Immune dysregulation and inflammation causing hypopigmentation in post kala-azar dermal leishmaniasis: partners in crime?

Post kala-azar dermal leishmaniasis (PKDL), a heterogeneous dermal sequela of visceral leishmaniasis (VL), is challenging in terms of its etiopathogenesis. Hypopigmentation is a consistent clinical feature in PKDL, but mechanisms contributing to the loss of melanocytes remains poorly defined. Like other hypopigmentary dermatoses - for example, vitiligo, psoriasis, and leprosy - the destruction of melanocytes is likely a multifactorial phenomenon, key players being immune dysregulation and inflammation. This review focuses on immunological mechanisms responsible for the 'murder' of melanocytes, prime suspects at the lesional sites being CD8+ T cells and keratinocytes and their criminal tools being proinflammatory cytokines, for example, IFN-γ, IL-6, and TNF-α. Collectively, these may cause decreased secretion of melanocyte growth factors, loss/attenuation of cell adhesion molecules and inflammasome activation, culminating in melanocyte death.

Mass spectrometry imaging identifies altered hepatic lipid signatures during experimental Leishmania donovani infection.

Introduction: Spatial analysis of lipids in inflammatory microenvironments is key to understand the pathogenesis of infectious disease. Granulomatous inflammation is a hallmark of leishmaniasis and changes in host and parasite lipid metabolism have been observed at the bulk tissue level in various infection models. Here, mass spectrometry imaging (MSI) is applied to spatially map hepatic lipid composition following infection with Leishmania donovani, an experimental mouse model of visceral leishmaniasis. Methods: Livers from naïve and L. donovani-infected C57BL/6 mice were harvested at 14- and 20-days post-infection (n=5 per time point). 12 µm transverse sections were cut and covered with norhamane, prior to lipid analysis using MALDI-MSI. MALDI-MSI was performed in negative mode on a Rapiflex (Bruker Daltonics) at 5 and 50 µm spatial resolution and data-dependent analysis (DDA) on an Orbitrap-Elite (Thermo-Scientific) at 50 µm spatial resolution for structural identification analysis of lipids. Results: Aberrant lipid abundances were observed in a heterogeneous distribution across infected mouse livers compared to naïve mouse liver. Distinctive localized correlated lipid masses were found in granulomas and surrounding parenchymal tissue. Structural identification revealed 40 different lipids common to naïve and d14/d20 infected mouse livers, whereas 15 identified lipids were only detected in infected mouse livers. For pathology-guided MSI imaging, we deduced lipids from manually annotated granulomatous and parenchyma regions of interests (ROIs), identifying 34 lipids that showed significantly different intensities between parenchyma and granulomas across all infected livers. Discussion: Our results identify specific lipids that spatially correlate to the major histopathological feature of Leishmania donovani infection in the liver, viz. hepatic granulomas. In addition, we identified a three-fold increase in the number of unique phosphatidylglycerols (PGs) in infected liver tissue and provide direct evidence that arachidonic acid-containing phospholipids are localized with hepatic granulomas. These phospholipids may serve as important precursors for downstream oxylipin generation with consequences for the regulation of the inflammatory cascade. This study provides the first description of the use of MSI to define spatial-temporal lipid changes at local sites of infection induced by Leishmania donovani in mice.

Upregulation of ß-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2-5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator ß-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS.

Early reduction in PD-L1 expression predicts faster treatment response in human cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is caused by Leishmania donovani in Sri Lanka. Pentavalent antimonials (e.g., sodium stibogluconate [SSG]) remain first-line drugs for CL with no new effective treatments emerging. We studied whole blood and lesion transcriptomes from Sri Lankan patients with CL at presentation and during SSG treatment. From lesions but not whole blood, we identified differential expression of immune-related genes, including immune checkpoint molecules, after onset of treatment. Using spatial profiling and RNA-FISH, we confirmed reduced expression of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) proteins on treatment in lesions of a second validation cohort and further demonstrated significantly higher expression of these checkpoint molecules on parasite-infected compared with noninfected lesional CD68+ monocytes and macrophages. Crucially, early reduction in PD-L1 but not IDO1 expression was predictive of rate of clinical cure (HR = 4.88) and occurred in parallel with reduction in parasite load. Our data support a model whereby the initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition on T cells, facilitating immune-drug synergism and clinical cure. Our findings demonstrate that PD-L1 expression can be used as a predictor of rapidity of clinical response to SSG treatment in Sri Lanka and support further evaluation of PD-L1 as a host-directed therapeutic in leishmaniasis.

Interferon-γ-Producing CD4+ T Cells Drive Monocyte Activation in the Bone Marrow During Experimental Leishmania donovani Infection.

