Background and Aims: Human innate lymphoid cells (ILCs) are critically involved in the modulation of homeostatic and inflammatory processes in various tissues. However, only little is known about the composition of the intrahepatic ILC pool and its potential role in chronic liver disease. Here, we performed a detailed characterization of intrahepatic ILCs in both healthy and fibrotic livers. Approach and Results: A total of 50 livers (nonfibrotic = 22, and fibrotic = 29) were analyzed and compared with colon and tonsil tissue (each N = 14) and peripheral blood (N = 32). Human intrahepatic ILCs were characterized ex vivo and on stimulation using flow cytometry and single-cell RNA sequencing. ILC differentiation and plasticity were analyzed by both bulk and clonal expansion experiments. Finally, the effects of ILC-derived cytokines on primary human HSteCs were studied. Unexpectedly, we found that an “unconventional” ILC3-like cell represented the major IL-13-producing liver ILC subset. IL-13⁺ ILC3-like cells were specifically enriched in the human liver, and increased frequencies of this cell type were found in fibrotic livers. ILC3-derived IL-13 production induced upregulation of proinflammatory genes in HSteCs, indicating a potential role in the regulation of hepatic fibrogenesis. Finally, we identified KLRG1-expressing ILC precursors as the potential progenitor of hepatic IL-13⁺ ILC3-like cells. Conclusions: We identified a formerly undescribed subset of IL-13–producing ILC3-like cells that is enriched in the human liver and may be involved in the modulation of chronic liver disease.

Key aspects of intestinal T cells, including their antigen specificity and their selection by the microbiota and other intestinal antigens, as well as the contribution of individual T cell clones to regulatory and effector functions, remain unresolved. Here we tracked adoptively transferred T cell populations to specify the interrelation of T cell receptor repertoire and the gut antigenic environment. We show that dominant TCRα clonotypes were shared between interferon-γ- and interleukin-17-producing but not regulatory Foxp3⁺ T cells. Identical TCRα clonotypes accumulated in the colon of different individuals, whereas antibiotics or defined colonization correlated with the expansion of distinct expanded T cell clonotypes. Our results demonstrate key aspects of intestinal CD4⁺ T cell activation and suggest that few microbial species exert a dominant effect on the intestinal T cell repertoire during colitis. We speculate that dominant proinflammatory T cell clones might provide a therapeutic target in human inflammatory bowel disease.

The periodontium comprising periodontal ligament (PDL), gingiva, and epithelium play crucial roles in maintaining tooth integrity and function. Understanding tissue cellular composition and gene expression is crucial for illuminating periodontal pathophysiology. This study aimed to identify tissue-specific markers via scRNA-Seq. Primary human PDL, gingiva, and epithelium tissues (n = 7) were subjected to cell hashing and sorting. scRNA-Seq library preparation using 10× Genomics protocol and Illumina sequencing was conducted. The analysis was performed using Cellranger (v3.1.0), with downstream analysis via R packages Seurat (v5.0.1) and SCORPIUS (v1.0.9). Investigations identified eight distinct cellular clusters, revealing the ubiquitous presence of epithelial and gingival cells. PDL cells evolved in two clusters with numerical superiority. The other clusters showed varied predominance regarding gingival and epithelial cells or an equitable distribution of both. The cluster harboring most cells mainly consisted of PDL cells and was present in all donors. Some of the other clusters were also tissue-inherent, while the presence of others was environmentally influenced, revealing variability across donors. Two clusters exhibited genetic profiles associated with tissue development and cellular integrity, respectively, while all other clusters were distinguished by genes characteristic of immune responses. Developmental trajectory analysis uncovered that PDL cells may develop after epithelial and gingival cells, suggesting the inherent PDL cell-dominated cluster as a final developmental stage. This single-cell RNA sequencing study delineates the hierarchical organization of periodontal tissue development, identifies tissue-specific markers, and reveals the influence of environmental factors on cellular composition, advancing our understanding of periodontal biology and offering potential insights for therapeutic interventions.

Chronic kidney disease (CKD) affects more than 10% of the global population. As kidney function negatively correlates with the presence of interstitial fibrosis, the development of new anti-fibrotic therapies holds promise to stabilize functional decline in CKD patients. The goal of the study was to generate a scalable bioprinted 3-dimensional kidney tubulo-interstitial disease model of kidney fibrosis. We have generated novel human PDGFRβ⁺ pericytes, CD10⁺ epithelial and CD31⁺ endothelial cell lines and compared their transcriptomic signature to their in vivo counterpart using bulk RNA sequencing in comparison to human kidney single cell RNA-sequencing datasets. This comparison indicated that the novel cell lines still expressed kidney cell specific genes and shared many features with their native cell-state. PDGFRβ⁺ pericytes showed three-lineage differentiation capacity and differentiated towards myofibroblasts following TGFβ treatment. We utilized a fibrinogen/gelatin-based hydrogel as bioink and confirmed a good survival rate of all cell types within the bioink after printing. We then combined all three cells in a bioprinted model using separately printed compartments for tubule epithelium, and interstitial endothelium and pericytes. We confirmed that this 3D printed model allows to recapitulate key disease driving epithelial-mesenchymal crosstalk mechanisms of kidney fibrosis since injury of epithelial cells prior to bioprinting resulted in myofibroblast differentiation and fibrosis driven by pericytes after bioprinting. The bioprinted model was also scalable up to a 96-well format.

Neuronal mechanisms that facilitate adaptive strategies to enable an animal to overcome anxiety in threatening situations remain unknown. Using single-cell calcium imaging and cell-type-specific activity manipulations in behaving mice, we identified leptin-sensitive neuronal subpopulations in the lateral hypothalamus (LH; LepR ^LH ) that encode anxiogenic stimuli. In high-anxiety animals, LepR ^LH neurons differentiated poorly among anxiogenic stimuli and were inhibited by input from the prefrontal cortex. The activity of LepR ^LH neurons predicted the anxiety level of individual animals, and the activation of LepR ^LH neurons enabled adaptive responses under anxiogenic conditions—exploration of new terrain, eating despite anxiogenic environment and limiting maladaptive excessive locomotion in an anorexia nervosa disease model. Thus, leptin-sensitive neuronal subpopulations in the LH enable adaptive fulfillment of vital needs despite anxiogenic conditions, both in healthy and pathological states.

A key challenge for the discovery of novel molecular targets and therapeutics against pediatric bone metastatic disease is the lack of bona fide in vitro cell models. Here, we show that a beta-tricalcium phosphate (β-TCP) multicellular 3D in vitro bone microtissue model reconstitutes key phenotypic and transcriptional patterns of native metastatic tumor cells while promoting their stemness and proinvasive features. Comparing planar with interconnected channeled scaffolds, we identified geometry as a dominant orchestrator of proangiogenic traits in neuroblastoma tumor cells. On the other hand, the β-TCP-determined gene signature was DNA replication related. Jointly, the geometry and chemical impact of β-TCP revealed a prometastatic landscape of the engineered tumor microenvironment. The proposed 3D multicellular in vitro model of pediatric bone metastatic disease may advance further analysis of the molecular, genetic and metabolic bases of the disease and allow more efficient preclinical target validations.