Mast cell-derived IL-10 protects intestinal barrier integrity during malaria in mice and regulates parasite transmission to Anopheles stephensi with a female-biased immune response.

Malaria is strongly predisposed to bacteremia, which is associated with increased gastrointestinal permeability and a poor clinical prognosis. We previously identified mast cells (MCs) as mediators of intestinal permeability in malaria and described multiple cytokines that rise with parasitemia, including interleukin (IL)-10, which could protect the host from an inflammatory response and alter parasite transmission to Anopheles mosquitoes. Here, we used the Cre-loxP system and non-lethal Plasmodium yoelii yoelii 17XNL to study the roles of MC-derived IL-10 in malaria immunity and transmission. Our data suggest a sex-biased and local inflammatory response mediated by MC-derived IL-10, supported by early increased number and activation of MCs in females relative to males. Increased parasitemia in female MC IL-10 (-) mice was associated with increased ileal levels of chemokines and plasma myeloperoxidase (MPO). We also observed increased intestinal permeability in female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice but no differences in blood bacterial 16S DNA levels. Transmission success of P. yoelii to A. stephensi was higher in female relative to male mice and from female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice. These patterns were associated with increased plasma levels of pro-inflammatory cytokines in female MC IL-10 (-) mice and increased plasma levels of chemokines and markers of neutrophil activation in male MC IL-10 (-) mice. Overall, these data suggest that MC-derived IL-10 protects intestinal barrier integrity, regulates parasite transmission, and controls local and systemic host immune responses during malaria, with a female bias.

Basophil Depletion Alters Host Immunity, Intestinal Permeability, and Mammalian Host-to-Mosquito Transmission in Malaria.

Malaria-induced bacteremia has been shown to result from intestinal mast cell (MC) activation. The appearance of MCs in the ileum and increased intestinal permeability to enteric bacteria are preceded by an early Th2-biased host immune response to infection, characterized by the appearance of IL-4, IL-10, mast cell protease (Mcpt)1 and Mcpt4, and increased circulating basophils and eosinophils. Given the functional similarities of basophils and MCs in the context of allergic inflammation and the capacity of basophils to produce large amounts of IL-4, we sought to define the role of basophils in increased intestinal permeability, in MC influx, and in the development of bacteremia in the context of malaria. Upon infection with nonlethal Plasmodium yoelii yoelii 17XNL, Basoph8 × ROSA-DTα mice or baso (-) mice that lack basophils exhibited increased intestinal permeability and increased ileal MC numbers, without any increase in bacterial 16S ribosomal DNA copy numbers in the blood, relative to baso (+) mice. Analysis of cytokines, chemokines, and MC-associated factors in the ileum revealed significantly increased TNF-α and IL-13 at day 6 postinfection in baso (-) mice compared with baso (+) mice. Moreover, network analysis of significantly correlated host immune factors revealed profound differences between baso (-) and baso (+) mice following infection in both systemic and ileal responses to parasites and translocated bacteria. Finally, basophil depletion was associated with significantly increased gametocytemia and parasite transmission to Anopheles mosquitoes, suggesting that basophils play a previously undescribed role in controlling gametocytemia and, in turn, mammalian host-to-mosquito parasite transmission.

The Basophil IL-18 Receptor Precisely Regulates the Host Immune Response and Malaria-Induced Intestinal Permeability and Alters Parasite Transmission to Mosquitoes without Effect on Gametocytemia.

We have recently demonstrated that basophils are protective against intestinal permeability during malaria and contribute to reduced parasite transmission to mosquitoes. Given that IL-18 is an early cytokine/alarmin in malaria and has been shown to activate basophils, we sought to determine the role of the basophil IL-18R in this protective phenotype. To address this, we infected control [IL18r flox/flox or basoIL-18R (+)] mice and mice with basophils lacking the IL-18R [IL18r flox/flox × Basoph8 or basoIL-18R (-)] with Plasmodium yoelii yoelii 17XNL, a nonlethal strain of mouse malaria. Postinfection (PI), intestinal permeability, ileal mastocytosis, bacteremia, and levels of ileal and plasma cytokines and chemokines were measured through 10 d PI. BasoIL-18R (-) mice exhibited greater intestinal permeability relative to basoIL-18R (+) mice, along with increased plasma levels of proinflammatory cytokines at a single time point PI, day 4 PI, a pattern not observed in basoIL-18R (+) mice. Surprisingly, mosquitoes fed on basoIL-18R (-) mice became infected less frequently than mosquitoes fed on basoIL-18R (+) mice, with no difference in gametocytemia, a pattern that was distinct from that observed previously with basophil-depleted mice. These findings suggest that early basophil-dependent protection of the intestinal barrier in malaria is mediated by IL-18, and that basophil IL-18R-dependent signaling differentially regulates the inflammatory response to infection and parasite transmission.

