Modulation by IL-17A and IL-17F of bovine mammary epithelial cell innate immune response to Staphylococcus aureus components

L’IL-17 est une cytokine essentielle dans la défense de l’organisme contre de nombreuses bactéries pathogènes extracellulaires, comme Siaphylococcus aureus qui est un agent majeur des mammites des ruminants, en agissant en particulier sur les cellules épithéliales. L’objectif du travail était de tester l’hypothèse d’un rôle de i’IL-17 dans la protection de la mamelle en caractérisant la réponse des cellules épithéliales mammaires bovines (CEMb) à l’IL-17A et à l’IL-17F. Ces cytokines bovines ont été utilisées seules ou en combinaison avec des agonistes du système immunitaire inné produits par S. aureus, le muramyl dipeptide (MDP) et l’acide lipotéichoïque (LTA). Une première étude a montré que le LTA et le MDP exercent un effet synergique dans la mamelle et sur les CEMb en culture. Le tissu mammaire et les CEMb expriment le récepteur pour l’IL-l7A et F, et les CEMb ont répondu à i’lL-l7A et l’IL-l7F, bien que de façon modérée. Les réponses ont été sensiblement augmentées par la combinaison de l’IL-17 avec le LTA ou le MDP. Les réponses se caractérisent par une production de chimiokines capables d’attirer les polynucléaires neutrophiles et les leucocytes mononucléés, ainsi que par une surexpression de gènes à activités antibactériennes. Par contre, les CEMb ne produisent pas de cytokines inflammatoires. Ces résultats indiquent que i’IL-l7 est adaptée à un fonctionnement en milieu septique et ales capacités pour jouer un rôle dans la protection de la mamelle contre les infections staphylococciques.Through its activity on epithelial cells, interleukin-17 is a pivotai cytokine for the host defense against several extracellular pathogens, such as Siaphylococcus aureus that is a major agent of mastitis of ruminants. The investigation aimed at evaluating the role of IL-17 in udder protection by characterizing the response of bovine mammary epithelial cells (bMEC) to IL-17A and IL-17F. These bovine cytokines were used either alone or in combination with one of two innate immune system agonists associated with S. aureus, muramyldipeptide (MDP) or lipoteichoic acid (LTA). The first study showed that LTA synergized with MDP to recruit neutrophils in the mammary gland and to stimulate bMEC. A second study established that mammary tissue and bMEC in culture express the IL-17 receptor, and that bMEC respond to IL-17A and IL-17F, although with a moderate intensity. The response was noticeably augmented by the combination of IL-17 with LTA or MDP. The response was characterized by the production of chemokines able to attract neutrophils but also mononuclear leukocytes, as well as by an overexpression of genes endowed with antibacterial activity. Remarkably, bMEC did not release pro inflammatory cytokines. These results indicate that IL-17A and IL-17F are well suited for operating in a septic environment and have the potential to play a role in udder protection against staphylococcal infections

T helper 17-associated cytokines are produced during antigen-specific inflammation in the mammary gland.

Infectious mastitis cuts down milk production profitability and is a major animal welfare problem. Bacteria-induced inflammation in the mammary gland (MG) is driven by innate immunity, but adaptive immunity can modulate the innate response. Several studies have shown that it is possible to elicit inflammation in the MG by sensitization to an antigen subsequently infused into the lumen of the gland. The objective of our study was to characterize the inflammation triggered in the MG of cows sensitized to ovalbumin, by identifying the cytokines and chemokines likely to play a part in the reaction. Among immunized cows, responders mobilized locally high numbers of leukocytes. An overexpression of the genes encoding IL-17a, IL-17F, IL-21, IL-22 and INF-γ was found in milk cell RNA extracts in the early phase of the inflammatory response. At the protein level, IL-17A was detected in milk as soon as the first sampling time (8 h post-challenge), and both IL-17A and IFN-γ concentrations peaked at 12 to 24 h post-challenge. In mammary tissue from challenged quarters, overexpression of the genes encoding IL-17A, IL-17F, IL-21, IL-22, IL-26 and IFN-γ was observed. Neutrophil-attracting chemokines (CXCL3 and CXCL8) were found in milk, and overexpressed transcripts of chemokines attracting lymphocytes and other mononuclear leukocytes (CXCL10, CCL2, CCL5, CCL20) were detected in mammary tissue. Expression of IL-17A, as revealed by immunohistochemistry, was located in epithelial cells, in leukocytes in the connective tissue and in association with the epithelium, and in migrated alveolar leukocytes of challenged quarters. Altogether, these results show that antigen-specific inflammation in the MG was characterized by the production of IL-17 and IFN-γ. The orientation of the inflammatory response induced by the antigen-specific response has the potential to strongly impact the outcome of bacterial infections of the MG

Concentrations of chemoattractants and cytokines in milk samples of the 9 responsive cows.

