Christian Kurts

Microglia are innate immune cells of the central nervous system (CNS). They extend their processes and migrate toward injuries in vivo. However, how the fractalkine receptor (CX3CR1) influences microglial migration remains unknown. Label‐free proteomic profiling predicted changes in Ras homology family (RHO)‐signaling activity that hint at dysregulated cytoskeleton signaling in Cx3cr1‐ deficient murine cortex tissue. To further investigate microglial migration, we carried out two‐photon in vivo imaging at 4‐h intervals for 72 h after a laser lesion in the cortex. Cx3cr1 ‐deficient microglia showed enhanced migration toward the lesion. Additionally, the length and velocity of microglial fine processes extending toward the lesion were increased in Cx3cr1‐ deficient microglia. Migration remained unchanged in Ccr2 ‐deficient mice, indicating that monocyte‐derived macrophages/microglia did not contribute to microglia accumulation around the lesion. These results demonstrate that CX3CR1 modulates microglia migration toward laser‐induced CNS injury. Manipulating microglia migration via the CX3CR1 signaling axis is therefore a potential target for the treatment of CNS injury.

Introduction: Acute pyelonephritis remains a major clinical problem. Relapses occur despite apparently appropriate antibiotic therapy, suggesting that uropathogenic Escherichia coli (UPEC) persist in intrarenal niches. Intracellular bacterial reservoirs are a plausible explanation, but the relevant host cells, entry mechanisms and therapeutic implications in the kidney remain undefined. In principle, such reservoirs should favor choosing intracellularly active antibiotics, but increasing resistance to many of these agents leaves β-lactams widely used in clinical practice, despite their predominantly extracellular activity. Methods: We analyzed murine pyelonephritis to identify the cellular reservoir of persistent UPEC. We generated mice genetically deficient for complement receptors CR3 and CR4 and tested their role in bacterial entry and persistence in vivo . Pharmacological complement receptor inhibition was applied to assess whether blocking bacterial re-entry into host cells improves antibiotic efficacy. Results: Renal MNP were identified as the major intracellular reservoir for UPEC in mice. Complement opsonization enabled bacterial entry into these cells through CR3 and CR4, allowing UPEC to evade neutrophil-mediated killing and extracellularly active antibiotics. Genetic deletion of CR3 and CR4 abolished intracellular bacterial persistence and reduced renal bacterial burden. Because MNP undergo physiological turnover, intracellular UPEC must periodically exit host cells and infect new ones. Pharmacological inhibition of complement receptors prevented such bacterial re-entry and enhanced the efficacy of β-lactam antibiotics which cannot penetrate cell membranes. Conclusions: Complement receptor-mediated entry into renal MNP establishes an intracellular UPEC reservoir that promotes persistence during experimental pyelonephritis. Blocking these receptors prevents renewal of the intracellular niche and improves β-lactam efficacy in mice.

Birds use a variety of navigational strategies, including the geomagnetic field, especially when other cues are not available, such as under overcast or nocturnal conditions. Magnetite particles in the beak, cryptochromes in the eye, cellular ion-channel alterations, and changes in the vestibular system have been proposed to explain magnetoreception, but the exact mechanisms remain debated. Here, we used physical, morphological, functional, and genomic assays to identify the presence of superparamagnetic macrophages in the liver. We found that after macrophage depletion, pigeons flying under overcast conditions lacked their usual orientation capabilities. Orientation was unimpaired in birds without macrophages when the sun was visible, suggesting that this was their primary cue. We propose that in homing pigeons, superparamagnetic macrophages in the liver are required for finding magnetic direction.

When cells are isolated for single‐cell RNA sequencing (scRNA‐seq), their positional information is inevitably lost. Here, an algorithm termed MERLIN is described that can reconstruct such information in organs with compartmentalized anatomy, such as the kidney. Several independent immune cell scRNA‐seq datasets from three renal compartments were generated to train different machine learning algorithms. A modified multi‐layer Perceptron approach most accurately predicted positions of resident macrophages, best, achieving over 75% accuracy in both murine and human kidney datasets. More motile immune cells, like lymphocytes, were not predictable. Positional transcriptomic fingerprints were enriched in pathways of microenvironmental responses and cellular adaptation, and showed a sex bias. MERLIN also predicted positions of resident and recently recruited macrophages in a crescentic glomerulonephritis mouse model. Analysis of published scRNA‐seq datasets from endotoxin‐ and ischemia/reperfusion‐induced models of acute kidney injury revealed proinflammatory responses predominantly in outer medullary macrophages, consistent with the known pathology. Moreover, the response of cortical macrophages to commonly used therapies for diabetic nephropathy aligned with the known clinical drug efficacy. Finally, MERLIN was successfully trained to predict the spatial distribution of brain microglia. Together, MERLIN enables spatial interpretation of scRNA‐seq datasets in organs with defined anatomical regions and enhances mechanistic insights into disease processes.

