Long read sequencing reveals novel isoforms and insights into splicing regulation during cell state changes.

BACKGROUND: Alternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Here, we utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation. RESULTS: We identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. We show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation. CONCLUSIONS: Our work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.

CX3CR1+ Cell-Mediated Salmonella Exclusion Protects the Intestinal Mucosa during the Initial Stage of Infection.

During Salmonella Typhimurium infection, intestinal CX3CR1+ cells can either extend transepithelial cellular processes to sample luminal bacteria or, very early after infection, migrate into the intestinal lumen to capture bacteria. However, until now, the biological relevance of the intraluminal migration of CX3CR1+ cells remained to be determined. We addressed this by using a combination of mouse strains differing in their ability to carry out CX3CR1-mediated sampling and intraluminal migration. We observed that the number of S. Typhimurium traversing the epithelium did not differ between sampling-competent/migration-competent C57BL/6 and sampling-deficient/migration-competent BALB/c mice. In contrast, in sampling-deficient/migration-deficient CX3CR1-/- mice the numbers of S. Typhimurium penetrating the epithelium were significantly higher. However, in these mice the number of invading S. Typhimurium was significantly reduced after the adoptive transfer of CX3CR1+ cells directly into the intestinal lumen, consistent with intraluminal CX3CR1+ cells preventing S. Typhimurium from infecting the host. This interpretation was also supported by a higher bacterial fecal load in CX3CR1+/gfp compared with CX3CR1gfp/gfp mice following oral infection. Furthermore, by using real-time in vivo imaging we observed that CX3CR1+ cells migrated into the lumen moving through paracellular channels within the epithelium. Also, we reported that the absence of CX3CR1-mediated sampling did not affect Ab responses to a noninvasive S. Typhimurium strain that specifically targeted the CX3CR1-mediated entry route. These data showed that the rapidly deployed CX3CR1+ cell-based mechanism of immune exclusion is a defense mechanism against pathogens that complements the mucous and secretory IgA Ab-mediated system in the protection of intestinal mucosal surface.

Age-associated modifications of intestinal permeability and innate immunity in human small intestine.

The physical and immunological properties of the human intestinal epithelial barrier in aging are largely unknown. Ileal biopsies from young (7-12 years), adult (20-40 years) and aging (67-77 years) individuals not showing symptoms of gastrointestinal (GI) pathologies were used to assess levels of inflammatory cytokines, barrier integrity and cytokine production in response to microbial challenges. Increased expression of interleukin (IL)-6, but not interferon (IFN)γ, tumour necrosis factor (TNF)-α and IL-1ß was observed during aging; further analysis showed that cluster of differentiation (CD)11c(+) dendritic cells (DCs) are one of the major sources of IL-6 in the aging gut and expressed higher levels of CD40. Up-regulated production of IL-6 was accompanied by increased expression of claudin-2 leading to reduced transepithelial electric resistance (TEER); TEER could be restored in in vitro and ex vivo cultures by neutralizing anti-IL-6 antibody. In contrast, expression of zonula occludens-1 (ZO-1), occludin and junctional-adhesion molecule-A1 did not vary with age and overall permeability to macromolecules was not affected. Finally, cytokine production in response to different microbial stimuli was assessed in a polarized in vitro organ culture (IVOC). IL-8 production in response to flagellin declined progressively with age although the expression and distribution of toll-like receptor (TLR)-5 on intestinal epithelial cells (IECs) remained unchanged. Also, flagellin-induced production of IL-6 was less pronounced in aging individuals. In contrast, TNF-α production in response to probiotics (VSL#3) did not decline with age; however, in our experimental model probiotics did not down-regulate the production of IL-6 and expression of claudin-2. These data suggested that aging affects properties of the intestinal barrier likely to impact on age-associated disturbances, both locally and systemically.

The impact of ageing on the intestinal epithelial barrier and immune system.

The vast mucosal surface of the intestine is patrolled by a large number of lymphocytes forming the intestinal immune system. Like any other system in the body, this branch of the immune system is affected by ageing. Although our knowledge on the age-associated changes of the systemic immune system has improved over the past few years, our understanding of the mechanisms of senescence of both adaptive and innate immune system of the gastrointestinal (GI) tract is still largely incomplete. However, recent advances in the field have shown that the identification of the events underlying the ageing process in the gut may have important consequences on health and wellbeing far beyond the GI-tract. The aim of this review is to summarise the impact of ageing on the intestinal immune system, including the gut epithelium and other components of the intestinal barrier that maintain intestinal immune homeostasis and shape antigen-specific immune responses.

Macrophage migration inhibitory factor plays a role in the regulation of microfold (M) cell-mediated transport in the gut.

