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.

M cells reside within the follicle-associated epithelium (FAE) overlying the gut-associated lymphoid tissues. These unique phagocytic epithelial cells enable the mucosal immune system to sample antigens within the lumen of the intestine. The differentiation of M cells from uncommitted precursors in the FAE is dependent on the production of receptor activator of nuclear factor-κB ligand (RANKL) by subepithelial stromal cells. The ligation of a variety of cell surface receptors activates the nuclear factor-κB (NF-κB) family of transcription factors which in-turn induce the transcription of multiple target genes. RANKL-stimulation can stimulate the nuclear translocation of the NF-κB subunit c-Rel. We therefore used c-Rel-deficient mice to determine whether the differentiation and functional maturation of M cells in the Peyer's patches was dependent on c-Rel. Our data show that c-Rel-deficiency does not influence the expression of RANKL or RANK in Peyer's patches, or the induction of M-cell differentiation in the FAE. RANKL-stimulation in the differentiating M cells induces the expression of SpiB which is essential for their subsequent maturation. However, SpiB expression in the FAE was also unaffected in the absence of c-Rel. As a consequence, the functional maturation of M cells was not impaired in the Peyer's patches of c-Rel-deficient mice. Although our data showed that the specific expression of CCL20 and ubiquitin D in the FAE was not impeded in the absence of c-Rel, the expression of ubiquitin D was dramatically reduced in the B cell-follicles of c-Rel-deficient mice. Coincident with this, we also observed that the status of follicular dendritic cells in the B cell-follicles was dramatically reduced in Peyer's patches from c-Rel-deficient mice. Taken together, our data show that c-Rel is dispensable for the RANKL-mediated differentiation and functional maturation of M cells.

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.

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.

Prion diseases are a unique, infectious, neurodegenerative disorders that can affect animals and humans. Data from mouse transmissions show that efficient infection of the host after intravenous (IV) prion exposure is dependent upon the early accumulation and amplification of the prions on stromal follicular dendritic cells (FDC) in the B cell follicles. How infectious prions are initially conveyed from the blood-stream to the FDC in the spleen is uncertain. Addressing this issue is important as susceptibility to peripheral prion infections can be reduced by treatments that prevent the early accumulation of prions upon FDC. The marginal zone (MZ) in the spleen contains specialized subsets of B cells and macrophages that are positioned to continuously monitor the blood-stream and remove pathogens, toxins and apoptotic cells. The continual shuttling of MZ B cells between the MZ and the B-cell follicle enables them to efficiently capture and deliver blood-borne antigens and antigen-containing immune complexes to splenic FDC. We tested the hypothesis that MZ B cells also play a role in the initial shuttling of prions from the blood-stream to FDC. MZ B cells were temporarily depleted from the MZ by antibody-mediated blocking of integrin function. We show that depletion of MZ B cells around the time of IV prion exposure did not affect the early accumulation of blood-borne prions upon splenic FDC or reduce susceptibility to IV prion infection. In conclusion, our data suggest that the initial delivery of blood-borne prions to FDC in the spleen occurs independently of MZ B cells.

Prion diseases or transmissible spongiform encephalopathies are fatal, progressive, neurodegenerative, protein-misfolding disorders. Prion diseases may arise spontaneously, be inherited genetically or be acquired by infection and affect a variety of mammalian species including humans. Prion infections in the central nervous system (CNS) cause extensive neuropathology, including abnormal accumulations of misfolded host prion protein, vacuolar change resulting in sponge-like (spongiform) appearance of CNS tissue, neurodegeneration and reactive glial responses. Many different prion agent strains exist and these can differ based on disease duration, clinical signs and the targeting and distribution of the neuropathology in distinct brain areas. Reactive astrocytes are a prominent feature in the prion disease affected CNS as revealed by distinct morphological changes and upregulation of glial fibrillary acidic protein (GFAP). The CD44 antigen is a transmembrane glycoprotein involved in cell-cell interactions, cell adhesion and migration. Here we show that CD44 is also highly expressed in a subset of reactive astrocytes in regions of the CNS targeted by prions. Astrocyte heterogeneity revealed by differential CD44 upregulation occurs coincident with the earliest neuropathological changes during the pre-clinical phase of disease, and is not affected by the route of infection. The expression and distribution of CD44 was compared in brains from a large collection of 15 distinct prion agent strains transmitted to mice of different prion protein (Prnp) genotype backgrounds. Our data show that the pattern of CD44 upregulation observed in the hippocampus in each prion agent strain and host Prnp genotype combination was unique. Many mouse-adapted prion strains and hosts have previously been characterized based on the pattern of the distribution of the spongiform pathology or the misfolded PrP deposition within the brain. Our data show that CD44 expression also provides a reliable discriminatory marker of prion infection with a greater dynamic range than misfolded prion protein deposition, aiding strain identification. Together, our data reveal CD44 as a novel marker to detect reactive astrocyte heterogeneity during CNS prion disease and for enhanced identification of distinct prion agent strains.