Extracting single-molecule lectin binding kinetics from primary cells has not been possible to date. Here, we present Glyco-PAINT-APP (Automated Processing Pipeline), an automated method that enables the extraction of subcellular glycan interaction kinetics using a Points Accumulation for Imaging in Nano-Topography (PAINT)-based approach. This approach leverages an algorithm for precise, high-throughput, subcellular analysis of glycan binding dynamics and facilitates functional correlation studies between glycoform binding patterns and immune cell polarization. Using synthetic glycans and glycosylated antigens, we demonstrate the ability of the technique to automatically correlate glycan binding parameters in subregions of dendritic cell membranes with increased uptake and cross-presentation efficiency of these antigens. Additionally, we show how the method can uncover subtle differences in MR-mediated glycan binding across various MR-expressing primary cells and cell lines. Taken together, Glyco-PAINT-APP enables insights into the cell-intrinsic heterogeneity of glycan-structure-activity relationships in myeloid immune cells.

High salt diet (HSD) is known to reduce cancer growth in some tumor models, which has been attributed to tissue accumulation of sodium that enhances local anti-tumor immunity. Here, we show that a HSD inhibits melanoma growth independent of sodium accumulation and immune cells in skin and lung. Melanoma cells from mice on a HSD upregulated the metabolic inhibitor Tuberous sclerosis complex 2 (TSC2), causing metabolic shutdown despite nutrient availability. Furthermore, Microphthalmia-associated transcription factor (MITF), a crucial regulator of melanoma metabolism and differentiation, was upregulated, resulting in enhanced melanogenesis and cell cycle arrest. Thus, a HSD reversed the de-differentiation of melanoma cells and promoted their re-differentiation into a “normal” melanocytic state. These findings suggest that the anti-tumor effect of HSD may be tumor-specific and in some cases immune cell-independent.

Glucocorticoids are widely used to treat autoimmune diseases like crescentic glomerulonephritis (cGN), but their immunosuppressive functions are not fully understood. Here, we generated a single immune cell sequencing atlas at different stages of experimental cGN. We identified a proinflammatory neutrophil subset as important for disease progression and as a glucocorticoid target. Such neutrophils produced proinflammatory cytokines known to drive cGN and expressed Siglec-F and decoy tumor necrosis factor–related apoptosis-inducing ligand receptor 1 (dcTRAIL-R1) in mice and SIGLEC8 in humans. Depleting such neutrophils attenuated disease in mice, whereas their adoptive transfer aggravated disease. They differentiated within the inflamed kidney in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by clonally expanded CD4 ⁺ T helper 17 (T _H 17) cells and persisted locally longer than normal neutrophils. Glucocorticoids decreased intrarenal numbers of T _H 17 cells and down-regulated GM-CSF receptor expression by neutrophils and reduced their cytokine production. Selective genetic reduction of glucocorticoid receptor expression in neutrophils reenabled their in vivo differentiation during glucocorticoid therapy and aggravated cGN. Low glucocorticoid doses were sufficient to prevent intrarenal neutrophil differentiation in mice, if applied repetitively, even without an initial high-dose steroid pulse. Spatial sequencing of kidney biopsies, especially from patients with high disease activity, uncovered similar neutrophils in intrarenal inflammatory niches, and their abundance was lower after repetitive low-dose glucocorticoid application. These findings identify proinflammatory neutrophils as progression drivers in cGN and suggest that low-dose glucocorticoid therapy may be sufficient to suppress them.

BK virus nephropathy is a severe, graft-threatening complication of kidney transplantation that requires an effective T cell response. It typically emerges in the kidney medulla. Elevated osmolyte concentrations that dynamically respond to loop diuretic therapy characterize this environment. Here, BK viremia development in kidney graft recipients negatively correlated with loop diuretic therapy. The association remained significant in multivariable and propensity score–matched analyses. Kidney function was better preserved and CD8 ⁺ T cell abundance higher in loop diuretic–exposed allografts. CD8 ⁺ T cell densities in healthy human and murine kidney medulla were lower than in cortex and increased upon loop diuretic therapy in mice. As a potential underlying mechanism, kidney medullary NaCl and urea concentrations decreased primary human CD8 ⁺ T cell numbers in vitro by induction of cell death and limitation of proliferation, respectively. Both osmolytes downregulated interferon-related gene expression. NaCl induced p53-dependent apoptosis and upregulated Na ⁺ -transporter SLC38A2 , which promoted caspase-3 activation. Both decreased T cell response and cytokine secretion in response to viral peptide and allogeneic tubular epithelial cell killing, components of anti-BK virus response in the kidney allograft. Our results propose osmolyte-mediated mitigation of CD8 ⁺ T cell function as a what we believe to be novel mechanism that impairs immune response to BK virus, the therapeutic potential of which is testable.