Développement d'un test de caractérisation de la réponse immunitaire de bovins suite à une vaccination

Maste

Induction of IL-17A production during an antigen-specific inflammatory response in the bovine mammary gland

International audienceMammary gland (MG) response to bacterial intrusion is driven by the innate immune system, but can be modulated by the adaptive immunity. The model antigen ovalbumin (OVA) has already been shown to induce an antigen-specific inflammatory response when infused into the MG of cows previously sensitized by a systemic immunization but not of control naive cows. The objective of our study was to characterize this immune response. An overexpression of the genes encoding IL-17A, IL-17F, IL-21, IL-22 and INF-γ was found in milk cells and mammary tissue RNA extracts in the early phase of the inflammatory response following intramammary infusion of OVA. At the protein level, IL-17A was detected in milk as soon as 8 h post-challenge, and both IL-17A and IFN-γ concentrations peaked at 12 to 24 h postchallenge. 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. In vitro, an OVA-restimulation of PBMC or whole blood of sensitized cows induced the production of IL-17A and IFN-γ. The characterization of the IL-17A-producing leukocytes is underway. These results suggest that a Th17-type immune response can be induced in the MG by immunization to protein antigens, in parallel to a Th1-type response. As IL-17A is known to be involved in the immune defense against extracellular bacteria, and as we previously showed that bovine mammary epithelial cells respond to IL-17A by producing chemokines and antimicrobial peptides, we hypothesize that this cytokine contributes to the defense of the mammary gland against mastitis-causing bacteria

Antigen specific mammary inflammation depends on the production of IL-17A and IFN-γ by bovine CD4+ T lymphocytes

Remerciements au LEGTA de Vendôme, Loir-et-CherIntramammary infusion of the antigen used to sensitize cows by the systemic route induces a local inflammation associated with neutrophil recruitment. We hypothesize that this form of delayed type hypersensitivity, which may occur naturally during infections or could be induced intentionally by vaccination, can impact the outcome of mammary gland infections. We immunized cows with ovalbumin to identify immunological correlates of antigen-specific mammary inflammation. Intraluminal injection of ovalbumin induced a mastitis characterized by a prompt tissue reaction (increase in teat wall thickness) and an intense influx of leukocytes into milk of 10 responder cows out of 14 immunized animals. The magnitude of the local inflammatory reaction, assessed through milk leukocytosis, correlated with antibody titers, skin thickness test, and production of IL-17A and IFN-γ in a whole-blood antigen stimulation assay (WBA). The production of these two cytokines significantly correlated with the magnitude of the milk leukocytosis following the ovalbumin intramammary challenge. The IL-17A and IFN-γ production in the WBA was dependent on the presence of CD4+ cells in blood samples. In vitro stimulation of peripheral blood lymphocytes with ovalbumin followed by stimulation with PMA/ionomycin allowed the identification by flow cytometry of CD4+ T cells producing either IL-17A, IFN-γ, or both cytokines. The results indicate that the antigen-specific WBA, and specifically IL-17A and IFN-γ production by circulating CD4+ cells, can be used as a predictor of mammary hypersensitivity to protein antigens. This prompts further studies aiming at determining how Th17 and/or Th1 lymphocytes modulate the immune response of the mammary gland to infection

Ultrasonographic assessment of the teat cistern volume following ovalbumin infusion.

<p><b>(</b>A) Graph representing the evolution of the teat cistern volume (mean values from 10 responders and 4 low responders, ± standard deviations) relative to T0 (before ovalbumin infusion) showing a significant reduction of the teat cistern volume 14 hours after ovallbumin infusion into teat cisterns of high-responder cows (*p<0.05); (B) Ultrasounds photographs representing an ovalbumin-infused teat before infusion; (1): the cistern is filled with liquid (milk) therefore weakly echogenic; (2) 14 hpi, there was a thickening and permeabilization of the wall of the teat (inflammation) reducing the cistern volume. The white dashed line represents the initial position of the cistern wall; (3) 96 hpi, the teat has regained its original appearance. The bar represents 1 cm.</p

Concentrations of cytokines in milk samples of the 10 responder cows.

<p>Time-course of concentration variation (median and interquartiles) of CXCL8 (A), IL-17A (B) and IFN- γ (C) in the milk of quarters infused with ovalbumin at 0 hpi.</p

Kinetics of increases in skin thickness following intradermal inoculation with ovalbumin of the 14 immunized cows.

<p>(A) Increases in skinfold thickness calculated by subtracting the thickness value measured before inoculation from the values measured after injection. (B) Spearman correlation between Peak SCC and skin test values measured at 24 or 48 h post-inoculation, considering either the 14 immunized cows (All) or only the 10 responders (R). ns: not significant (p > 0.05).</p

Correlations (Spearman’s rank test) between skin thickness, Peak SCC and OVA-specific WBA

<p>Data based on the 14 cows under experiment. The skin tests values read 24 h or 48 h after ovalbumin intradermal inoculation were correlated with the concentrations of IL-17A or IFN-γ yielded by the OVA-specific WBA performed 45 (D45) or 60 (D60) days after the first immunization.</p><p>Correlations (Spearman’s rank test) between skin thickness, Peak SCC and OVA-specific WBA</p

Intracellular expression of IL-17A and IFN-γ by CD4+ T lymphocytes.

<p>PBMC were isolated one month after ovalbumin booster injection, stimulated in vitro with ovalbumin for 3 days, rested for 2 days and finally stimulated with PMA/ionomycin for 5 h with Brefeldin A for the last 3 hours. Cells were labeled for surface CD4 and intracellular IL-17A and IFN-γ. The numbers in the plots indicate the percentages of labeled cells in comparison to the isotype control. (A) Production of viable CD4+ T lymphoblasts after culture of PBMC with (OVA) or without (NS) ovalbumin. Left panels depict PBMC from a responder cow, right panels PBMC from a low-responder. (B) PBMC from two responder cows (R1 & R2) were labeled for surface CD4 and intracellular IL-17A or IFN-γ, showing CD4+ and CD4- IL-17A- and IFN-γ-producing cells. (C) labeling of CD4+ cells with anti-IL-17A and anti-IFN-γ antibodies, showing single-producing and double-producing cells. D) Double labeling of CD4+ cells from two low-responders (R3 and R4). Percentages of labeled cells are indicated in the quadrants. Results are from a representative experiment.</p

Principal component analysis of the inflammatory and immune parameters variability measured from the 14 immunized cows.

<p>The two components retained explained respectively 53.5% (p < 0.001) a,d 16.8% (p = 0.16) of the total inertia. (A) The ellipses representing the LR (Low-responders) and R (Responders) groups are clearly separated (p = 0.004). (B) The 1.5 standard deviation-inertia ellipses of the Montanide (red ellipse and cow numbers) and the Montanide + curdlan (blue ellipse and cow numbers) overlap extensively. The between-group test was not significant (p = 0.58).</p

Monitoring of the bodily temperature with an intravaginal sensor system.

<p>Median values of body temperature of high responder and low-responder cows before and after the intramammary challenge with OVA at 48 h (red arrow). Minor ticks indicate 4-hour intervals.</p