It has been shown previously that certain bacteria rapidly (3 h) up-regulated in vivo microfold cell (M cell)-mediated transport of Ag across the follicle-associated epithelium of intestinal Peyer's patch. Our aim was to determine whether soluble mediators secreted following host-bacteria interaction were involved in this event. A combination of proteomics and immunohistochemical analyses was used to identify molecules produced in the gut in response to bacterial challenge in vivo; their effects were then tested on human intestinal epithelial cells in vitro. Macrophage migration inhibitory factor (MIF) was the only cytokine produced rapidly after in vivo bacterial challenge by CD11c(+) cells located beneath the M cell-rich area of the follicle-associated epithelium of the Peyer's patch. Subsequently, in vitro experiments conducted using human Caco-2 cells showed that, within hours, MIF induced the appearance of cells that showed temperature-dependent transport of microparticles and M cell-specific bacterium Vibrio cholerae, and acquired biochemical features of M cells. Furthermore, using an established in vitro human M cell model, we showed that anti-MIF Ab blocked Raji B cell-mediated conversion of Caco-2 cells into Ag-sampling cells. Finally, we report that MIF(-/-) mice, in contrast to wild-type mice, failed to show increased M cell-mediated transport following in vivo bacterial challenge. These data show that MIF plays a role in M cell-mediated transport, and cross-talk between bacteria, gut epithelium, and immune system is instrumental in regulating key functions of the gut, including M cell-mediated Ag sampling.

Faecal microbiota transplant from aged donor mice affects spatial learning and memory via modulating hippocampal synaptic plasticity- and neurotransmission-related proteins in young recipients.

BACKGROUND: The gut-brain axis and the intestinal microbiota are emerging as key players in health and disease. Shifts in intestinal microbiota composition affect a variety of systems; however, evidence of their direct impact on cognitive functions is still lacking. We tested whether faecal microbiota transplant (FMT) from aged donor mice into young adult recipients altered the hippocampus, an area of the central nervous system (CNS) known to be affected by the ageing process and related functions. RESULTS: Young adult mice were transplanted with the microbiota from either aged or age-matched donor mice. Following transplantation, characterization of the microbiotas and metabolomics profiles along with a battery of cognitive and behavioural tests were performed. Label-free quantitative proteomics was employed to monitor protein expression in the hippocampus of the recipients. We report that FMT from aged donors led to impaired spatial learning and memory in young adult recipients, whereas anxiety, explorative behaviour and locomotor activity remained unaffected. This was paralleled by altered expression of proteins involved in synaptic plasticity and neurotransmission in the hippocampus. Also, a strong reduction of bacteria associated with short-chain fatty acids (SCFAs) production (Lachnospiraceae, Faecalibaculum, and Ruminococcaceae) and disorders of the CNS (Prevotellaceae and Ruminococcaceae) was observed. Finally, the detrimental effect of FMT from aged donors on the CNS was confirmed by the observation that microglia cells of the hippocampus fimbria, acquired an ageing-like phenotype; on the contrary, gut permeability and levels of systemic and local (hippocampus) cytokines were not affected. CONCLUSION: These results demonstrate that age-associated shifts of the microbiota have an impact on protein expression and key functions of the CNS. Furthermore, these results highlight the paramount importance of the gut-brain axis in ageing and provide a strong rationale to devise therapies aiming to restore a young-like microbiota to improve cognitive functions and the declining quality of life in the elderly. Video Abstract.

Mapping specific adhesive interactions on living human intestinal epithelial cells with atomic force microscopy.

Specific molecular-receptor interactions with gut epithelium cells are important in understanding bioactivity of food components and drugs, binding of commensal microflora, attachment and initiation of defense mechanisms against pathogenic bacteria and for development of targeted delivery systems to the gut. However, methods for probing such interactions are lacking. Methodology has been developed and validated to measure specific molecular-receptor interactions on living human colorectal cancer cells as in vitro models for the gut epithelium. Atomic force microscopy (AFM) was used to measure ligand-receptor interactions and to map receptor locations on cell surfaces. Measurements were made using silica beads attached to the AFM tip-cantilever assembly, which were functionalized by coupling of ligands to the bead surface. Wheat germ agglutinin (WGA) binds to the glycosylated extracellular domain III of the epidermal growth factor receptor. Methodology was tested by measuring binding of WGA to the surface of confluent monolayers of living Caco-2 human intestinal epithelial cells. The measured modal detachment force of 125 pN is typical of values expected for single molecule interactions. Adhesive events were used to map the location of binding sites on the cell surface revealing heterogeneity in their distribution within and between cells within the monolayer.

Differential regulation of dendritic cell-T cell cross talk in the gut-associated lymphoid tissue.

Dendritic cells (DC) play a central role in the regulation of immune responses by processing and presenting antigens to naïve T cells. It has been proposed that after the initial interaction between DC and T cells, T cell-induced DC apoptosis serves as a down-regulatory mechanism that prevents the otherwise continuous activation of T cells by antigen-bearing DC. Our aim was to investigate and compare the susceptibility of Peyer's patch (PP)-derived and systemic (splenic) DC to antigen-specific T cell-mediated apoptosis in mice of different genetic background. Freshly isolated CD11c(+/hi)B220(-) DC from intestinal Peyer's patch and spleen from Balb/c and C3H/HeJ mice were co-cultured with syngeneic antigen-specific T cells in the presence or absence of the relevant antigen. In both mouse strains PP-DC showed higher susceptibility to T cell-mediated apoptosis compared to splenic ones, but levels of DC apoptosis were overall higher in C3H/HeJ mice compared to Balb/c. DC apoptosis was induced by both Th1 and Th2 antigen-specific clones and was strictly MHC class II-dependent in both strains, and interestingly we observed that although CD95-CD95L ligation played an overall minor part in T cell-induced DC apoptosis its role varied according to the mouse strain. Here, we demonstrated that PP-DC and splenic DC significantly differed in regard to their susceptibility to T cell-mediated killing. We interpreted these data as showing that the reciprocal regulation between DC and T cells in the gastrointestinal immune system is under stricter control compared to the systemic immune system and we hypothesized that these events are likely to contribute to the generation of fine balanced responses to intestinal antigens.

