Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells

Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis

Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis

Mastitis remains a major disease of cattle with a strong impact on the dairy industry. There is a growing interest in understanding how cell mediated immunity contributes to the defence of the mammary gland against invading mastitis causing bacteria. Cytokines belonging to the IL-17 family, and the cells that produce them, have been described as important modulators of the innate immunity, in particular that of epithelial cells. We report here that expression of IL-17A and IL-17F genes, encoding two members of the IL-17 family, are induced in udder tissues of cows experimentally infected with Escherichia coli. The impact of IL-17A on the innate response of bovine mammary epithelial cells was investigated using a newly isolated cell line, the PS cell line. We first showed that PS cells, similar to primary bovine mammary epithelial cells, were able to respond to agonists of TLR2 and to LPS, provided CD14 was added to the culture medium. We then showed that secretion of CXCL8 and transcription of innate immunity related-genes by PS cells were increased by IL-17A, in particular when these cells were stimulated with live E. coli bacteria. Together with data from the literature, these results support the hypothesis that IL-17A and IL-17 F could play an important role in mediating of host-pathogen interactions during mastitis

Inhibition of histamine-mediated signaling confers significant protection against severe malaria in mouse models of disease

From the inoculation of Plasmodium sporozoites via Anopheles mosquito bites to the development of blood-stage parasites, a hallmark of the host response is an inflammatory reaction characterized by elevated histamine levels in the serum and tissues. Given the proinflammatory and immunosuppressive activities associated with histamine, we postulated that this vasoactive amine participates in malaria pathogenesis. Combined genetic and pharmacologic approaches demonstrated that histamine binding to H1R and H2R but not H3R and H4R increases the susceptibility of mice to infection with Plasmodium. To further understand the role of histamine in malaria pathogenesis, we used histidine decarboxylase–deficient (HDC−/−) mice, which are free of histamine. HDC−/− mice were highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. HDC−/− mice displayed resistance to two lethal strains: Plasmodium berghei (Pb) ANKA, which triggers cerebral malaria (CM), and Pb NK65, which causes death without neurological symptoms. The resistance of HDC−/− mice to CM was associated with preserved blood–brain barrier integrity, the absence of infected erythrocyte aggregation in the brain vessels, and a lack of sequestration of CD4 and CD8 T cells. We demonstrate that histamine-mediated signaling contributes to malaria pathogenesis. Understanding the basis for these biological effects of histamine during infection may lead to novel therapeutic strategies to alleviate the severity of malaria

Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria

FcεR1-expressing neutrophils accumulate in the brain of mice infected with Plasmodium berghei (PbANKA) and promote the development of experimental cerebral malaria

Highlighting variability within<em> Escherichia coli</em> strains responsible for distinct outcomes of mastitis

International audienceIn France and Europe the first source of financial impairment in bovine dairy cattle is mastitis, an inflammation of the mammary gland generally induced by bacteria. When Escherichia coli (E. coli) infects the udder, the gravity of the clinical signs are variable, but the infection is generally brief, and the pathogen rapidly cleared. In many cases, E. coli infection results in systemic reaction in the cow, that alters the udder functionality. However in some cases, it leads to death. Thus, this study tends to characterize and understand how some E. coli strains can trigger such diverse responses. Three E. coli strains responsible for mastitis showed, in vivo and in vitro, very distinct inflammatory responses, as for cellular recruitment and pro-inflammatory cytokines, chemokines and anti-microbial peptides production. In a mouse model, P4 and B117 induced a rapid and massive recruitment of leucocytes, especially neutrophils, the key cellular population for an efficient bacterial clearance. As for K08, it induced a milder inflammation associated with weaker production of chemokines and inflammatory cytokines. Intrinsic characteristics like resistance to bovine serum, acute multiplication in the milk and a weak stimulatory effect on mammary epithelial cells may explain how some E. coli strains like P4 and B117 induce hyper acute and acute mastitis. On the contrary, strains like K08 showing serum sensitivity, weak multiplication potential in milk and strong inflammatory responses would be implicated in subacute mastitis. This study underlines the importance of considering mastitis in terms of clinical signs, but also of taking into account the bacterial strains implied

