Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-β and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations

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Not AvailableThe limited efficacy of DNA vaccines against foot-and-mouth disease (FMD) in cattle and other natural hosts has prompted a search for a more effective vaccination regimen. In this study we tested a DNA prime-protein boost vaccination strategy against FMD in bovine calves. We used purified recombinant FMDV specific multi-epitope protein (rMEG990) and an optimized sindbis virus replicase-based DNA vaccine expressing this protein (pSinCMV-Vac-MEG990). We demonstrate that vaccination with a low dose of pSinCMV-Vac-MEG990 (10 μg/animal) and subsequently boosting with rMEG990 resulted in induction of neutralizing antibodies, IFN-γ production and protection against homologous virus challenge. However, vaccination with a high dose of pSinCMV-Vac-MEG990 (100 μg/animal) and boosting with rMEG990 resulted in significantly lower immune responses and more severity to the challenge test. Additionally, we show that the post-vaccinal IFN-γ levels in animals correlated positively to their protection against FMDV challenge. These findings suggest that a replicase-based DNA vaccine in proper prime-boost combination may offer an efficient vaccine strategy against FMDV and that IFN-γ could be used as an additional immune parameter to predict protection against FMDV infection.Not Availabl

Expression, purification, and functional characterisation of Flagellin, a TLR5-ligand

Flagellin, a Toll-like receptor 5 (TLR5)-ligand, is known for its activities like adjuvant, induction of pro-inflammatory cytokines and innate immunity. In this context, fliC gene of Salmonella Typhimurium was cloned into pET32a expression plasmid using in-house designed gene specific primers. The frame and orientation of the inserted fliC gene was confirmed upon colony PCR, restriction enzyme analysis and sequencing. Sequence analysis of fliC revealed proper orientation of the gene and had 1,485 nucleotides. Following transformation of pET-fliC plasmid into Escherichia coli BL21 (DE3) cells, the gene was expressed after inducing with IPTG (Isopropylβ-D-1-thiogalactopyranoside). The polyHis-tag-fliC was ~70kDa as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The identity/authenticity of the recombinant-fliC was confirmed by its specific reactivity with commercial anti-fliC MAb of S. Typhimurium. Further, the antigenic and functional properties of recombinant-fliC were determined espousing its ability to induce antigen specific antibodies in G pigs and increased m-RNA expression of certain pro-inflammatory mediators like TNF-α and GM-CSF in vitro

Morphological and phenotypic characterization of immature MoDCs.

<p>(A) Light microscopy (20X) of freshly isolated monocyte. (B) Light microscopy (20X) of 4^th day culture of presumably immature MoDCs showing clusters of veiled cells with pseudopodia. (C) Freshly isolated monocytes and 5 day culture of MoDCs were analysed for phenotypic changes by evaluating mRNA expression levels of various TLRs and costimulatory genes by qRT-PCR assay. Results are expressed as fold change (log10) in mRNA transcription of monocytes and MoDCs. Data presented are mean ± standard deviation of values obtained from three independent experiments involving two cattle of Hallikar breed. *<i>p</i>< 0.05.**<i>p</i>< 0.01.</p

Kinetics of cytokine mRNA transcription from MoDCs treated with either BGs or LPS.

<p>RNA was extracted and gene transcription was quantified by qRT-PCR at 0, 6 and 12 h. Results are expressed as fold induction (log10) of cytokine mRNA transcription by BGs or LPS stimulated cells compared to the media treated (Med) cells. GAPDH was used as an internal control and mRNA levels at 0 h was used as calibrator. Histograms represent mean cytokine levels and bars represent standard deviation of three independent experiments. *p < 0.05, **p<0.01, ns = non-significant.</p

Characterization of <i>E</i>. <i>coli</i> ghosts by electron microscopy.

<p>(A) Field emission scanning electron micrograph (FE-SEM) of protein E lysed <i>E</i>. <i>coli</i> ghosts showing transmembrane tunnels, indicated by arrow heads. (B) FESEM of intact <i>E</i>. <i>coli</i> before lysis. (C) Transmission electron micrograph (TEM) of <i>E</i>. <i>coli</i> ghosts with a loss of cytoplasmic contents but intact cellular morphology. (D) TEM of intact <i>E</i>. <i>coli</i> prior to gene <i>E</i> induction.</p

Antigen presenting capacity of MoDCs determined by mixed lymphocyte reaction.

<p>(A) Bovine MoDCs (1.5 x 10⁴) were cultured with allogenic T cells (1.1 x 10⁵) and treated either with ConA, BGs, LPS or media (Med). The T cells alone stimulated either with LPS or media were kept as controls. (B) MoDCs were cultured with allogenic T cells (1.1 x 10⁵) of a different breed in a graded manner. The cultures were maintained for three days and subsequently, T cell proliferation was measured by the uptake of MTT dye and expressed as stimulation index. Data presented are mean ± standard deviation of values for MoDCs and allogenic T cells isolated from three animals. *p<0.05.**p<0.01.</p

Morphological and phenotypical features of mature bovine MoDCs.

<p>(A) Light microscopy (20X) of MoDCs after stimulation with BGs demonstrated large dendrite like pseudopodia (B) qRT-PCR analysis of MoDCs after stimulation with either BGs or LPS or treated with media (Med). Results are expressed as fold induction (log10) in mRNA transcription after BGs or LPS stimulation compared to the media treated (non-stimulated) cells. Gene expressions were normalized to GAPDH and mRNA levels at 0 h were used as calibrator. Data presented are mean ± standard deviation of three independent experiments involving two cattle of Hallikar breed. **p < 0.01, ***p<0.001, ns = non-significant.</p

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Not AvailableFoot and mouth disease virus (FMDV) causes an economically devastating, contagious viral disease in cloven-hoofed animals. Macrophages are potent mediators of innate immune responses against viruses. The macrophage-FMDV interaction is not studied in detail, especially the innate immune responses to the virus. Here, we investigated the effects of experimental infection of FMDV on bovine monocyte-derived macrophages (MDM). The detection of negative-strand FMDV RNA by strand specific RT-PCR and demonstration of viral antigen by immunofluorescence indicated the replication of FMDV RNA and synthesis of viral proteins, respectively in the infected MDM. However, replication kinetic experiments revealed that the copy number of FMDV transcripts and virus titration peaked at 12 hpi with subsequent reduction, indicating non-progressive replication. FMDV infection upregulated the expression of RIG1 and MDA5 in MDM, suggests that the FMDV RNA are sensed by RLRs. In addition, nearly 75% of the infected MDM differentiated into M1 phenotype, characterized by the increased expression of M1-specific markers; CD80, iNOS and proinflammatory cytokines such as TNFα and IL12. The infection induced the expression of Type I IFNs, which coincided with the decline in the viral RNA and progeny virus after 12 hpi. In addition, infected MDM abundantly expressed ISGs such as PKR, OAS1, Mx1 and viperin against FMDV infection at 12 hpi. In conclusion, FMDV underwent a non-progressive replication in the bovine MDM that might be due to induction of M1 polarization and upregulation of antiviral genes.ICAR-IVR