findings have useful implications for the clinical management of VL patients under anti-TNF immunosuppression.

Berardinelli–Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals’ premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways.

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L . infantum , the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L . infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 –putative) form a LinfCRL1 complex structurally similar to the H . sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1–6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L . infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L . infantum . Thus, a new class of E3 ubiquitin ligases has been described in L . infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment.