Sestrins induce natural killer function in senescent-like CD8+ T cells.

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8+ T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27-CD28-CD8+ T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27-CD28-CD8+ T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27-CD28-CD8+ T cells to acquire a broad-spectrum, innate-like killing activity.

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronic Trypanosoma brucei infection.

The meningeal space is a critical brain structure providing immunosurveillance for the central nervous system (CNS), but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT) or sleeping sickness, accumulates in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, by combining single-cell transcriptomics and mass cytometry by time-of-flight (CyTOF) with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges. These infection-induced ELAs were defined by the presence of ER-TR7+ fibroblastic reticular cells, CD21/35+ follicular dendritic cells (FDCs), CXCR5+ PD1+ T follicular helper-like phenotype, GL7+ CD95+ GC-like B cells, and plasmablasts/plasma cells. Furthermore, the B cells found in the infected meninges produced high-affinity autoantibodies able to recognise mouse brain antigens, in a process dependent on LTß signalling. A mid-throughput screening identified several host factors recognised by these autoantibodies, including myelin basic protein (MBP), coinciding with cortical demyelination and brain pathology. In humans, we identified the presence of autoreactive IgG antibodies in the cerebrospinal fluid (CSF) of second stage HAT patients that recognised human brain lysates and MBP, consistent with our findings in experimental infections. Lastly, we found that the pathological B cell responses we observed in the meninges required the presence of T. brucei in the CNS, as suramin treatment before the onset of the CNS stage prevented the accumulation of GL7+ CD95+ GC-like B cells and brain-specific autoantibody deposition. Taken together, our data provide evidence that the meningeal immune response during chronic T. brucei infection results in the acquisition of lymphoid tissue-like properties, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders, including neuropsychiatric lupus and multiple sclerosis.

Defining the pig microglial transcriptome reveals its core signature, regional heterogeneity, and similarity with human and rodent microglia.

Microglia play key roles in brain homeostasis as well as responses to neurodegeneration and neuroinflammatory processes caused by physical disease and psychosocial stress. The pig is a physiologically relevant model species for studying human neurological disorders, many of which are associated with microglial dysfunction. Furthermore, pigs are an important agricultural species, and there is a need to understand how microglial function affects their welfare. As a basis for improved understanding to enhance biomedical and agricultural research, we sought to characterize pig microglial identity at genome-wide scale and conduct inter-species comparisons. We isolated pig hippocampal tissue and microglia from frontal cortex, hippocampus, and cerebellum, as well as alveolar macrophages from the lungs and conducted RNA-sequencing (RNAseq). By comparing the transcriptomic profiles between microglia, macrophages, and hippocampal tissue, we derived a set of 239 highly enriched genes defining the porcine core microglial signature. We found brain regional heterogeneity based on 150 genes showing significant (adjusted p < 0.01) regional variations and that cerebellar microglia were most distinct. We compared normalized gene expression for microglia from human, mice and pigs using microglia signature gene lists derived from each species and demonstrated that a core microglial marker gene signature is conserved across species, but that species-specific expression subsets also exist. Our data provide a valuable resource defining the pig microglial transcriptome signature that validates and highlights pigs as a useful large animal species bridging between rodents and humans in which to study the role of microglia during homeostasis and disease.

Temporal transcriptome profiling of floating apical out chicken enteroids suggest stability and reproducibility.

Enteroids are miniature self-organising three-dimensional (3D) tissue cultures which replicate much of the complexity of the intestinal epithelium. We recently developed an apical-out leukocyte-containing chicken enteroid model providing a novel physiologically relevant in vitro tool to explore host-pathogen interactions in the avian gut. However, the replicate consistency and culture stability have not yet been fully explored at the transcript level. In addition, causes for the inability to passage apical-out enteroids were not determined. Here we report the transcriptional profiling of chicken embryonic intestinal villi and chicken enteroid cultures using bulk RNA-seq. Comparison of the transcriptomes of biological and technical replicate enteroid cultures confirmed their high level of reproducibility. Detailed analysis of cell subpopulation and function markers revealed that the mature enteroids differentiate from late embryonic intestinal villi to recapitulate many digestive, immune and gut-barrier functions present in the avian intestine. These transcriptomic results demonstrate that the chicken enteroid cultures are highly reproducible, and within the first week of culture they morphologically mature to appear similar to the in vivo intestine, therefore representing a physiologically-relevant in vitro model of the chicken intestine.

Microglia deficiency accelerates prion disease but does not enhance prion accumulation in the brain.

Prion diseases are transmissible, neurodegenerative disorders associated with misfolding of the prion protein. Previous studies show that reduction of microglia accelerates central nervous system (CNS) prion disease and increases the accumulation of prions in the brain, suggesting that microglia provide neuroprotection by phagocytosing and destroying prions. In Csf1rΔFIRE mice, the deletion of an enhancer within Csf1r specifically blocks microglia development, however, their brains develop normally and show none of the deficits reported in other microglia-deficient models. Csf1rΔFIRE mice were used as a refined model in which to study the impact of microglia-deficiency on CNS prion disease. Although Csf1rΔFIRE mice succumbed to CNS prion disease much earlier than wild-type mice, the accumulation of prions in their brains was reduced. Instead, astrocytes displayed earlier, non-polarized reactive activation with enhanced phagocytosis of neuronal contents and unfolded protein responses. Our data suggest that rather than simply phagocytosing and destroying prions, the microglia instead provide host-protection during CNS prion disease and restrict the harmful activities of reactive astrocytes.

Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids.

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Pre/pro-B cells generate macrophage populations during homeostasis and inflammation.

Most tissue-resident macrophages (Mφs) are believed to be derived prenatally and are assumed to maintain themselves throughout life by self-proliferation. However, in adult mice we identified a progenitor within bone marrow, early pro-B cell/fraction B, that differentiates into tissue Mφs. These Mφ precursors have non-rearranged B-cell receptor genes and coexpress myeloid (GR1, CD11b, and CD16/32) and lymphoid (B220 and CD19) lineage markers. During steady state, these precursors exit bone marrow, losing Gr1, and enter the systemic circulation, seeding the gastrointestinal system as well as pleural and peritoneal cavities but not the brain. While in these tissues, they acquire a transcriptome identical to embryonically derived tissue-resident Mφs. Similarly, these Mφ precursors also enter sites of inflammation, gaining CD115, F4/80, and CD16/32, and become indistinguishable from blood monocyte-derived Mφs. Thus, we have identified a population of cells within the bone marrow early pro-B cell compartment that possess functional plasticity to differentiate into either tissue-resident or inflammatory Mφs, depending on microenvironmental signals. We propose that these precursors represent an additional source of Mφ populations in adult mice during steady state and inflammation.

The Effects of Immune System Modulation on Prion Disease Susceptibility and Pathogenesis.

Prion diseases are a unique group of infectious chronic neurodegenerative disorders to which there are no cures. Although prion infections do not stimulate adaptive immune responses in infected individuals, the actions of certain immune cell populations can have a significant impact on disease pathogenesis. After infection, the targeting of peripherally-acquired prions to specific immune cells in the secondary lymphoid organs (SLO), such as the lymph nodes and spleen, is essential for the efficient transmission of disease to the brain. Once the prions reach the brain, interactions with other immune cell populations can provide either host protection or accelerate the neurodegeneration. In this review, we provide a detailed account of how factors such as inflammation, ageing and pathogen co-infection can affect prion disease pathogenesis and susceptibility. For example, we discuss how changes to the abundance, function and activation status of specific immune cell populations can affect the transmission of prion diseases by peripheral routes. We also describe how the effects of systemic inflammation on certain glial cell subsets in the brains of infected individuals can accelerate the neurodegeneration. A detailed understanding of the factors that affect prion disease transmission and pathogenesis is essential for the development of novel intervention strategies.

Oral Prion Neuroinvasion Occurs Independently of PrPC Expression in the Gut Epithelium.