Sensing of coliforms by the bovine mammary gland and epithelial cells

International audienceDespite continuous efforts to reduce its incidence, mastitis remains a major disease in dairy cows. Main mastitis pathogens include Gram-positive as well as Gram-negative bacteria such as Escherichia coli, Klebsiella pneumonia and Serratia marcescens. Mammary Epithelial Cells (MEC) are likely key players in the initial response of the udder to intra-mammary bacterial infections. In this study we describe how these cells can sense the presence of mastitis pathogens. Our data show that MECs are equiped with Pattern-Recognition Receptors (PRR) that allow them to respond to LPS as well as to lipoproteins and peptidoglycan degradation products but, surprisingly, not to flagellin. These data are in agreement with in vivo data showing that infusion in the udder of these molecules, with the notable exception of flagellin, triggers an inflammatory response. In order to investigate how expression of PRR and reponse to purified bacterial agonists translate in terms of response to live bacteria, we compared the innate immune response of bovine mammary epithelial cells to infection by different strains of Enterobacteriacae. The response was analyzed in terms of IL-8 production. Altogether, our results will allow a better understanding of the initial steps of the interaction between mammary epithelial cells and Gram-negative mastitis pathogens

Reconnaissance des bactéries coliformes dans la mamelle et modulation de la réponse pro-inflammatoire

National audienc

Escherichia coli mastitis strains: In vitro phenotypes and severity of infection in vivo.

Mastitis remains a major infection of dairy cows and an important issue for dairy farmers and the dairy industry, in particular infections due to Escherichia coli strains. So far, properties specific to E. coli causing mastitis remain ill defined. In an attempt to better understand the properties required for E. coli to trigger mastitis, we used a range of in vitro assays to phenotypically characterize four E. coli strains, including the prototypical E. coli mastitis strain P4, possessing different relative abilities to cause mastitis in a mouse model. Our results indicate that a certain level of serum resistance might be required for colonization of the mammary gland. Resistance to neutrophil killing is also likely to contribute to a slower clearance of bacteria and higher chances to colonize the udder. In addition, we show that the four different strains do induce a pro-inflammatory response by mammary epithelial cells but with different intensities. Interestingly, the prototypical mastitis strain P4 actually induces the less intense response while it is responsible for the most severe infections in vivo. Altogether, our results suggest that different strategies can be used by E. coli strains to colonize the mammary gland and cause mastitis

Highlighting variability within <em>Escherichia coli</em> strains responsible for distinct outcomes of mastitis

National audienc

Diversité des réponses immunitaires de la glande mammaire lors de mammites induites par différentes souches d’ <em>Escherichia coli</em>

National audienceEn France et en Europe, les mammites constituent la première source de pertes financières des cheptels bovins laitiers et allaitants. Il s’agit d’une inflammation de la glande mammaire, essentiellement d’origine bactérienne. Les infections induites par Escherichia coli (E. coli) s’accompagnent de signes cliniques de gravité variable, et sont en général brèves, du fait d’une clairance rapide du pathogène. Néanmoins dans certains cas, la bactérie entraîne une réaction systémique chez la vache, pouvant altérer la fonctionnalité de la mamelle voire induire la mort de l’animal. Ainsi la présente étude vise-t-elle à caractériser et à comprendre comment des souches colibacillaires peuvent induire des réponses de la glande mammaire diverses. Les résultats portant sur quatre souches d’E. coli isolées de cas de mammites tendent à montrer que les réponses inflammatoires déclenchées par ces souches sont très différentes en termes de recrutement cellulaire et de production de cytokines pro-inflammatoires, de chimiokines et de peptides antimicrobiens. Des caractéristiques intrinsèques telles que la résistance au sérum bovin, une capacité accrue de multiplication dans le lait et un faible pouvoir inducteur de réponse immunitaire des cellules épithéliales mammaires pourraient expliquer comment des souches d’E. coli sont capables d’induire des mammites aiguës et suraiguës. Au contraire, des souches hautement sensibles au sérum, se multipliant faiblement dans le lait et déclenchant des réponses inflammatoires fortes seraient rapidement éliminées de la glande mammaire, sans induction de signe clinique. Cette étude souligne ainsi l’importance de considérer les mammites en fonction des signes cliniques observés, et en regard des souches bactériennes impliquées