Ly6Chi inflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chi monocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chi monocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected with Leishmania donovani, the number of bone marrow Ly6Chi monocytes increased over time. Ly6Chi monocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2-/- mice <10% of bone marrow monocytes were MHCII+ at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+ T cells. Depletion of CD4+ T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFNγ produced by CD4+ T cells. In B6.Il10-/- mice, L. donovani infection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+ T cell production of IFNγ and resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10-/- mice and drug (AmBisome®) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that during L. donovani infection, Ly6Chi monocytes are primed in the bone marrow in a process driven by CD4+ T cells and whereby IFNγ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation.

Spatially Resolved Immunometabolism to Understand Infectious Disease Progression.

Infectious diseases, including those of viral, bacterial, fungal, and parasitic origin are often characterized by focal inflammation occurring in one or more distinct tissues. Tissue-specific outcomes of infection are also evident in many infectious diseases, suggesting that the local microenvironment may instruct complex and diverse innate and adaptive cellular responses resulting in locally distinct molecular signatures. In turn, these molecular signatures may both drive and be responsive to local metabolic changes in immune as well as non-immune cells, ultimately shaping the outcome of infection. Given the spatial complexity of immune and inflammatory responses during infection, it is evident that understanding the spatial organization of transcripts, proteins, lipids, and metabolites is pivotal to delineating the underlying regulation of local immunity. Molecular imaging techniques like mass spectrometry imaging and spatially resolved, highly multiplexed immunohistochemistry and transcriptomics can define detailed metabolic signatures at the microenvironmental level. Moreover, a successful complementation of these two imaging techniques would allow multi-omics analyses of inflammatory microenvironments to facilitate understanding of disease pathogenesis and identify novel targets for therapeutic intervention. Here, we describe strategies for downstream data analysis of spatially resolved multi-omics data and, using leishmaniasis as an exemplar, describe how such analysis can be applied in a disease-specific context.

Relish plays a dynamic role in the niche to modulate Drosophila blood progenitor homeostasis in development and infection.

Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. Here, we show that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-κB (NF-κB), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase-dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program toward precocious differentiation, thereby bolstering the cellular arm of the immune response.

Malat1 Suppresses Immunity to Infection through Promoting Expression of Maf and IL-10 in Th Cells.

Despite extensive mapping of long noncoding RNAs in immune cells, their function in vivo remains poorly understood. In this study, we identify over 100 long noncoding RNAs that are differentially expressed within 24 h of Th1 cell activation. Among those, we show that suppression of Malat1 is a hallmark of CD4+ T cell activation, but its complete deletion results in more potent immune responses to infection. This is because Malat1-/- Th1 and Th2 cells express lower levels of the immunosuppressive cytokine IL-10. In vivo, the reduced CD4+ T cell IL-10 expression in Malat1-/- mice underpins enhanced immunity and pathogen clearance in experimental visceral leishmaniasis (Leishmania donovani) but more severe disease in a model of malaria (Plasmodium chabaudi chabaudi AS). Mechanistically, Malat1 regulates IL-10 through enhancing expression of Maf, a key transcriptional regulator of IL-10 Maf expression correlates with Malat1 in single Ag-specific Th cells from P. chabaudi chabaudi AS-infected mice and is downregulated in Malat1-/- Th1 and Th2 cells. The Malat1 RNA is responsible for these effects, as antisense oligonucleotide-mediated inhibition of Malat1 also suppresses Maf and IL-10 levels. Our results reveal that through promoting expression of the Maf/IL-10 axis in effector Th cells, Malat1 is a nonredundant regulator of mammalian immunity.

Dpp dependent Hematopoietic stem cells give rise to Hh dependent blood progenitors in larval lymph gland of Drosophila.

Drosophila hematopoiesis bears striking resemblance with that of vertebrates, both in the context of distinct phases and the signaling molecules. Even though, there has been no evidence of Hematopoietic stem cells (HSCs) in Drosophila, the larval lymph gland with its Hedgehog dependent progenitors served as an invertebrate model of progenitor biology. Employing lineage-tracing analyses, we have now identified Notch expressing HSCs in the first instar larval lymph gland. Our studies clearly establish the hierarchical relationship between Notch expressing HSCs and the previously described Domeless expressing progenitors. These HSCs require Decapentapelagic (Dpp) signal from the hematopoietic niche for their maintenance in an identical manner to vertebrate aorta-gonadal-mesonephros (AGM) HSCs. Thus, this study not only extends the conservation across these divergent taxa, but also provides a new model that can be exploited to gain better insight into the AGM related Hematopoietic stem cells (HSCs).