The early replication of certain prion strains within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain after oral exposure. Our data show that orally acquired prions utilize specialized gut epithelial cells known as M cells to enter Peyer's patches. M cells express the cellular isoform of the prion protein, PrPC, and this may be exploited by some pathogens as an uptake receptor to enter Peyer's patches. This suggested that PrPC might also mediate the uptake and transfer of prions across the gut epithelium into Peyer's patches in order to establish infection. Furthermore, the expression level of PrPC in the gut epithelium could influence the uptake of prions from the lumen of the small intestine. To test this hypothesis, transgenic mice were created in which deficiency in PrPC was specifically restricted to epithelial cells throughout the lining of the small intestine. Our data clearly show that efficient prion neuroinvasion after oral exposure occurred independently of PrPC expression in small intestinal epithelial cells. The specific absence of PrPC in the gut epithelium did not influence the early replication of prions in Peyer's patches or disease susceptibility. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease pathogenesis and susceptibility. However, our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences the risk of oral prion disease susceptibility.IMPORTANCE The accumulation of orally acquired prions within Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. Little is known of how the prions initially establish infection within Peyer's patches. Some gastrointestinal pathogens utilize molecules, such as the cellular prion protein PrPC, expressed on gut epithelial cells to enter Peyer's patches. Acute mucosal inflammation can enhance PrPC expression in the intestine, implying the potential to enhance oral prion disease susceptibility. We used transgenic mice to determine whether the uptake of prions into Peyer's patches was dependent upon PrPC expression in the gut epithelium. We show that orally acquired prions can establish infection in Peyer's patches independently of PrPC expression in gut epithelial cells. Our data suggest that the magnitude of PrPC expression in the epithelium lining the small intestine is unlikely to be an important factor which influences oral prion disease susceptibility.

Effects of host-derived chemokines on the motility and viability of Trypanosoma brucei.

African trypanosomes (Trypanosoma brucei spp.) are extracellular, hemoflagellate, protozoan parasites. Mammalian infection begins when the tsetse fly vector injects trypanosomes into the skin during blood feeding. The trypanosomes then reach the draining lymph nodes before disseminating systemically. Intravital imaging of the skin post-tsetse fly bite revealed that trypanosomes were observed both extravascularly and intravascularly in the lymphatic vessels. Whether host-derived cues play a role in the attraction of the trypanosomes towards the lymphatic vessels to aid their dissemination from the site of infection is not known. Since chemokines can mediate the attraction of leucocytes towards the lymphatics, in vitro chemotaxis assays were used to determine whether chemokines might also act as chemoattractants for trypanosomes. Although microarray data suggested that the chemokines CCL8, CCL19, CCL21, CCL27 and CXCL12 were highly expressed in mouse skin, they did not stimulate the chemotaxis of T brucei. Certain chemokines also possess potent antimicrobial properties. However, none of the chemokines tested exerted any parasiticidal effects on T brucei. Thus, our data suggest that host-derived chemokines do not act as chemoattractants for T brucei. Identification of the mechanisms used by trypanosomes to establish host infection will aid the development of novel approaches to block disease transmission.

Enhancement of cutaneous immunity during aging by blocking p38 mitogen-activated protein (MAP) kinase-induced inflammation.

BACKGROUND: Immunity decreases with age, which leads to reactivation of varicella zoster virus (VZV). In human subjects age-associated immune changes are usually measured in blood leukocytes; however, this might not reflect alterations in tissue-specific immunity. OBJECTIVES: We used a VZV antigen challenge system in the skin to investigate changes in tissue-specific mechanisms involved in the decreased response to this virus during aging. METHODS: We assessed cutaneous immunity based on the extent of erythema and induration after intradermal VZV antigen injection. We also performed immune histology and transcriptomic analyses on skin biopsy specimens taken from the challenge site in young (<40 years) and old (>65 years) subjects. RESULTS: Old human subjects exhibited decreased erythema and induration, CD4+ and CD8+ T-cell infiltration, and attenuated global gene activation at the site of cutaneous VZV antigen challenge compared with young subjects. This was associated with increased sterile inflammation in the skin in the same subjects related to p38 mitogen-activated protein kinase-related proinflammatory cytokine production (P < .0007). We inhibited systemic inflammation in old subjects by means of pretreatment with an oral small-molecule p38 mitogen-activated protein kinase inhibitor (Losmapimod; GlaxoSmithKline, Brentford, United Kingdom), which reduced both serum C-reactive protein levels and peripheral blood monocyte secretion of IL-6 and TNF-α. In contrast, cutaneous responses to VZV antigen challenge were increased significantly in the same subjects (P < .0003). CONCLUSION: Excessive inflammation in the skin early after antigen challenge retards antigen-specific immunity. However, this can be reversed by inhibition of inflammatory cytokine production that can be used to promote vaccine efficacy and the treatment of infections and malignancy during aging.