Prion infections in the central nervous system (CNS) can cause extensive neurodegeneration. Systemic inflammation can affect the progression of some neurodegenerative disorders. Therefore, we used the gastrointestinal helminth pathogen Trichuris muris to test the hypothesis that a chronic systemic inflammatory response to a gastrointestinal infection would similarly affect CNS prion disease pathogenesis. Mice were injected with prions directly into the CNS and subsequently orally co-infected with T. muris before the onset of clinical signs. We show that co-infection with a low dose of T. muris that leads to the development of a chronic T helper cell type 1-polarized systemic immune response accelerated the onset of clinical prion disease. In contrast, co-infection with a high dose of T. muris that induces a T helper cell type 2-polarized immune response did not affect prion disease pathogenesis. The reduced survival times in mice co-infected with a low dose of T. muris on d 105 after CNS prion infection coincided with enhanced astrocyte activation in the brain during the preclinical phase. These data aid our understanding of how systemic inflammation may augment the progression of neurodegeneration in the CNS.

Infection of the mammalian host with African trypanosomes begins when the tsetse fly vector injects the parasites into the skin dermis during blood feeding. After injection into the skin, trypanosomes first accumulate in the draining lymph node before disseminating systemically. Whether this early accumulation within the draining lymph node is important for the trypanosomes to establish infection was not known. Lymphotoxin-β-deficient mice (LTβ-/- mice) lack most secondary lymphoid tissues, but retain the spleen and mesenteric lymph nodes. These mice were used to test the hypothesis that the establishment of infection after intradermal (ID) T. brucei infection would be impeded in the absence of the skin draining lymph nodes. However, LTβ-/- mice revealed greater susceptibility to ID T. brucei infection than wild-type mice, indicating that the early accumulation of the trypanosomes in the draining lymph nodes was not essential to establish systemic infection. Although LTβ-/- mice were able to control the first parasitemia wave as effectively as wild-type mice, they were unable to control subsequent parasitemia waves. LTβ-/- mice also lack organized B cell follicles and germinal centers within their remaining secondary lymphoid tissues. As a consequence, LTβ-/- mice have impaired immunoglobulin (Ig) isotype class-switching responses. When the disturbed microarchitecture of the B cell follicles in the spleens of LTβ-/- mice was restored by reconstitution with wild-type bone marrow, their susceptibility to ID T. brucei infection was similar to that of wild-type control mice. This effect coincided with the ability to produce significant serum levels of Ig isotype class-switched parasite-specific antibodies. Thus, our data suggest that organized splenic microarchitecture and the production of parasite-specific Ig isotype class-switched antibodies are essential for the control of ID African trypanosome infections.

Mammalian three-dimensional (3D) enteroids mirror in vivo intestinal organisation and are powerful tools to investigate intestinal cell biology and host-pathogen interactions. We have developed complex multilobulated 3D chicken enteroids from intestinal embryonic villi and adult crypts. These avian enteroids develop optimally in suspension without the structural support required to produce mammalian enteroids, resulting in an inside-out enteroid conformation with media-facing apical brush borders. Histological and transcriptional analyses show these enteroids comprise of differentiated intestinal epithelial cells bound by cell-cell junctions, and notably, include intraepithelial leukocytes and an inner core of lamina propria leukocytes. The advantageous polarisation of these enteroids has enabled infection of the epithelial apical surface with Salmonella Typhimurium, influenza A virus and Eimeria tenella without the need for micro-injection. We have created a comprehensive model of the chicken intestine which has the potential to explore epithelial and leukocyte interactions and responses in host-pathogen, food science and pharmaceutical research.

