Mesenchymal stromal cells modulate infection and inflammation in the uterus and mammary gland.

The use of mesenchymal stromal cells (MSCs) is emerging as an efficacious and safe treatment for many infectious and non-infectious inflammatory diseases in human and veterinary medicine. Such use could be done to treat mastitis and metritis, which are the most common disease conditions affecting dairy cows leading to considerable economic losses and reduced animal welfare. Currently, both disease conditions are commonly treated using local and systemic administration of antibiotics. However, this strategy has many disadvantages including low cure rates and the public health hazards. Looking for alternative approaches, we investigated the properties of MSCs using in-vitro mammary and endometrial cell systems and in-vivo mastitis and metritis murine model systems. In-vitro, co-culture of mammary and uterus epithelial cells constructed with NF-kB reporter system, the master regulator of inflammation, demonstrated their anti-inflammatory effects in response to.LPS. In vivo, we challenge animals with field strains of mammary and utero pathogenic Escherichia coli and evaluated the effects of local and systemic application of MSC in the animal models. Disease outcome was evaluated using histological analysis, bacterial counts and gene expression of inflammatory markers. We show that MSC treatment reduced bacterial load in metritis and significantly modulated the inflammatory response of the uterus and mammary gland to bacterial infection. Most notably are the immune modulatory effects of remotely engrafted intravenous MSCs, which open new avenues to the development of MSC-based cell-free therapies.

Correction of Defective T-Regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor-α.

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4ß7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4ß7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4ß7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.

The role of O-polysaccharide chain and complement resistance of Escherichia coli in mammary virulence.

Mastitis, inflammation of the mammary gland, is a common disease of dairy animals. The disease is caused by bacterial infection ascending through the teat canal and mammary pathogenic Escherichia coli (MPEC) are common etiology. In the first phase of infection, virulence mechanisms, designated as niche factors, enable MPEC bacteria to resist innate antimicrobial mechanisms, replicate in milk, and to colonize the mammary gland. Next, massive replication of colonizing bacteria culminates in a large biomass of microbe-associated molecular patterns (MAMPs) recognized by pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) mediating inflammatory signaling in mammary alveolar epithelial cells (MAEs) and macrophages. Bacterial lipopolysaccharides (LPSs), the prototypical class of MAMPs are sufficient to elicit mammary inflammation mediated by TLR4 signaling and activation of nuclear factor kB (NF-kB), the master regulator of inflammation. Using in vivo mastitis model, in low and high complements mice, and in vitro NF-kB luminescence reporter system in MAEs, we have found that the smooth configuration of LPS O-polysaccharides in MPEC enables the colonizing organisms to evade the host immune response by reducing inflammatory response and conferring resistance to complement. Screening a collection of MPEC field strains, we also found that all strains were complement resistant and 94% (45/48) were smooth. These results indicate that the structure of LPS O-polysaccharides chain is important for the pathogenesis of MPEC mastitis and provides protection against complement-mediated killing. Furthermore, we demonstrate a role for complement, a key component of innate immunity, in host-microbe interactions of the mammary gland.

Type Three Secretion System-Dependent Microvascular Thrombosis and Ischemic Enteritis in Human Gut Xenografts Infected with Enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe intestinal disease and infant mortality in developing countries. Virulence is mediated by a type three secretion system (T3SS), causing the hallmark attaching and effacing (AE) lesions and actin-rich pedestal formation beneath the infecting bacteria on the apical surface of enterocytes. EPEC is a human-specific pathogen whose pathogenesis cannot be studied in animal models. We therefore established an EPEC infection model in human gut xenografts in SCID mice and used it to study the role of T3SS in the pathogenesis of the disease. Following EPEC O127:H6 strain E2348/69 infection, T3SS-dependent AE lesions and pedestals were demonstrated in all infected xenografts. We report here the development of T3SS-dependent intestinal thrombotic microangiopathy (iTMA) and ischemic enteritis in ∼50% of infected human gut xenografts. Using species-specific CD31 immunostaining, we showed that iTMA was limited to the larger human-mouse chimeric blood vessels, which are located between the muscularis mucosa and circular muscular layer of the human gut. These blood vessels were massively invaded by bacteria, which adhered to and formed pedestals on endothelial cells and aggregated with mouse neutrophils in the lumen. We conclude that endothelial infection, iTMA, and ischemic enteritis might be central mechanisms underlying severe EPEC-mediated disease.

Immune profiling of experimental murine mastitis reveals conserved response to mammary pathogenic Escherichia coli, Mycoplasma bovis, and Streptococcus uberis.

