A major cause of enteric infection, Gram-negative pathogenic bacteria activate mucosal inflammation through lipopolysaccharide (LPS) binding to intestinal toll-like receptor 4 (TLR4). Breast feeding ...lowers risk of disease, and human milk modulates inflammation.
This study tested whether human milk oligosaccharides (HMOSs) influence pathogenic Escherichia coli-induced interleukin (IL)-8 release by intestinal epithelial cells (IECs), identified specific proinflammatory signalling molecules modulated by HMOSs, specified the active HMOS and determined its mechanism of action.
Models of inflammation were IECs invaded by type 1 pili enterotoxigenic E. coli (ETEC) in vitro: T84 modelled mature, and H4 modelled immature IECs. LPS-induced signalling molecules co-varying with IL-8 release in the presence or absence of HMOSs were identified. Knockdown and overexpression verified signalling mediators. The oligosaccharide responsible for altered signalling was identified.
HMOSs attenuated LPS-dependent induction of IL-8 caused by ETEC, uropathogenic E. coli, and adherent-invasive E. coli (AIEC) infection, and suppressed CD14 transcription and translation. CD14 knockdown recapitulated HMOS-induced attenuation. Overexpression of CD14 increased the inflammatory response to ETEC and sensitivity to inhibition by HMOSs. 2'-fucosyllactose (2'-FL), at milk concentrations, displayed equivalent ability as total HMOSs to suppress CD14 expression, and protected AIEC-infected mice.
HMOSs and 2'-FL directly inhibit LPS-mediated inflammation during ETEC invasion of T84 and H4 IECs through attenuation of CD14 induction. CD14 expression mediates LPS-TLR4 stimulation of portions of the 'macrophage migration inhibitory factors' inflammatory pathway via suppressors of cytokine signalling 2/signal transducer and activator of transcription 3/NF-κB. HMOS direct inhibition of inflammation supports its functioning as an innate immune system whereby the mother protects her vulnerable neonate through her milk. 2'-FL, a principal HMOS, quenches inflammatory signalling.
Toll-like receptor (TLR) signaling is central to innate immunity. Aberrant expression of TLRs is found in neonatal inflammatory diseases. Several bioactive components of human milk modulate TLR ...expression and signaling pathways, including soluble toll-like receptors (sTLRs), soluble cluster of differentiation (sCD) 14, glycoproteins, small peptides, and oligosaccharides. Some milk components, such as sialyl (α2,3) lactose and lacto-N-fucopentaose III, are reported to increase TLR signaling; under some circumstances this might contribute toward immunologic balance. Human milk on the whole is strongly anti-inflammatory, and contains abundant components that depress TLR signaling pathways: sTLR2 and sCD14 inhibit TLR2 signaling; sCD14, lactadherin, lactoferrin, and 2′-fucosyllactose attenuate TLR4 signaling; 3′-galactosyllactose inhibits TLR3 signaling, and β-defensin 2 inhibits TLR7 signaling. Feeding human milk to neonates decreases their risk of sepsis and necrotizing enterocolitis. Thus, the TLR regulatory components found in human milk hold promise as benign oral prophylactic and therapeutic treatments for the many gastrointestinal inflammatory disorders mediated by abnormal TLR signaling.
Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are ...associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type-specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis.
Pancreatic islet dysfunction and beta cell failure are hallmarks of type 2 diabetes mellitus (T2DM) pathogenesis. In this review, we discuss how genome-wide association studies (GWASs) and recent ...developments in islet (epi)genome and transcriptome profiling (particularly single cell analyses) are providing novel insights into the genetic, environmental, and cellular contributions to islet (dys)function and T2DM pathogenesis. Moving forward, study designs that interrogate and model genetic variation e.g., allelic profiling and (epi)genome editing will be critical to dissect the molecular genetics of T2DM pathogenesis, to build next-generation cellular and animal models, and to develop precision medicine approaches to detect, treat, and prevent islet (dys)function and T2DM.
Detecting multiplets in single nucleus (sn)ATAC-seq data is challenging due to data sparsity and limited dynamic range. AMULET (ATAC-seq MULtiplet Estimation Tool) enumerates regions with greater ...than two uniquely aligned reads across the genome to effectively detect multiplets. We evaluate the method by generating snATAC-seq data in the human blood and pancreatic islet samples. AMULET has high precision, estimated via donor-based multiplexing, and high recall, estimated via simulated multiplets, compared to alternatives and identifies multiplets most effectively when a certain read depth of 25K median valid reads per nucleus is achieved.