Macrophages and dendritic cells (DCs) in peripheral tissue interact closely with their local microenvironment by scavenging protein and nucleic acids released by neighboring cells. Material transfer between cell types is necessary for pathogen detection and antigen presentation, but thought to be relatively limited in scale. Recent reports, however, demonstrate that the quantity of transferred material can be quite large when DCs are in direct contact with live cells. This observation may be problematic for conditional gene deletion models that assume gene products will remain in the cell they are produced in. Here, we investigate whether conditional gene deletions induced by the widely used Cre/Lox system can be overcome at the protein level in DCs. Of concern, using the human Langerin Cre mouse model, we find that epidermal Langerhans cells and CD11b+CD103+ mesenteric DCs can overcome gene deletion if the deleted gene is expressed by neighboring cells. Surprisingly, we also find that the mechanism of material transfer does not resemble known mechanisms of antigen uptake, is dependent on extra- and intracellular calcium, PI3K, and scavenger receptors, and mediates a majority of material transfer to DCs. We term this novel process intracellular monitoring, and find that it is specific to DCs, but occurs in all murine DC subsets tested, as well as in human DCs. Transferred material is successfully presented and cross presented on MHC-II and MHC-I, and occurs between allogeneic donor and acceptors cells-implicating this widespread and unique process in immunosurveillance and organ transplantation.

Prions are a unique group of pathogens, which are considered to comprise solely of an abnormally folded isoform of the cellular prion protein. The accumulation and replication of prions within secondary lymphoid organs is important for their efficient spread from the periphery to the brain where they ultimately cause neurodegeneration and death. Mononuclear phagocytes (MNP) play key roles in prion disease pathogenesis. Some MNP appear to facilitate the propagation of prions to and within lymphoid tissues, whereas others may aid their clearance by phagocytosis and by destroying them. Our recent data show that an intact splenic marginal zone is important for the efficient delivery of prions into the B-cell follicles where they subsequently replicate upon follicular dendritic cells before infecting the nervous system. Sialoadhesin is an MNP-restricted cell adhesion molecule that binds sialylated glycoproteins. Sialoadhesin is constitutively expressed upon splenic marginal zone metallophilic and lymph node sub-capsular sinus macrophage populations, where it may function to bind sialylated glycoproteins, pathogens and exosomes in the blood and lymph via recognition of terminal sialic acid residues. As the prion glycoprotein is highly sialylated, we tested the hypothesis that sialoadhesin may influence prion disease pathogenesis. We show that after peripheral exposure, prion pathogenesis was unaltered in sialoadhesin-deficient mice; revealing that lymphoid sequestration of prions is not mediated via sialoadhesin. Hence, although an intact marginal zone is important for the efficient uptake and delivery of prions into the B-cell follicles of the spleen, this is not influenced by sialoadhesin expression by the MNP within it.

In mice, three major B-cell subsets have been identified with distinct functionalities: B1 B cells, marginal zone B cells and follicular B2 B cells. Here, we used the growing body of publicly available transcriptomics data to create an expression atlas of 84 gene expression microarray data sets of distinct mouse B-cell subsets. These data were subjected to network-based cluster analysis using BioLayout Express(3D). Using this analysis tool, genes with related functions clustered together in discrete regions of the network graph and enabled the identification of transcriptional networks that underpinned the functional activity of distinct cell populations. Some gene clusters were expressed highly by most of the cell populations included in this analysis (such as those with activity related to house-keeping functions). Others contained genes with expression patterns specific to distinct B-cell subsets. While these clusters contained many genes typically associated with the activity of the cells they were specifically expressed in, many novel B-cell-subset-specific candidate genes were identified. A large number of uncharacterized genes were also represented in these B-cell lineage-specific clusters. Further analysis of the activities of these uncharacterized candidate genes will lead to the identification of novel B-cell lineage-specific transcription factors and regulators of B-cell function. We also analysed 36 microarray data sets from distinct human B-cell populations. These data showed that mouse and human germinal centre B cells shared similar transcriptional features, whereas mouse B1 B cells were distinct from proposed human B1 B cells.

