Human-relevant near-organ neuromodulation of the immune system via the splenic nerve.

Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve-induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

Mammalian lectin arrays for screening host-microbe interactions.

Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen-receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.

Activation of Dendritic Cells in Tonsils Is Associated with CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus.

Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163+CD14+ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-γ+ CD8 T cell response in C-strain vaccinated pigs, but not in pigs that received Alfort-187. Moreover, the frequency of CSFV-specific IFN-γ+ CD8 T cells was inversely correlated to the viral load in the tonsils of individual animals. Accordingly, we hypothesise that the activation of cDC1 is key in initiating local CSFV-specific CD8 T cell responses which curtail early virus replication and dissemination.

CD23 is a glycan-binding receptor in some mammalian species.

CD23, the low-affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors. In most mammalian species, the CD23 residues required to form a sugar-binding site are present, although binding of CD23 to IgE does not involve sugars. Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, GlcNAc, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions. Crystal structures of the cow CRD in the presence of α-methyl mannoside and GlcNAcß1-2Man reveal that a range of oligosaccharide ligands can be accommodated in an open binding site in which most interactions are with a single terminal sugar residue. Although mouse CD23 shows a pattern of monosaccharide and glycoprotein binding similar to cow CD23, the binding is weaker. In contrast, no sugar binding was observed in similar experiments with human CD23. The absence of sugar-binding activity correlates with accumulation of mutations in the gene for CD23 in the primate lineage leading to humans, resulting in loss of key sugar-binding residues. These results are consistent with a role for CD23 in many species as a receptor for potentially pathogenic microorganisms as well as IgE. However, the ability of CD23 to bind several different ligands varies between species, suggesting that it has distinct functions in different organisms.

Live and inactivated Salmonella enterica serovar Typhimurium stimulate similar but distinct transcriptome profiles in bovine macrophages and dendritic cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of gastroenteritis in cattle and humans. Dendritic cells (DC) and macrophages (Mø) are major players in early immunity to Salmonella, and their response could influence the course of infection. Therefore, the global transcriptional response of bovine monocyte-derived DC and Mø to stimulation with live and inactivated S. Typhimurium was compared. Both cell types mount a major response 2 h post infection, with a core common response conserved across cell-type and stimuli. However, three of the most affected pathways; inflammatory response, regulation of transcription and regulation of programmed cell death, exhibited cell-type and stimuli-specific differences. The expression of a subset of genes associated with these pathways was investigated further. The inflammatory response was greater in Mø than DC, in the number of genes and the enhanced expression of common genes, e.g., interleukin (IL) 1B and IL6, while the opposite pattern was observed with interferon gamma. Furthermore, a large proportion of the investigated genes exhibited stimuli-specific differential expression, e.g., Mediterranean fever. Two-thirds of the investigated transcription factors were significantly differentially expressed in response to live and inactivated Salmonella. Therefore the transcriptional responses of bovine DC and Mø during early S. Typhimurium infection are similar but distinct, potentially due to the overall function of these cell-types. The differences in response of the host cell will influence down-stream events, thus impacting on the subsequent immune response generated during the course of the infection.

Identifying and addressing barriers and opportunities for bovine respiratory disease complex vaccination: a consensus paper on practical recommendations for best practise vaccination.

Many questions are raised, and challenges faced in the new era of (intranasal) bovine respiratory disease complex vaccination. An increase in vaccination rate is expected, due to its positive impact on cattle health, reduction of antimicrobial use and economic factors. However, engagement of farmers and veterinarians with regard to vaccination is often affected by limitations, resulting in the development of barriers to vaccination, but also opportunities to overcome these. The objective of the report is to provide practical recommendations and a consensus on best practises for BRDC vaccination, addressing barriers faced by veterinarians and farmers. The report combines an evidence review with expert opinions and includes discussions on different vaccination approaches, such as intranasal and systemic protocols. As result of the discussions, several barriers to BRDC vaccination were identified, including a lack of knowledge or visibility of the disease's impact, the preference for blanket antibiotic use over vaccination, resistance to change, the need for visible success, uncertainty about the best time to vaccinate, and concerns about adverse reactions and vaccine efficacy in the presence of maternal antibodies. While these barriers seem substantial, they provide opportunities for the veterinary sector. Indeed, veterinarians are encouraged to use the argumentation presented, along with local case studies and diagnostic testing to highlight the impact of disease, while conducting calf health audits, ensuring expectations are managed to achieve visible success. Overall, this consensus paper aims to provide practical recommendations and support for veterinarians and farmers to overcome barriers and increase BRDC vaccination rates in cattle.