Mastitis is one of the most prevalent and economically important diseases of dairy animals. The disease is caused by ascending bacterial infection through the teat canal. Among the most common mastitis-causing bacteria are Gram-negative coliforms, Gram-positive streptococci and staphylococci, and mycoplasma. The most prominent cellular hallmark of acute mammary infection is a massive recruitment of blood neutrophils into the tubular and alveolar milk spaces. The complex biological processes of leukocyte recruitment, activation, adhesion, and migration in the mammary gland remain largely elusive to date. While field research of mastitis in dairy animals contributed a lot to the development of mitigation, control, and even eradication programs, little progress was made toward understanding the molecular mechanisms underlying the pathogenesis of the disease. We report here experimental mastitis model systems in lactating mice challenged with field strains of common udder pathogens in dairy cows. We used these model systems to apply recently developed multiplex gene expression technology (Nanostring nCounter), which enabled us to study the expression of over 700 immune genes. Our analysis revealed a core of 100 genes that are similarly regulated and functionally or physically interacting in E. coli, M. bovis, and Strep uberis murine mastitis. Common significantly enriched gene sets include TNFɑ signaling via NFkB, Interferon gamma and alpha response, and IL6-JAK-STAT3 signaling. In addition, we show a significantly enriched expression of genes associated with neutrophil extracellular traps (NET) in glands challenged by the three pathogens. Ligand-receptor analysis revealed interactions shared by the three pathogens, including the interaction of the cytokines IL1ß, IL1ɑ, and TNFɑ with their receptors, and proteins involved in immune cell recruitment such as complement C3 and ICAM1 (with CD11b), chemokines CCL3 and CCL4 (with CCR1), and CSF3 (with CSF3R). Taken together, our results show that mammary infection with E. coli, M. bovis, and Strep uberis culminated in the activation of a conserved core of immune genes and pathways including NET formation.

Foxa2 regulates the expression of Nato3 in the floor plate by a novel evolutionarily conserved promoter.

The development of the neural tube into a complex central nervous system involves morphological, cellular and molecular changes, all of which are tightly regulated. The floor plate (FP) is a critical organizing center located at the ventral-most midline of the neural tube. FP cells regulate dorsoventral patterning, differentiation and axon guidance by secreting morphogens. Here we show that the bHLH transcription factor Nato3 (Ferd3l) is specifically expressed in the spinal FP of chick and mouse embryos. Using in ovo electroporation to understand the regulation of the FP-specific expression of Nato3, we have identified an evolutionarily conserved 204 bp genomic region, which is necessary and sufficient to drive expression to the chick FP. This promoter contains two Foxa2-binding sites, which are highly conserved among distant phyla. The two sites can bind Foxa2 in vitro, and are necessary for the expression in the FP in vivo. Gain and loss of Foxa2 function in vivo further emphasize its role in Nato3 promoter activity. Thus, our data suggest that Nato3 is a direct target of Foxa2, a transcription activator and effector of Sonic hedgehog, the hallmark regulator of FP induction and spinal cord development. The identification of the FP-specific promoter is an important step towards a better understanding of the molecular mechanisms through which Nato3 transcription is regulated and for uncovering its function during nervous system development. Moreover, the promoter provides us with a powerful tool for conditional genetic manipulations in the FP.

Lipoproteins Are Potent Activators of Nuclear Factor Kappa B in Mammary Epithelial Cells and Virulence Factors in Mycoplasma bovis Mastitis.

Mastitis due to Mycoplasma bovis is a worldwide problem, which leads to significant economic losses and affects animal welfare. However, the mechanisms by which M. bovis establishes and maintains intra-mammary infections (IMI) in dairy cows are largely unknown. To study in further detail the pathogenesis of M. bovis IMI, time- and cost-effective experimental models are needed. To this end, we established and characterized an in vitro murine mammary alveolar epithelial (EpH4) cell-based model and an in vivo murine mastitis model. Our results showed that live and UV-treated M. bovis field strain 161791 and its lipid-associated membrane proteins (LAMP) activated nuclear factor kappa B (NF-kB) in EpH4 cells in a dose-dependent manner. In the murine mastitis model, temporal and spatial dynamics of inflammation in the mammary tissues were evident. Live M. bovis elicited diffuse inflammation affecting the whole challenged gland peaking at 48 h post infection (pi) in contrast to LAMP challenge, which elicited only focal inflammation peaking at 24 h and resolving at 48 h pi. Inflammation was characterized by massive neutrophil recruitment into the milk spaces and by elevated expression of the inflammatory mediators TNF-α, KC, iNOS and NF-kB dependent genes: A20 and IkBα. Moreover, the presence of intraepithelial bacterial communities in glands challenged with live M. bovis bacteria was shown. The developed models can be used efficiently for future characterization of M. bovis virulence factors and host immune response to IMI.

NF-kappa-B activation unveils the presence of inflammatory hotspots in human gut xenografts.

