Effect of FTA on apoptosis of PBMCs infected with BVDV.

<p>PBMCs were collected from the indicated cultures at 24 h after BVDV challenge. (A) Representative two-dimensional scatter plots of annexin V versus propidium iodide, the percentage of early apoptotic cells and the percentage of late apoptotic cells. (B) Relative mRNA expression of Bcl-xL and Bim in bovine PBMCs after simulation. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Effect of FTA on the expression of TRAF1 and TRAF2 in bovine PBMCs infected with BVDV.

<p>(A) Relative mRNA expression of TRAF-1 and TRAF-2 cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The TRAF-2 protein express by western blot (left panels) and ratio of TRAF-2 band intensity to that of GAPDH (right panels). TRAF-2 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Primers used for real-time PCR, length of the respective PCR product and gene accession number.

<p>Primers used for real-time PCR, length of the respective PCR product and gene accession number.</p

Effect of FTA on the expression of CD28 and CTLA-4 in bovine PBMCs infected with BVDV.

<p>(A) Relative mRNA expression of CD28/CTLA-4 cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The CD28 protein express by western blot (left panels) and ratio of CD28 band intensity to that of GAPDH (right panels). CD28 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Effect of FTA on the expression of OX40, 4-1BB, and 4-1BBL in bovine PBMCs infected with BVDV.

<p>(A) Relative mRNA expression of OX40, 4-1BB, and 4-1BBL cultured with medium alone, FTA, BVDV, and BVDV plus FTA at 24, 48, 72 h in bovine PBMCs. (B) The 4-1BB protein express by western blot (left panels) and ratio of 4-1BB band intensity to that of GAPDH (right panels). 4-1BB protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. Data are presented as the means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Concentrations of IFN-γ, IL-2, and IgG2a in cell culture supernatants.

<p>Bovine PBMCs cultured with medium alone, FTA, BVDV, and BVDV plus FTA. Concentrations of IFN-γ (A), IL-2 (B) and IgG2a (C) were detected by ELISA. Data are presented as means ± SEM of three independent experiments. Mean values at the same time point without a common superscript (^{a, b, c}) differ significantly (P <0.05).</p

Effect of FTA on BVDV replication and virion infectivity.

<p>(A) The number of living cell (left panels) and virus copies per cell (right panels) at 24, 48, and 72 h after simulation. The number were counted with Trypan blue stain and the virus copies per living cells were measured by absolute quantitative real-time PCR for amplifying the 5’ UTR. (B) The BVDV-E2 protein express by western blot (left panels) and ratio of E2 band intensity to that of GAPDH (right panels). BVDV E2 protein in PBMCs was collected from the indicated PBMCs cultures at 24, 48, and 72 h after stimulation. Expression of GAPDH was measured as an internal control. (C) BVDV titers in supernatants of PBMCs treated with BVDV or BVDV and FTA at 24, 48, and 72h. Data are presented as the means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Proliferation of PBMCs after stimulation.

<p>Bovine PBMCs were labeled with CFSE before stimulation with FTA alone, BVDV alone or BVDV plus FTA, and were assessed for their ability to proliferate at 72 (A)and 96 h (B) later by flow cytometry.</p

Image_2_Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Activation of NLRP3 and NLRC4 Inflammasomes With Differential Requirement for ASC.TIF

<p>Escherichia coli is a common cause of mastitis in dairy cows. The adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) synergizes with caspase-1 to regulate inflammasome activation during pathogen infection. Here, the ASC gene was knocked out in bovine mammary epithelial (MAC-T) cells using clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated (Cas)-9 technology. MAC-T cells were pre-incubated with and without Lactobacillus rhamnosus GR-1 and then exposed to E. coli. Western blot analysis demonstrated increased expression of NLRP3 and NLRC4 following E. coli infection, but this increase was attenuated by pre-incubation with L. rhamnosus GR-1, regardless of ASC knockout. Western blot and immunofluorescence analyses revealed that pre-incubation with L. rhamnosus GR-1 decreased E. coli-induced caspase-1 activation at 6 h after E. coli infection, as also observed in ASC-knockout MAC-T cells. The E. coli-induced increase in caspase-4 mRNA expression was inhibited by pre-incubation with L. rhamnosus GR-1. ASC knockout diminished, but did not completely prevent, increased production of IL-1β and IL-18 and cell pyroptosis associated with E. coli infection, whereas pre-incubation with L. rhamnosus GR-1 inhibited this increase. Our data indicate that L. rhamnosus GR-1 suppresses activation of ASC-dependent NLRP3 and NLRC4 inflammasomes and production of downstream IL-lβ and IL-18 during E. coli infection. L. rhamnosus GR-1 also inhibited E. coli-induced cell pyroptosis, in part through attenuation of NLRC4 and non-canonical caspase-4 activation independently of ASC.</p

Concentrations of TNF-α, IL-10, and PGE₂ in IPEC-J2 cell culture supernatants.

<p>Supernatants were collected from the indicated cultures at 3 h after F4^<b>+</b> ETEC challenge. The concentrations of (A) TNF-α, (B) IL-10, and (C) PGE_<b>2</b> were determined by ELISA. Data are presented as means ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p