Multi-omics analysis reveals regime shifts in the gastrointestinal ecosystem in chickens following anticoccidial vaccination and Eimeria tenella challenge.

Coccidiosis, caused by Eimeria parasites, significantly impacts poultry farm economics and animal welfare. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and caecal metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial Bifidobacterium. Using a multi-omics factor analysis, data on caecal microbiota and metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offering potential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.IMPORTANCEAdvances in anticoccidial vaccines have garnered significant attention in poultry health management. However, the intricacies of vaccine-induced alterations in the chicken gut microbiome and its subsequent impact on host metabolism remain inadequately explored. This study delves into the metabolic and microbiotic shifts in chickens post-vaccination, employing a multi-omics integration analysis. Our findings highlight a notable synergy between the microbiome composition and host-microbe interacted metabolic pathways in vaccinated chickens, differentiating them from infected or non-vaccinated cohorts. These insights pave the way for more targeted and efficient approaches in poultry disease control, enhancing both the efficacy of vaccines and the overall health of poultry populations.

First detection and characterisation of Eimeria zaria in European chickens.

The global poultry industry has experienced dramatic growth in recent decades, increasing the significance of pathogens of chickens. Protozoan parasites of the genus Eimeria can cause the disease coccidiosis, compromising animal health and welfare, and incurring significant annual costs. Seven Eimeria species have long been recognised to infect chickens, supplemented by three new candidate species first reported from Australia in 2007/8. Named Eimeria lata, Eimeria nagambie and Eimeria zaria, one or more of these new species have been reported in Australia, several countries in sub-Saharan Africa, India, Venezuela, and most recently the United States of America, but none have been detected in Europe. Here, a panel of 56 unvaccinated broiler chicken farms were sampled in the final week of production from France, Greece, Italy, the Netherlands, the Republic of Ireland, and the United Kingdom to assess the occurrence of all ten Eimeria species using specific polymerase chain reaction (PCR). Overall, 39 of 56 (69.6%) farms were found to host at least one species. Eimeria acervulina, E. tenella, and E. maxima were most common, with E. mitis and E. praecox also widespread. Eimeria necatrix was detected on one farm in France, while E. brunetti was not detected. Eimeria zaria was detected for the first time in Europe, appearing in Greece and Italy (one occurrence each). New primers were designed to confirm detection of E. zaria and provide template for phylogenetic comparison with the reference isolate from Australia. Detection of E. zaria in Europe reinforces the importance of integrated control for coccidiosis given the lack of protection induced by current anticoccidial vaccines.

Characterization of canine intestinal microRNA expression in inflammatory bowel disease and T-cell lymphoma.

Differentiating between canine inflammatory bowel disease (IBD) and intestinal T-cell lymphoma by histopathological examination of endoscopically-derived intestinal biopsies can be challenging and involves an invasive procedure requiring specialized equipment and training. A rapid, non-invasive method of diagnosis, such as blood or faecal analysis for a conserved and stable biomarker, would be a useful adjunct or replacement. Studies on dogs and humans with various types of lymphoma have shown altered microRNA (miRNA) expression patterns in blood, faeces and tissues indicating their potential use as biomarkers of disease. The present study used residual archived endoscopically-derived, formalin-fixed, paraffin-embedded (FFPE) duodenal tissue taken from pet dogs undergoing routine investigation of gastrointestinal disease. The dogs had previously been diagnosed with either normal/minimal intestinal inflammation, severe IBD or intestinal T-cell lymphoma. Next generation sequencing with qPCR validation was used to elucidate differentially expressed miRNAs between groups. Our results show that miRNA can be extracted from archived endoscopically-derived FFPE tissues from the canine duodenum and used to differentiate normal/minimally inflamed canine duodenal tissue from severe lymphoplasmacytic IBD and T-cell lymphoma.

Characterisation of the bovine C-type lectin receptor Mincle and potential evidence for an endogenous ligand.

Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6'-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macrophages. However, boMincle mRNA transcripts were also detected in granulocytes, B cells, and T cells. Finally, we show that boMincle binds to isolated bovine CD4+ T cells in a specific manner, indicating the potential to recognise endogenous ligands. This suggests that the receptor might also play a role in homeostasis in cattle.

Adaptive evolution of Toll-like receptor 5 in domesticated mammals.

BACKGROUND: Previous studies have proposed that mammalian toll like receptors (TLRs) have evolved under diversifying selection due to their role in pathogen detection. To determine if this is the case, we examined the extent of adaptive evolution in the TLR5 gene in both individual species and defined clades of the mammalia. RESULTS: In support of previous studies, we find evidence of adaptive evolution of mammalian TLR5. However, we also show that TLR5 genes of domestic livestock have a concentration of single nucleotide polymorphisms suggesting a specific signature of adaptation. Using codon models of evolution we have identified a concentration of rapidly evolving codons within the TLR5 extracellular domain a site of interaction between host and the bacterial surface protein flagellin. CONCLUSIONS: The results suggest that interactions between pathogen and host may be driving adaptive change in TLR5 by competition between species. In support of this, we have identified single nucleotide polymorphisms (SNP) in sheep and cattle TLR5 genes that are co-localised and co-incident with the predicted adaptive codons suggesting that adaptation in this region of the TLR5 gene is on-going in domestic species.

TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility.

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.

Inhibition of cytosolic Phospholipase A2 prevents prion peptide-induced neuronal damage and co-localisation with Beta III Tubulin.

BACKGROUND: Activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites. RESULTS: Exposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3) reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death. CONCLUSIONS: Collectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

East Coast fever, a neglected tropical disease with an outdated vaccine approach?

Since its discovery, bovine theileriosis has caused major socioeconomic losses in sub-Saharan Africa. Acaricide resistance of the intermediate host, paucity of therapeutics, and lack of sufficiently cross-protective vaccines increase the risk of parasite spread due to global warming. Here, we highlight three important areas that require investigation to develop next-generation vaccines.

Transcriptomic Analysis of Circulating Leukocytes Obtained during the Recovery from Clinical Mastitis Caused by Escherichia coli in Holstein Dairy Cows.

The risk and severity of clinical infection with Escherichia coli as a causative pathogen for bovine mastitis is influenced by the hosts' phenotypic and genotypic variables. We used RNA-Seq analysis of circulating leukocytes to investigate global transcriptomic profiles and genetic variants from Holstein cows with naturally occurring cases of clinical mastitis, diagnosed using clinical symptoms and milk microbiology. Healthy lactation-matched cows served as controls (CONT, n = 6). Blood samples were collected at two time periods during the recovery phase post diagnosis: EARLY (10.3 ± 1.8 days, n = 6) and LATE (46.7 ± 11 days, n = 3). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21 and subjected to enrichment analysis. Variant calling was performed following GATKv3.8 best practice. The comparison of E. coli(+) EARLY and CONT cows found the up-regulation of 1090 DEGs, mainly with immune and inflammatory functions. The key signalling pathways involved NOD-like and interleukin-1 receptors and chemokines. Many up-regulated DEGs encoded antimicrobial peptides including cathelicidins, beta-defensins, S100 calcium binding proteins, haptoglobin and lactoferrin. Inflammation had largely resolved in the E. coli(+) LATE group, with only 29 up-regulated DEGs. Both EARLY and LATE cows had up-regulated DEGs encoding ATP binding cassette (ABC) transporters and haemoglobin subunits were also up-regulated in LATE cows. Twelve candidate genetic variants were identified in DEGs between the infected and CONT cows. Three were in contiguous genes WIPI1, ARSG and SLC16A6 on BTA19. Two others (RAC2 and ARHGAP26) encode a Rho-family GTPase and Rho GTPase-activating protein 26. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite prompt treatment and provide preliminary evidence for genetic differences between cows that may predispose them to infection.

A Novel Whole Yeast-Based Subunit Oral Vaccine Against Eimeria tenella in Chickens.