Immune cell activation assays have been widely used for immune monitoring and for understanding disease mechanisms. However, these assays are typically limited in scope. A holistic study of ...circulating immune cell responses to different activators is lacking. Here we developed a cost-effective high-throughput multiplexed single-cell RNA-seq combined with epitope tagging (CITE-seq) to determine how classic activators of T cells (anti-CD3 coupled with anti-CD28) or monocytes (LPS) alter the cell composition and transcriptional profiles of peripheral blood mononuclear cells (PBMCs) from healthy human donors. Anti-CD3/CD28 treatment activated all classes of lymphocytes either directly (T cells) or indirectly (B and NK cells) but reduced monocyte numbers. Activated T and NK cells expressed senescence and effector molecules, whereas activated B cells transcriptionally resembled autoimmune disease- or age-associated B cells (e.g., CD11c, T-bet). In contrast, LPS specifically targeted monocytes and induced two main states: early activation characterized by the expression of chemoattractants and a later pro-inflammatory state characterized by expression of effector molecules. These data provide a foundation for future immune activation studies with single cell technologies (https://czi-pbmc-cite-seq.jax.org/).
β cells may participate and contribute to their own demise during Type 1 diabetes (T1D). Here we report a role of their expression of Tet2 in regulating immune killing. Tet2 is induced in murine and ...human β cells with inflammation but its expression is reduced in surviving β cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate β cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells.Tet2-KO β cells show reduced expression of IFNγ-induced inflammatory genes that are needed to activate diabetogenic T cells. Here we show that Tet2 regulates pathologic interactions between β cells and immune cells and controls damaging inflammatory pathways. Our data suggests that eliminating TET2 in β cells may reduce activating pathologic immune cells and killing of β cells.
Pancreatic Islets of Langerhans are heterogeneous tissues consisting of multiple endocrine cell types that carry out distinct yet coordinated roles to regulate blood glucose homeostasis. Islet ...dysfunction and specifically failure of the beta cells to secrete adequate insulin are known precursors to type 2 diabetes (T2D) onset. However, the exact genetic, (epi)genomic, and environmental mechanisms that contribute to islet failure, and ultimately to T2D pathogenesis, require further elucidation.
This review summarizes efforts and advances in dissection of the complex genetic underpinnings of islet function and resilience in T2D pathogenesis. In this review, we will highlight results of the latest T2D genome-wide association study (GWAS) and discuss how these data are being combined with clinical measures in patients to uncover putative T2D subtypes and with functional (epi)genomic studies in islets to understand the genetic programming of islet cell identity, function, and adaptation. Finally, we discuss new and important opportunities to address major knowledge gaps in our understanding of islet (dys)function in T2D risk and progression.
Genetic variation exerts clear effects on the islet epigenome, regulatory element usage, and gene expression. Future (epi)genomic comparative analyses between T2D and normal islets should incorporate genetics to distinguish patient-specific from disease-specific differences. Incorporating genotype information into future analyses and studies will also enable more precise insights into the molecular genetics of islet deficiency and failure in T2D risk, and should ultimately contribute to a stratified view of T2D and more precise treatment strategies. Islet cellular heterogeneity continues to remain a challenge for understanding the associations between islet failure and T2D development. Further efforts to obtain purified islet cell type populations and determine the specific genetic and environmental effects on each will help address this. Beyond observation of islets at steady state conditions, more research of islet stress and stimulation responses are needed to understand the transition of these tissues from a healthy to diseased state. Together, focusing on these objectives will provide more opportunities to prevent, treat, and manage T2D.
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Acute myeloid leukemia (AML) is an aggressive blood cancer with poor clinical outcomes. Emerging data suggest that mitochondrial oxidative phosphorylation (mtOXPHOS) plays a significant role in AML ...tumorigenesis, progression, and resistance to chemotherapies. However, how the mtOXPHOS is regulated in AML cells is not well understood. In this study, we investigated the oncogenic functions of ERRα in AML by combining in silico, in vitro, and in vivo analyses and showed ERRα is a key regulator of mtOXPHOS in AML cells. The increased ERRα level was associated with worse clinical outcomes of AML patients. Single cell RNA-Seq analysis of human primary AML cells indicated that ERRα-expressing cancer cells had significantly higher mtOXPHOS enrichment scores. Blockade of ERRα by pharmacologic inhibitor (XCT-790) or gene silencing suppressed mtOXPHOS and increased anti-leukemic effects in vitro and in xenograft mouse models.
Clustering is carried out to identify patterns in transcriptomics profiles to determine clinically relevant subgroups of patients. Feature (gene) selection is a critical and an integral part of the ...process. Currently, there are many feature selection and clustering methods to identify the relevant genes and perform clustering of samples. However, choosing an appropriate methodology is difficult. In addition, extensive feature selection methods have not been supported by the available packages. Hence, we developed an integrative R-package called multiClust that allows researchers to experiment with the choice of combination of methods for gene selection and clustering with ease. Using multiClust, we identified the best performing clustering methodology in the context of clinical outcome. Our observations demonstrate that simple methods such as variance-based ranking perform well on the majority of data sets, provided that the appropriate number of genes is selected. However, different gene ranking and selection methods remain relevant as no methodology works for all studies.
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