Muscarinic receptor agonist-induced ßPix binding to ß-catenin promotes colon neoplasia.

M3 muscarinic receptors (M3R) modulate ß-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, ßPix, binds to ß-catenin in colon cancer cells, augmenting ß-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of ßPix and ß-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of ßPix with ß-catenin. ßPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing ßPix dose-dependently augmented ß-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of ßPix. Expression levels of ß-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from ßPix-deficient conditional knockout mice. Targeting the M3R/ßPix/ß-catenin axis may have therapeutic potential.

Multiomics integration of 22 immune-mediated monogenic diseases reveals an emergent axis of human immune health.

Monogenic diseases are often studied in isolation due to their rarity. Here we utilize multiomics to assess 22 monogenic immune-mediated conditions with age- and sex-matched healthy controls. Despite clearly detectable disease-specific and "pan-disease" signatures, individuals possess stable personal immune states over time. Temporally stable differences among subjects tend to dominate over differences attributable to disease conditions or medication use. Unsupervised principal variation analysis of personal immune states and machine learning classification distinguishing between healthy controls and patients converge to a metric of immune health (IHM). The IHM discriminates healthy from multiple polygenic autoimmune and inflammatory disease states in independent cohorts, marks healthy aging, and is a pre-vaccination predictor of antibody responses to influenza vaccination in the elderly. We identified easy-to-measure circulating protein biomarker surrogates of the IHM that capture immune health variations beyond age. Our work provides a conceptual framework and biomarkers for defining and measuring human immune health.

TNF inhibition in vasculitis management in adenosine deaminase 2 deficiency (DADA2).

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations. OBJECTIVE: We sought to explore the pathophysiology and the underlying mechanisms of TNF-inhibitor response in these patients. METHODS: We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in patients with DADA2 and studied their response to TNF-inhibitor treatment. RESULTS: We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and nuclear factor kappa B pathways in patients' primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation toward the proinflammatory M1 macrophage subset, and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2. CONCLUSIONS: Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells, and the endothelial cell damage is rescued with anti-TNF treatment.

The Spectrum of the Deficiency of Adenosine Deaminase 2: An Observational Analysis of a 60 Patient Cohort.

The deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessively inherited disease that has undergone extensive phenotypic expansion since being first described in patients with fevers, recurrent strokes, livedo racemosa, and polyarteritis nodosa in 2014. It is now recognized that patients may develop multisystem disease that spans multiple medical subspecialties. Here, we describe the findings from a large single center longitudinal cohort of 60 patients, the broad phenotypic presentation, as well as highlight the cohort's experience with hematopoietic cell transplantation and COVID-19. Disease manifestations could be separated into three major phenotypes: inflammatory/vascular, immune dysregulatory, and hematologic, however, most patients presented with significant overlap between these three phenotype groups. The cardinal features of the inflammatory/vascular group included cutaneous manifestations and stroke. Evidence of immune dysregulation was commonly observed, including hypogammaglobulinemia, absent to low class-switched memory B cells, and inadequate response to vaccination. Despite these findings, infectious complications were exceedingly rare in this cohort. Hematologic findings including pure red cell aplasia (PRCA), immune-mediated neutropenia, and pancytopenia were observed in half of patients. We significantly extended our experience using anti-TNF agents, with no strokes observed in 2026 patient months on TNF inhibitors. Meanwhile, hematologic and immune features had a more varied response to anti-TNF therapy. Six patients received a total of 10 allogeneic hematopoietic cell transplant (HCT) procedures, with secondary graft failure necessitating repeat HCTs in three patients, as well as unplanned donor cell infusions to avoid graft rejection. All transplanted patients had been on anti-TNF agents prior to HCT and received varying degrees of reduced-intensity or non-myeloablative conditioning. All transplanted patients are still alive and have discontinued anti-TNF therapy. The long-term follow up afforded by this large single-center study underscores the clinical heterogeneity of DADA2 and the potential for phenotypes to evolve in any individual patient.

Deficiency of Adenosine Deaminase 2 (DADA2): Hidden Variants, Reduced Penetrance, and Unusual Inheritance.

PURPOSE: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that manifests with fever, early-onset vasculitis, strokes, and hematologic dysfunction. This study aimed to identify disease-causing variants by conventional Sanger and whole exome sequencing in two families suspected to have DADA2 and non-confirmatory genotypes. ADA2 enzymatic assay confirmed the clinical diagnosis of DADA2. Molecular diagnosis was important to accurately identify other family members at risk. METHODS: We used a variety of sequencing technologies, ADA2 enzymatic testing, and molecular methods including qRT-PCR and MLPA. RESULTS: Exome sequencing identified heterozygosity for the known pathogenic variant ADA2: c.1358A>G, p.Tyr453Cys in a 14-year-old female with a history of ischemic strokes, livedo, and vasculitis. No second pathogenic variant could be identified. ADA2 enzymatic testing in combination with quantitative RT-PCR suggested a loss-of-function allele. Subsequent genome sequencing identified a canonical splice site variant, c.-47+2T>C, within the 5'UTR of ADA2. Two of her unaffected siblings were found to carry the same two pathogenic variants. A homozygous 800-bp duplication comprising exon 7 of ADA2 was identified in a 5-year-old female with features consistent with Diamond-Blackfan anemia (DBA). The duplication was missed by Sanger sequencing of ADA2, chromosomal microarray, and exome sequencing but was detected by MLPA in combination with long-read PCR sequencing. The exon 7 duplication was also identified in her non-symptomatic father and younger sister. CONCLUSIONS: ADA2 pathogenic variants may not be detected by conventional sequencing and genetic testing and may require the incorporation of additional diagnostic methods. A definitive molecular diagnosis is crucial for all family members to make informed treatment decisions.

Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.