Generation of complex bone marrow organoids from human induced pluripotent stem cells.

The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.

Human MD2 deficiency-an inborn error of immunity with pleiotropic features.

BACKGROUND: Toll-like receptors (TLRs) are important pattern recognition receptors that sense microbes and control host defense. Myeloid differentiation protein 2 (MD2) is the indispensable coreceptor for TLR4, facilitating the binding to the gram-negative bacterial cell wall component LPS and activation of downstream signaling. OBJECTIVE: We sought to provide phenotypic and mechanistic insights into human MD2 deficiency. METHODS: To elucidate the genetic cause in a patient with very early onset inflammatory bowel disease, we performed whole-exome sequencing and studied the functional consequences of the identified mutation in LY96 (encoding for MD2) in genetically engineered induced pluripotent stem cell-derived macrophages with knockout of MD2 or knockin of the patient-specific mutation, including TLR4-mediated signaling, cytokine production, and bacterial handling. RESULTS: Whole-exome sequencing identified a homozygous in-frame deletion in the LY96 gene (c.347_349delCAA; p.Thr116del) in a patient with very early onset inflammatory bowel disease and a sibling presenting with pneumonia and otitis media. Induced pluripotent stem cell-derived macrophages with knockout of MD2 or expression of the Thr116del mutation showed impaired activation of nuclear factor kappa B and mitogen-activated protein kinase signaling as well as TLR4 endocytosis on challenge with LPS or bacteria. In addition, MD2-deficient macrophages showed decreased cytokine expression (eg, IL-6, TNF, and IL-10) in response to LPS or gram-negative but not gram-positive bacteria. CONCLUSIONS: Human MD2 deficiency causes defective TLR4 signaling in response to LPS or gram-negative bacteria. The clinical manifestations and expressivity might be variable due to unknown secondary risk factors. Because TLR4 represents a therapeutic target for multiple inflammatory conditions, our study may provide insights into potential side effects of pharmacological TLR4 targeting.

Dysregulated inflammasome activity in intestinal inflammation - Insights from patients with very early onset IBD.

Inflammatory bowel disease (IBD) is a multifactorial disorder triggered by imbalances of the microbiome and immune dysregulations in genetically susceptible individuals. Several mouse and human studies have demonstrated that multimeric inflammasomes are critical regulators of host defense and gut homeostasis by modulating immune responses to pathogen- or damage-associated molecular patterns. In the context of IBD, excessive production of pro-inflammatory Interleukin-1ß has been detected in patient-derived intestinal tissues and correlated with the disease severity or failure to respond to anti-tumor necrosis factor therapy. Correspondingly, genome-wide association studies have suggested that single nucleotide polymorphisms in inflammasome components might be associated with risk of IBD development. The relevance of inflammasomes in controlling human intestinal homeostasis has been further exemplified by the discovery of very early onset IBD (VEO-IBD) patients with monogenic defects affecting different molecules in the complex regulatory network of inflammasome activity. This review provides an overview of known causative monogenic entities of VEO-IBD associated with altered inflammasome activity. A better understanding of the molecular mechanisms controlling inflammasomes in monogenic VEO-IBD may open novel therapeutic avenues for rare and common inflammatory diseases.

Spatial and temporal domain filtering for underwater lidar.

Combined spatial and temporal processing techniques are presented to enhance optical ranging in underwater environments. The performance of underwater light detection and ranging (lidar) is often limited by scattering. Previous work has demonstrated that both hybrid lidar-radar, which temporally modulates the amplitude of light, and optical spatial coherence filtering, which spatially modulates the phase of light, have independently reduced the effects of scattering, improving performance. The combined performance of the processing methods is investigated, and experimental results demonstrate that the combined filtering improves the performance of underwater lidar systems beyond what either method provides independently.

NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency.