Impact of Zostavax Vaccination on T-Cell Accumulation and Cutaneous Gene Expression in the Skin of Older Humans After Varicella Zoster Virus Antigen-Specific Challenge.

Background: The live attenuated vaccine Zostavax was developed to prevent varicella zoster virus (VZV) reactivation that causes herpes zoster (shingles) in older humans. However, the impact of vaccination on the cutaneous response to VZV is not known. Methods: We investigated the response to intradermal VZV antigen challenge before and after Zostavax vaccination in participants >70 years of age by immunohistological and transcriptomic analyses of skin biopsy specimens collected from the challenge site. Results: Vaccination increased the proportion of VZV-specific CD4+ T cells in the blood and promoted the accumulation of both CD4+ and CD8+ T cells in the skin after VZV antigen challenge. However, Zostavax did not alter the proportion of resident memory T cells (CD4+ and CD8+) or CD4+Foxp3+ regulatory T cells in unchallenged skin. After vaccination, there was increased cutaneous T-cell proliferation at the challenge site and also increased recruitment of T cells from the blood, as indicated by an elevated T-cell migratory gene signature. CD8+ T-cell-associated functional genes were also highly induced in the skin after vaccination. Conclusion: Zostavax vaccination does not alter the abundance of cutaneous resident memory T cells but instead increases the recruitment of VZV-specific T cells from the blood and enhances T-cell activation, particularly cells of the CD8+ subset, in the skin after VZV antigen challenge.

Oral Prion Disease Pathogenesis Is Impeded in the Specific Absence of CXCR5-Expressing Dendritic Cells.

After oral exposure, the early replication of certain prion strains upon stromal cell-derived follicular dendritic cells (FDC) in the Peyer's patches in the small intestine is essential for the efficient spread of disease to the brain. However, little is known of how prions are initially conveyed from the gut lumen to establish infection on FDC. Our previous data suggest that mononuclear phagocytes such as CD11c+ conventional dendritic cells play an important role in the initial propagation of prions from the gut lumen into Peyer's patches. However, whether these cells conveyed orally acquired prions toward FDC within Peyer's patches was not known. The chemokine CXCL13 is expressed by FDC and follicular stromal cells and modulates the homing of CXCR5-expressing cells toward the FDC-containing B cell follicles. Here, novel compound transgenic mice were created in which a CXCR5 deficiency was specifically restricted to CD11c+ cells. These mice were used to determine whether CXCR5-expressing conventional dendritic cells propagate prions toward FDC after oral exposure. Our data show that in the specific absence of CXCR5-expressing conventional dendritic cells the early accumulation of prions upon FDC in Peyer's patches and the spleen was impaired, and disease susceptibility significantly reduced. These data suggest that CXCR5-expressing conventional dendritic cells play an important role in the efficient propagation of orally administered prions toward FDC within Peyer's patches in order to establish host infection.IMPORTANCE Many natural prion diseases are acquired by oral consumption of contaminated food or pasture. Once the prions reach the brain they cause extensive neurodegeneration, which ultimately leads to death. In order for the prions to efficiently spread from the gut to the brain, they first replicate upon follicular dendritic cells within intestinal Peyer's patches. How the prions are first delivered to follicular dendritic cells to establish infection was unknown. Understanding this process is important since treatments which prevent prions from infecting follicular dendritic cells can block their spread to the brain. We created mice in which mobile conventional dendritic cells were unable to migrate toward follicular dendritic cells. In these mice the early accumulation of prions on follicular dendritic cells was impaired and oral prion disease susceptibility was reduced. This suggests that prions exploit conventional dendritic cells to facilitate their initial delivery toward follicular dendritic cells to establish host infection.

Increased Abundance of M Cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility.

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases.

Increased susceptibility to oral Trichuris muris infection in the specific absence of CXCR5+ CD11c+ cells.