Ectopic lymphoid structures form in a wide range of inflammatory conditions, including infection, autoimmune disease, and cancer. In the context of infection, this response can be beneficial for the host: influenza A virus infection-induced pulmonary ectopic germinal centers give rise to more broadly cross-reactive antibody responses, thereby generating cross-strain protection. However, despite the ubiquity of ectopic lymphoid structures and their role in both health and disease, little is known about the mechanisms by which inflammation is able to convert a peripheral tissue into one that resembles a secondary lymphoid organ. Here, we show that type I IFN produced after viral infection can induce CXCL13 expression in a phenotypically distinct population of lung fibroblasts, driving CXCR5-dependent recruitment of B cells and initiating ectopic germinal center formation. This identifies type I IFN as a novel inducer of CXCL13, which, in combination with other stimuli, can promote lung remodeling, converting a nonlymphoid tissue into one permissive to functional tertiary lymphoid structure formation.

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.

Prion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue, we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk of infection and the preclinical diagnosis of disease.

Marginal zone (MZ) B cells are positioned within the spleen to capture blood-borne antigen and immune complexes and deliver them to follicular dendritic cells in the B-cell follicles. We show that within the spleens of aged mice antigen capture by MZ B cells, and their ability to shuttle between the follicle and MZ, were impaired. The ability of aged MZ B cells to migrate towards the MZ chemoattractant sphingosine-1-phosphate was increased, suggesting that aged MZ B cells had a greater propensity to be retained within the MZ. An extrinsic impairment in aged B-cell migration towards the MZ was demonstrated using bone marrow chimeras. The follicular shuttling of MZ B cells derived from either young or aged bone marrow was similarly reduced in aged recipient spleens, showing that ageing effects on splenic stromal cells were responsible for the impaired follicular shuttling of MZ B cells. MZ B cells rapidly mount T-cell-independent (TI) antibody-responses to microbial polysaccharide antigen. In aged mice the ability to produce immunoglobulins in response to the TI type 1 antigen TNP-LPS was impaired. These ageing-related changes to the MZ and MZ B cells have implications for the clearance of blood-borne pathogens. Indeed elderly people have increased susceptibility to Streptococcus pneumoniae, a TI antigen, and decreased responses to vaccination. A thorough analysis of the mechanisms that underpin the ageing-related decline in the status of the MZ and MZ B cells will help the design of novel treatments to improve immunity in the elderly.

Prion diseases are a unique group of transmissible, typically sub-acute, neurodegenerative disorders. During central nervous system (CNS) prion disease, the microglia become activated and are thought to provide a protective response by scavenging and clearing prions. The mammalian intestine is host to a large burden of commensal micro-organisms, especially bacteria, termed the microbiota. The commensal microbiota has beneficial effects on host health, including through the metabolism of essential nutrients, regulation of host development and protection against pathogens. The commensal gut microbiota also constitutively regulates the functional maturation of microglia in the CNS, and microglial function is impaired when it is absent in germ-free mice. In the current study, we determined whether the absence of the commensal gut microbiota might also affect prion disease pathogenesis. Our data clearly show that the absence of the commensal microbiota in germ-free mice did not affect prion disease duration or susceptibility after exposure to prions by intraperitoneal or intracerebral injection. Furthermore, the magnitude and distribution of the characteristic neuropathological hallmarks of terminal prion disease in the CNS, including the development of spongiform pathology, accumulation of prion disease-specific protein (PrP), astrogliosis and microglial activation, were similar in conventionally housed and germ-free mice. Thus, although the commensal gut microbiota constitutively promotes the maintenance of the microglia in the CNS under steady-state conditions in naïve mice, our data suggest that dramatic changes to the abundance or complexity of the commensal gut microbiota are unlikely to influence CNS prion disease pathogenesis.

Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages.CSF1R signaling has been implicated in the maintenance of the intestinal stem cell niche and differentiation of Paneth cells, but evidence of expression of CSF1R within the crypt is equivocal. Here we show that CSF1R-dependent macrophages influence intestinal epithelial differentiation and homeostasis. In the intestinal lamina propria CSF1R mRNA expression is restricted to macrophages which are intimately associated with the crypt epithelium, and is undetectable in Paneth cells. Macrophage ablation following CSF1R blockade affects Paneth cell differentiation and leads to a reduction of Lgr5+ intestinal stem cells. The disturbances to the crypt caused by macrophage depletion adversely affect the subsequent differentiation of intestinal epithelial cell lineages. Goblet cell density is enhanced, whereas the development of M cells in Peyer's patches is impeded. We suggest that modification of the phenotype or abundance of macrophages in the gut wall alters the development of the intestinal epithelium and the ability to sample gut antigens.

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.

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.