BACKGROUND: Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1. METHODS: Functional characterization of the NOX1 variant included ROS generation, wound healing, 2-dimensional collective chemotactic migration, single-cell planktonic migration in heterologous cell lines, and RNA scope and immunohistochemistry of paraffin-embedded patient tissue samples. RESULTS: Using exome sequencing, we identified a stop-gain mutation in NOX1 (c.160C>T, p.54R>*) in patients with pediatric-onset IBD. Our studies confirmed that loss-of-function of NOX1 causes abrogated ROS activity, but they also provided novel mechanistic insights into human NOX1 deficiency. Cells that were NOX1-mutant showed impaired wound healing and attenuated 2-dimensional collective chemotactic migration. High-resolution microscopy of the migrating cell edge revealed a reduced density of filopodial protrusions with altered focal adhesions in NOX1-deficient cells, accompanied by reduced phosphorylation of p190A. Assessment of single-cell planktonic migration toward an epidermal growth factor gradient showed that NOX1 deficiency is associated with altered migration dynamics with loss of directionality and altered cell-cell interactions. CONCLUSIONS: Our studies on pediatric-onset IBD patients with a rare sequence variant in NOX1 highlight that human NOX1 is involved in regulating wound healing by altering epithelial cytoskeletal dynamics at the leading edge and directing cell migration.

CARMIL2 Deficiency Presenting as Very Early Onset Inflammatory Bowel Disease.

BACKGROUND: Children with very early onset inflammatory bowel diseases (VEO-IBD) often have a refractory and severe disease course. A significant number of described VEO-IBD-causing monogenic disorders can be attributed to defects in immune-related genes. The diagnosis of the underlying primary immunodeficiency (PID) often has critical implications for the treatment of patients with IBD-like phenotypes. METHODS: To identify the molecular etiology in 5 patients from 3 unrelated kindred with IBD-like symptoms, we conducted whole exome sequencing. Immune workup confirmed an underlying PID. RESULTS: Whole exome sequencing revealed 3 novel CARMIL2 loss-of-function mutations in our patients. Immunophenotyping of peripheral blood mononuclear cells showed reduction of regulatory and effector memory T cells and impaired B cell class switching. The T cell proliferation and activation assays confirmed defective responses to CD28 costimulation, consistent with CARMIL2 deficiency. CONCLUSION: Our study highlights that human CARMIL2 deficiency can manifest with IBD-like symptoms. This example illustrates that early diagnosis of underlying PID is crucial for the treatment and prognosis of children with VEO-IBD.

Alternative Splicing Rescues Loss of Common Gamma Chain Function and Results in IL-21R-like Deficiency.

Inborn errors in interleukin 2 receptor, gamma (IL2RG) perturb signaling of the common gamma chain family cytokines and cause severe combined immunodeficiency (SCID). Here, we report two brothers suffering from chronic cryptosporidiosis, severe diarrhea, and cholangitis. Pan T, B, and NK cell numbers were normal, but immunophenotyping revealed defective B cell differentiation. Using whole exome sequencing, we identified a base pair deletion in the first exon of IL2RG predicted to cause a frameshift and premature stop. However, flow cytometry revealed normal surface expression of the IL-2Rγ chain. While IL-2, IL-7, and IL-15 signaling showed only mild defects of STAT5 phosphorylation in response to the respective cytokines, IL-4- and IL-21-induced phosphorylation of STAT3 and STAT6 was markedly reduced. Examination of RNA isoforms detected alternative splicing downstream of IL2RG exon 1 in both patients resulting in resolution of the predicted frameshift and 16 mutated amino acids. In silico modeling suggested that the IL-2Rγ mutation reduces the stabilization of IL-4 and IL-21 cytokine binding by affecting the N-terminal domain of the IL-2Rγ. Thus, our study shows that IL2RG deficiency can be associated with differential signaling defects. Confounding effects of alternative splicing may partially rescue genetic defects and should be considered in patients with inborn errors of immunity.

Independent component analysis for enhancement of an FMCW optical ranging technique in turbid waters.

Optical detection and ranging in turbid waters are challenged by the effects of absorption and scattering. In particular, backscatter creates a clutter return, which can mask the presence of weak underwater targets. This work explores the use of independent component analysis (ICA), a statistical signal processing approach, to recover weak targets from strong backscatter in turbid waters using a frequency-modulated continuous-wave optical rangefinder. ICA uses statistical differences between target and backscatter returns to suppress the backscatter return. In laboratory test tank experiments, the use of ICA is observed to improve probability of detection at various turbidities and extend target detection range by four optical attenuation lengths.