Improving M cell mediated transport across mucosal barriers: do certain bacteria hold the keys?

Specialized microfold (M) cells of the follicle-associated epithelium (FAE) of the mucosal-associated lymphoid tissue (MALT) in gut and the respiratory system play an important role in the genesis of both mucosal and systemic immune responses by delivering antigenic substrate to the underlying lymphoid tissue where immune responses start. Although it has been shown that dendritic cells (DC) also have the ability to sample antigens directly from the gut lumen, M cells certainly remain the most important antigen-sampling cell to be investigated in order to devise novel methods to improve mucosal delivery of biologically active compounds. Recently, novel information on the interactions between bacteria and FAE have come to light that unveil further the complex cross-talk taking place at mucosal interfaces between bacteria, epithelial cells and the immune system and which are central to the formation and function of M cells. In particular, it has been shown that M cell mediated transport of antigen across the FAE is improved rapidly by exposure to certain bacteria, thus opening the way to identify new means to achieve a more effective mucosal delivery. Here, these novel findings and their potential in mucosal immunity are analysed and discussed, and new approaches to improve antigen delivery to the mucosal immune system are also proposed.

Antigen-specific T cell-mediated apoptosis of dendritic cells is impaired in a mouse model of food allergy.

BACKGROUND: Dendritic cells (DCs) play a pivotal role in antigen presentation and regulation of immune responses, and strong evidence suggests their involvement in the pathogenesis of allergy. However, hitherto, DC-T-cell cross-talk in relation to IgE-mediated allergic reactions to food has not been investigated. OBJECTIVE: Our aim was to investigate T cell-mediated apoptosis of myeloid DCs from spleen and Peyer's patches of mice with cow's milk (CM) allergy after cognate interaction with antigen (CM)-specific T cells. METHODS: Freshly isolated myeloid CD11c(+/hi)/B220(-) DCs from spleen and Peyer's patches of mice with CM allergy and control mice were cultured with CM-specific T cells in the presence or absence of CM or unrelated antigen as a control. Levels of apoptosis in DCs were evaluated by assessing propidium iodide uptake and annexin V expression by means of flow cytometry. RESULTS: We observed that both systemic and gastrointestinal-derived DCs showed an increased resistance to T cell-mediated cell death compared with DCs from control but not allergic donors. Further experiments demonstrated that in both allergic and control mice, T cell-mediated DC apoptosis takes place exclusively in the presence of the specific antigen, is MHC II dependent, and is only partially CD95-CD95 ligand dependent. CONCLUSION: Here we demonstrate, for the first time, that the reciprocal, finely balanced regulation between these 2 cell types, which plays a central role in controlling immune responses, is altered in allergy. We hypothesize that these events are likely to have a profound influence on the genesis and maintenance of adverse reaction to food.

Adoptive transfer of dendritic cells from allergic mice induces specific immunoglobulin E antibody in naïve recipients in absence of antigen challenge without altering the T helper 1/T helper 2 balance.

Dendritic cells (DCs) are important in the regulation of immune responses and it has been proposed that these cells play an important role in asthma; however, their role in food allergy is still largely unknown. Our aim was to study specific immunoglobulin E (IgE) and immunoglobulin G (IgG) responses in naïve recipients following adoptive transfer of myeloid DCs from allergic and control mice. The phenotypic features and lymphokine production of DCs were also investigated. CD11c+/hi B220- DCs isolated from spleen and Peyer's patches (PP) of cow's milk (CM) allergic and control mice were transferred intravenously (i.v.) into naïve syngeneic recipients, and IgE- and IgG-specific responses were evaluated. Experiments were also carried out to determine the levels of interferon-gamma (IFN-gamma) and interleukin (IL)-4 produced by splenocytes from naïve recipients following the adoptive transfer, and CD40 ligand (CD40L)-mediated IL-10 production by DCs from allergic and control mice. DCs isolated from spleen and PP of allergic mice, but not control groups, induced CM-specific IgG and IgE antibody production in naïve recipients in the absence of previous immunization, but did not modify the T helper 1 (Th1) and T helper 2 (Th2) balance. Furthermore, although no difference was observed in the expression of canonical DC surface markers, PP DCs from allergic mice produced less IL-10 than DCs from controls. We interpret these data as showing that DCs play a pivotal role in allergen-specific IgE responses and that a Th2-skewed response may not be involved in the early phase of allergic responses. The identification of the mechanisms underlying these events may help to design novel strategies of therapeutic intervention in food allergy.