Trichuris muris is a natural mouse helminth pathogen which establishes infection specifically in the caecum and proximal colon. The rapid expulsion of T. muris in resistant mouse strains is associated with the induction of a protective T helper cell type 2 (Th2)-polarized immune response. Susceptible mouse strains, in contrast, mount an inappropriate Th1 response to T. muris infection. Expression of the chemokine CXCL13 by stromal follicular dendritic cells attracts CXCR5-expressing cells towards the B-cell follicles. Previous studies using a complex in vivo depletion model have suggested that CXCR5-expressing conventional dendritic cells (cDC) help regulate the induction of Th2-polarized responses. Here, transgenic mice with CXCR5 deficiency specifically restricted to CD11c+ cells were used to determine whether the specific absence CXCR5 on CD11c+ cells such as cDC would influence susceptibility to oral T. muris infection by affecting the Th1/Th2 balance. We show that in contrast to control mice, those which lacked CXCR5 expression on CD11c+ cells failed to clear T. muris infection and developed cytokine and antibody responses that suggested a disturbed Th1/Th2 balance with enhanced IFN-γ expression. These data suggest an important role of CXCR5-expressing CD11c+ cells such as cDC in immunity to oral T. muris infection.

Development of in vitro enteroids derived from bovine small intestinal crypts.

Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host-pathogen interactions in the bovine intestine. However, these cultures lack the cellular diversity encountered in the intestinal epithelium, and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium, and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid, or "mini gut", cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes, bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells, enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics, morphology or transcriptome. Furthermore, the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel, physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function, and host-pathogen interactions in the bovine small intestine can be studied.

Derivation of marker gene signatures from human skin and their use in the interpretation of the transcriptional changes associated with dermatological disorders.

Numerous studies have explored the altered transcriptional landscape associated with skin diseases to understand the nature of these disorders. However, data interpretation represents a significant challenge due to a lack of good maker sets for many of the specialized cell types that make up this tissue, whose composition may fundamentally alter during disease. Here we have sought to derive expression signatures that define the various cell types and structures that make up human skin, and demonstrate how they can be used to aid the interpretation of transcriptomic data derived from this organ. Two large normal skin transcriptomic datasets were identified, one RNA-seq (n = 578), the other microarray (n = 165), quality controlled and subjected separately to network-based analyses to identify clusters of robustly co-expressed genes. The biological significance of these clusters was then assigned using a combination of bioinformatics analyses, literature, and expert review. After cross comparison between analyses, 20 gene signatures were defined. These included expression signatures for hair follicles, glands (sebaceous, sweat, apocrine), keratinocytes, melanocytes, endothelia, muscle, adipocytes, immune cells, and a number of pathway systems. Collectively, we have named this resource SkinSig. SkinSig was then used in the analysis of transcriptomic datasets for 18 skin conditions, providing in-context interpretation of these data. For instance, conventional analysis has shown there to be a decrease in keratinization and fatty metabolism with age; we more accurately define these changes to be due to loss of hair follicles and sebaceous glands. SkinSig also highlighted the over-/under-representation of various cell types in skin diseases, reflecting an influx in immune cells in inflammatory disorders and a relative reduction in other cell types. Overall, our analyses demonstrate the value of this new resource in defining the functional profile of skin cell types and appendages, and in improving the interpretation of disease data. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Structural and functional changes to lymph nodes in ageing mice.

Lymph nodes (LN) are secondary lymphoid organs spread throughout the lymphatic system. They function to filter pathogenic material from the lymphatic fluid to maintain the health of the organism. Subcapsular sinus macrophages (SCSM) are among the first-responders within the LN due to their strategic location within the subcapsular sinus region. These macrophages aid the delivery of immune complexes to B cells and follicular dendritic cells (FDC) within the LN. Here we show an increase in SCSM and other macrophage populations within aged LN. However, immune complex uptake by macrophages within LN was not altered with age, nor was immune complex uptake by B cells. LN stromal cell populations, important in immune responses and the localization and survival of leucocytes, were altered in their representation and distribution in aged LN. In particular, FDC regions were decreased in size and had decreased chemokine CXCL13 expression. Furthermore, the retention of immune complexes by FDC was decreased in aged LN at 24 hr post-injection. As FDC are important in the maintenance of germinal centre responses, the decreased retention of immune complex in aged LN may contribute to the reduced germinal centre responses observed in aged mice.