Mast Cell Chymase/Mcpt4 Suppresses the Host Immune Response to Plasmodium yoelii, Limits Malaria-Associated Disruption of Intestinal Barrier Integrity and Reduces Parasite Transmission to Anopheles stephensi.

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4-/-) mice and Mcpt4+/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4-/- mice compared with Mcpt4+/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4-/- mice. Relative to infected Mcpt4+/+ mice, ileal MC accumulation in Mcpt4-/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4+/+ but not Mcpt4-/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4-/- mice, while levels of IL-2, IL-10 and MIP1ß (CCL4) were increased over the same period in Mcpt4+/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4-/- mice and type-2 response in Mcpt4+/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4-/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4+/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4-/- mice compared with infected Mcpt4+/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.

Advantages of Single-Nucleus over Single-Cell RNA Sequencing of Adult Kidney: Rare Cell Types and Novel Cell States Revealed in Fibrosis.

BACKGROUND: A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses. METHODS: We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc-seq, and 10X Chromium platforms on adult mouse kidney. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery. RESULTS: A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation-induced stress response genes. By contrast, snRNA-seq from all three platforms captured a diversity of kidney cell types that were not represented in the scRNA-seq dataset, including glomerular podocytes, mesangial cells, and endothelial cells. No stress response genes were detected. Our snRNA-seq protocol yielded 20-fold more podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, respectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast cell states, and previously unidentified tubulointerstitial signaling pathways. CONCLUSIONS: snRNA-seq achieves comparable gene detection to scRNA-seq in adult kidney, and it also has substantial advantages, including reduced dissociation bias, compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and successful performance on inflamed fibrotic kidney.

A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition.

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-ß (TGF-ß). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-ß-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

Comparative Analysis and Refinement of Human PSC-Derived Kidney Organoid Differentiation with Single-Cell Transcriptomics.

Kidney organoids derived from human pluripotent stem cells have great utility for investigating organogenesis and disease mechanisms and, potentially, as a replacement tissue source, but how closely organoids derived from current protocols replicate adult human kidney is undefined. We compared two directed differentiation protocols by single-cell transcriptomics of 83,130 cells from 65 organoids with single-cell transcriptomes of fetal and adult kidney cells. Both protocols generate a diverse range of kidney cells with differing ratios, but organoid-derived cell types are immature, and 10%-20% of cells are non-renal. Reconstructing lineage relationships by pseudotemporal ordering identified ligands, receptors, and transcription factor networks associated with fate decisions. Brain-derived neurotrophic factor (BDNF) and its cognate receptor NTRK2 were expressed in the neuronal lineage during organoid differentiation. Inhibiting this pathway improved organoid formation by reducing neurons by 90% without affecting kidney differentiation, highlighting the power of single-cell technologies to characterize and improve organoid differentiation.

Single-Cell Transcriptomics of a Human Kidney Allograft Biopsy Specimen Defines a Diverse Inflammatory Response.

Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen.Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection.Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database.Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.

Nitrite Therapy Ameliorates Myocardial Dysfunction via H2S and Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2)-Dependent Signaling in Chronic Heart Failure.

BACKGROUND: Bioavailability of nitric oxide (NO) and hydrogen sulfide (H2S) is reduced in heart failure (HF). Recent studies suggest cross-talk between NO and H2S signaling. We previously reported that sodium nitrite (NaNO2) ameliorates myocardial ischemia-reperfusion injury and HF. Nuclear factor-erythroid-2-related factor 2 (Nrf2) regulates the antioxidant proteins expression and is upregulated by H2S. We examined the NaNO2 effects on endogenous H2S bioavailability and Nrf2 activation in mice subjected to ischemia-induced chronic heart failure (CHF). METHODS AND RESULTS: Mice underwent 60 minutes of left coronary artery occlusion and 4 weeks of reperfusion. NaNO2 (165 µg/kgic) or vehicle was administered at reperfusion and then in drinking water (100 mg/L) for 4 weeks. Left ventricular (LV), ejection fraction (EF), LV end diastolic (LVEDD) and systolic dimensions (LVESD) were determined at baseline and at 4 weeks of reperfusion. Myocardial tissue was analyzed for oxidative stress and respective gene/protein-related assays. We found that NaNO2 therapy preserved LVEF, LVEDD and LVSD at 4 weeks during ischemia-induced HF. Myocardial malondialdehyde and protein carbonyl content were significantly reduced in NaNO2-treated mice as compared to vehicle, suggesting a reduction in oxidative stress. NaNO2 therapy markedly increased expression of Cu,Zn-superoxide dismutase, catalase, and glutathione peroxidase during 4 weeks of reperfusion. Furthermore, NaNO2 upregulated the activity of Nrf2, as well as H2S-producing enzymes, and ultimately increased H2S bioavailability in ischemia-induced CHF in mice as compared with vehicle. CONCLUSIONS: Our results demonstrate that NaNO2 therapy significantly improves LV function via increasing H2S bioavailability, Nrf2 activation, and antioxidant defenses.