The single-epithelial cell layer of the gut mucosa serves as an essential barrier between the host and luminal microflora and plays a major role in innate immunity against invading pathogens. Nuclear factor kB (NF-κB), a central component of the cellular signaling machinery, regulates immune response and inflammation. NF-κB proteins are activated by signaling pathways downstream to microbial recognition receptors and cytokines receptors. Highly regulated NF-κB activity in intestinal epithelial cells (IEC) is essential for normal gut homeostasis; dysregulated activity has been linked to a number of disease states, including inflammatory bowel diseases (IBD) such as Crohn's Disease (CD). Our aim was to visualize and quantify spatial and temporal dynamics of NF-κB activity in steady state and inflamed human gut. Lentivirus technology was used to transduce the IEC of human gut xenografts in SCID mice with a NF-κB luminescence reporter system. NF-κB signaling was visualized and quantified using low resolution, intravital imaging of the whole body and high resolution, immunofluorescence microscopic imaging of the tissues. We show that NF-κB is activated in select subset of IEC with low "leaky" NF-κB activity. These unique inflammatory epithelial cells are clustered in the gut into discrete hotspots of NF-κB activity that are visible in steady state and selectively activated by systemic LPS and human TNFα or luminal bacteria. The presence of inflammatory hotspots in the normal and inflamed gut might explain the patchy mucosal lesions characterizing CD and thus could have important implications for diagnosis and therapy.

Homeostasis of mucosal glial cells in human gut is independent of microbiota.

In mammals, neural crest cells populate the gut and form the enteric nervous system (ENS) early in embryogenesis. Although the basic ENS structure is highly conserved across species, we show important differences between mice and humans relating to the prenatal and postnatal development of mucosal enteric glial cells (mEGC), which are essential ENS components. We confirm previous work showing that in the mouse mEGCs are absent at birth, and that their appearance and homeostasis depends on postnatal colonization by microbiota. In humans, by contrast, a network of glial cells is already present in the fetal gut. Moreover, in xenografts of human fetal gut maintained for months in immuno-compromised mice, mEGCs persist following treatment with antibiotics that lead to the disappearance of mEGCs from the gut of the murine host. Single cell RNAseq indicates that human and mouse mEGCs differ not only in their developmental dynamics, but also in their patterns of gene expression.

Transplantation of Human Intestine Into the Mouse: A Novel Platform for Study of Inflammatory Enterocutaneous Fistulas.

BACKGROUND AND AIMS: Enteric fistulas represent a severe and medically challenging comorbidity commonly affecting Crohn's disease [CD] patients. Gut fistulas do not develop in animal models of the disease. We have used transplantation of the human fetal gut into mice as a novel platform for studying inflammatory enterocutaneous fistulas. METHODS: Human fetal gut segments were transplanted subcutaneously into mature SCID mice, where they grew and fully developed over the course of several months. We first analysed the resident immune cells and inflammatory response elicited by systemic lipopolysaccharide [LPS] in normal, fully developed human gut xenografts. Thereafter, we used immunostaining to analyse fully developed xenografts that spontaneously developed enterocutaneous fistulas. RESULTS: Resident human innate and adaptive immune cells were demonstrated in gut xenografts during steady state and inflammation. The expression of human IL-8, IL-1ß, IL-6, TNF-α, A20, and IkBα was significantly elevated in response to LPS, with no change in IL-10 gene expression. Approximately 17% [19/110] of fully developed subcutaneous human gut xenografts spontaneously developed enterocutaneous fistulas, revealing striking histopathological similarities with CD fistula specimens. Immunohistochemical analyses of fistulating xenografts revealed transmural lymphocytic enteritis associated with massive expansion of resident human CD4+ lymphocytes and their migration into the intraepithelial compartment. Regionally, mucosal epithelial cells assumed spindle-shaped mesenchymal morphology and formed fistulous tracts towards chronic non-healing wounds in the host mouse skin overlying the transplants. CONCLUSIONS: Inflammation and fistulas developed in human gut xenografts lacking IL-10 gene response. This novel model system will enable systematic studies of the inflamed and fistulating human gut in live animals.

Nato3 integrates with the Shh-Foxa2 transcriptional network regulating the differentiation of midbrain dopaminergic neurons.

Mesencephalic dopaminergic (mesDA) neurons originate from the floor plate of the midbrain, a transient embryonic organizing center located at the ventral-most midline. Since the loss of mesDA leads to Parkinson's disease, the molecular mechanisms controlling the genesis and differentiation of dopaminergic progenitors are extensively studied and the identification and characterization of new genes is of interest. Here, we show that the expression of the basic helix-loop-helix transcription factor Nato3 (Ferd3l) increases in parallel to the differentiation of SN4741 dopaminergic cells in vitro. Nato3 transcription is directly regulated by the transcription factor Foxa2, a target and effector of the Sonic hedgehog (Shh) signaling cascade. Moreover, pharmacological inhibition of Shh signaling downregulated the expression of Nato3, thus defining Nato3 as a novel component of one of the major pathways controlling cell patterning and generation of mesDA. Furthermore, we show that Nato3 regulated Shh and Foxa2 through a novel feed-backward loop. Up- and downregulation of Nato3 further affected the transcription of Nurr1, implicated in the genesis of mesDA, but not of TH. Taken together, these data shed new light on the transcriptional networks controlling the generation of mesDA and may be utilized in the efforts to direct stem cells towards a dopaminergic fate.