Cheap, easy-to-produce oral vaccines are needed for control of coccidiosis in chickens to reduce the impact of this disease on welfare and economic performance. Saccharomyces cerevisiae yeast expressing three Eimeria tenella antigens were developed and delivered as heat-killed, freeze-dried whole yeast oral vaccines to chickens in four separate studies. After vaccination, E. tenella replication was reduced following low dose challenge (250 oocysts) in Hy-Line Brown layer chickens (p<0.01). Similarly, caecal lesion score was reduced in Hy-Line Brown layer chickens vaccinated using a mixture of S. cerevisiae expressing EtAMA1, EtIMP1 and EtMIC3 following pathogenic-level challenge (4,000 E. tenella oocysts; p<0.01). Mean body weight gain post-challenge with 15,000 E. tenella oocysts was significantly increased in vaccinated Cobb500 broiler chickens compared to mock-vaccinated controls (p<0.01). Thus, inactivated recombinant yeast vaccines offer cost-effective and scalable opportunities for control of coccidiosis, with relevance to broiler production and chickens reared in low-and middle-income countries (LMICs).

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations.

Introduction: The interfascicular matrix (IFM; also known as the endotenon) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Methods: Immunolabelling of equine superficial digital flexor tendon was used to identify the interfascicular matrix niche, localising expression patterns of CD31 (endothelial cells), Desmin (smooth muscle cells and pericytes), CD146 (interfascicular matrix cells) and LAMA4 (interfascicular matrix basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. Results: Labelling for CD146 using standard histological and 3D imaging of large intact 3D segments revealed an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms extensive, interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralisation (osteogenesis) and lipid production (adipogenesis). Discussion: This study demonstrates that the interfascicular matrix is a unique tendon cell niche, containing a vascular-rich network of basement membrane, CD31+ endothelial cells, Desmin+ mural cells, and CD146+ cell populations that are likely essential to tendon structure and/or function. Contrary to our hypothesis, interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes. Instead, our results indicate CD146 as a pan-vascular marker within the tendon interfascicular matrix. Together with previous work demonstrating that endogenous tendon CD146+ cells migrate to sites of injury, our data suggest that their mobilisation to promote intrinsic repair involves changes in their relationships with local interfascicular matrix vascular and basement membrane constituents.

Defining the Genes Required for Survival of Mycobacterium bovis in the Bovine Host Offers Novel Insights into the Genetic Basis of Survival of Pathogenic Mycobacteria.

Tuberculosis has severe impacts on both humans and animals. Understanding the genetic basis of survival of both Mycobacterium tuberculosis, the human-adapted species, and Mycobacterium bovis, the animal-adapted species, is crucial to deciphering the biology of both pathogens. There are several studies that identify the genes required for survival of M. tuberculosis in vivo using mouse models; however, there are currently no studies probing the genetic basis of survival of M. bovis in vivo. In this study, we utilize transposon insertion sequencing in M. bovis AF2122/97 to determine the genes required for survival in cattle. We identify genes encoding established mycobacterial virulence functions such as the ESX-1 secretion system, phthiocerol dimycocerosate (PDIM) synthesis, mycobactin synthesis, and cholesterol catabolism that are required in vivo. We show that, as in M. tuberculosis H37Rv, phoPR is required by M. bovis AF2122/97 in vivo despite the known defect in signaling through this system. Comparison to studies performed in species that are able to use carbohydrates as an energy source, such as M. bovis BCG and M. tuberculosis, suggests that there are differences in the requirement for genes involved in cholesterol import (mce4 operon) and oxidation (hsd). We report a good correlation with existing mycobacterial virulence functions but also find several novel virulence factors, including genes involved in protein mannosylation, aspartate metabolism, and glycerol-phosphate metabolism. These findings further extend our knowledge of the genetic basis of survival in vivo in bacteria that cause tuberculosis and provide insight for the development of novel diagnostics and therapeutics. IMPORTANCE This is the first report of the genetic requirements of an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC) in a natural host. M. bovis has devastating impacts on cattle, and bovine tuberculosis is a considerable economic, animal welfare, and public health concern. The data highlight the importance of mycobacterial cholesterol catabolism and identify several new virulence factors. Additionally, the work informs the development of novel differential diagnostics and therapeutics for TB in both human and animal populations.

Corrigendum: Escape of TLR5 recognition by Leptospira spp.: A rationale for atypical endoflagella.

[This corrects the article DOI: 10.3389/fimmu.2020.02007.].