<p>Concentrations were measured by ELISA in the milk samples of the 9 responder cows. Quarters were infused with 25 µg ovalbumin at time 0 and milk samples taken at indicated times. Median values (Q1, Q3) are shown. Concentrations varied significantly (Friedman test) as a function of hpi for C5a, CXCL8, IL-1β, IL-6, IFN-γ, IL-17A (p<0.001) and CXCL3 (p = 0.002).</p

Deciphering the complexities of cancer cell immune evasion: Mechanisms and therapeutic implications

Cancer immune evasion is one of the principal mechanisms leading to the progression and metastatization of the disease. Despite the migration and infiltration at the tumor site of immune cells, multiple factors can influence the composition of hot or “immune-sensitive” tumors and cold or “immune-resistant” tumors. Among the multiple mechanisms responsible for the make-up of the tumor microenvironment are the expression levels of major histocompatibility molecules (MHC) and of the antigen processing machinery, the metabolic network, hypoxia, and the secretion of pro-inflammatory molecules (e.g., cytokines, chemokines, and growth factors). Moreover, the different triggered pathways can mediate the reprogramming of activated, memory, effector, or regulatory/tolerogenic subtypes of immune cells (T, NK, dendritic cells, and macrophages). Recent studies have focused on the role of cancer metabolism in evading immune surveillance through the action of the active tryptophan catabolic enzyme indoleamine 2,3-dioxygenase (IDO). Immune suppression and evasion mechanisms in cancer cells are now being extensively studied with a special focus on developing immunotherapy strategies, such as the targeting of immune checkpoints (programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1), Cytotoxic T-lymphocyte antigen-4 (CTLA-4)), adoptive cell therapy or cancer vaccines. In this review, an overview of the underlying mechanisms of cancer immune evasion and the efficacy of the therapeutic targets and agents to overcome the immune escape are described

Concentrations of antibodies to ovalbumin following immunization.

<p>Data are presented as a function of the immunization group (A) or of the mammary ASR category following intramammary ovalbumin challenge (B). Antibodies were quantified in serum by ELISA before immunization (D0), on the day of first recall (D35/R1), on the day of second recall (D77/R2), two weeks later (D90), just before the intramammary challenge with ovalbumin (D104/IM) and eight days later (D112). Antibody concentrations did not differ significantly between the OVA+IFA and OVA+IFA+MAMP groups. Concentrations differed significantly between the high and low responder groups when indicated (*; Mann & Whitney test).</p

Characterization of cytokine expression in milk cells following intramammary challenge with ovalbumin.

<p>Five responder cows were challenged in one quarter with 25 µg ovalbumin and milk cells were collected for RT-qPCR. A) Kinetics of cellular influx in milk of challenged quarters. B) Kinetics of the relative mRNA expression of the indicated cytokines or cytokine components. Median values (Q1, Q3) are shown. It is important to keep in mind that relative expressions, contrary to absolute expression, do not allow comparisons between genes.</p

Correlations between milk cell concentrations (MCC) and concentrations of the chemoattractants and pro-inflammatory cytokines that were detected in the milk of all the responder cows.

<p>MCC 0–24 hpi: milk cell concentrations during the onset of the inflammatory response (0, 8, 12 and 24 h post-infusion). MCC 0–92 hpi: milk cell concentrations during the whole monitoring period. Correlations were calculated with the Bravais-Pearson test over two periods of time: at the onset of inflammation (0–24 hpi) and throughout the observation period (0–96 hpi).</p

Analysis by immunohistochemistry of representative tissue sections of uninfused, healthy mammary glands.

<p>A–B) Immunoreactivity of the apical side of the epithelial cells lining the alveoli to the N-term antibody to IL-17A of cows #1018 and 2014, respectively; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #1018); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase (cow #1018); E–F) Immunoreactivity of the epithelial lining the alveoli to abcam antibody (cows #1018 and 2014, respectively); G–H) Immunoreactivity of mammary tissue of healthy uninfused quarters of cows 1018 and 2014 to the C-term antibody, respectively. Scale bars indicate 25 µm.</p

Influx of leukocytes following the intramammary challenge of 21 cows with ovalbumin.

<p>A) Kinetics of cell concentrations in milk of unimmunized control cows, cows immunized with ovalbumin in incomplete Freund adjuvant, or cows immunized with ovalbumin in IFA supplemented with MAMP (MDP and LTA) (n = 7 per group); B) Individual values of milk cell concentrations at the peak of the cellular recruitment (3×7 cows); C) percentage of neutrophils (median values), determined by examination of cytospin slides stained with May-Grünwald-Giemsa, of the cows that reacted following immunization with ovalbumin in IFA (IFA; n = 4) or immunization with ovalbumin in IFA supplemented with MAMP (IFA+MAMP; n = 5); D) Kinetics of the individual values of the 9 immunized cows that reacted to ovalbumin challenge without distinction of groups.</p