Innate Cytokine Responses and Toll-Like Receptor Induced by Recombinant Porcine Rotavirus VP6 and VP7 Proteins Expressing in Lactobacillus plantarum NC8 Strain Colonization in Mice

The significant function of Toll-like receptors (TLR) is the detection of mi-crobes by host guard cells that guide to the innate immune responses and to the successive adaptive. The current study patterns of TLR2, TLR3 and TLR9 expressing antigen presenting cells (APCs) in blood of mice after colonization with L. plantarum NC8 strain were assessed. The power of L. plantarum on serum innate cytokine and TLR responses stimulated by recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep and NC8-pSIP409-pgsA were also assessed. We confirmed that L. plantarum NC8 stimulated powerful TLR2 expressing APC responses in blood Recombinant strain stimulated a TLR3 response in spleen, and TLR9 responses were stimulated in blood or in spleen. Recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep on TLR2 and TLR9 expressing APC responses has a preservative outcome, reliable with the DCpep adjuvant outcome. In serum the recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep has increased the IL-4 and IFN-γ responses, except that on the TLR3 and TLR9 expressing CD14 APC responses it had an oppressive con-sequence in spleen and the IFN-α response in serum-stimulated by PRV. Our results give details that following PRV infection after immunization with NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep, the systemic TLR2, TLR3, and TLR9 expressing cDC and macrophage/monocyte responses.


Introduction
The significant function of Toll like receptor (TLRs), as kind of pattern-recognition receptor (PRR), is connection inborn and adaptive immune and inborn immunity responses and viral antigen identification [1] [2] [3]. On many cell types the TLRs can be expressed e.g. macrophages/monocytes and DCs. Through several signaling pathways the toll like receptor activation induces type I interferon production that leads to anti-viral and proinflammatory cytokine responses and induction of adaptive immune responses [4] [5] [6]. The recognition of different microbe-associated molecular patterns (MAMPs) is one of the roles of different TLRs [5]. The recognition of lipoteichoic acids, peptidoglycans and lipopeptides from bacteria is the role of TLR1in association with TLR2 and TLR6 [7] [8]. Toll like receptor 3 recognizes dsRNA, which is found many viruses like in rotavirus genome during their replication cycles. For worldwide the mainly vital etiologic agent of severe gastroenteritis in young animal and young children is rotaviruses [9]. Therefore, in systemic lymphoid tissues the TLR may contribute to the anti-viral immunity.
In animals and humans Lactic acid bacteria, including lactobacilli are broadly appraised as probiotics [10] [11] and have been shown in young animals and have been shown to significantly stimulate gut epithelial cell proliferation enhance innate and acquired immunity in young animals and children and suppress intestinal inflammation [12] [13] [14]. The severity of acute rotavirus gastroenteritis in children has been shown to reduce by different LAB strains [15] [16]. In the current study we focused on the effect of L. plantarum NC8 on adaptive and innate immune responses to porcine rotavirus infection and the effect of DCpep adjuvant on porcine rotavirus vaccines. The effect of different adjuvants of several LAB strains has been reported in pigs and in humans [17] [18] [19] [20] [21]. However, the mechanism is undefined.
Our study focused on the effect of recombinant L. Plantarum NC8 expressing VP6 and VP7 on early cytokine responses as via pattern recognition receptor (PRR); they are indicators of innate immune cells activation and function in the innate immune response. It directly interacts with intracellular TLR3 and enhances dsRNA-mediated TLR3 activation by supporting uptake of dsRNA into cells [22].
In this study to elucidate the effect of L. Plantarum NC8 on different APC in systemic lymphoid tissues the influences of LAB on frequencies of TLR2, TLR3 and TLR9 expressing CD14+ versus CD14− DCs and macrophages/monocytes in the blood and spleen of PRV infected mice were evaluated. Thus, the understanding of treatment and prevention of viral diseases in animals and humans on how

Chemical and Enzyme
All chemicals for cloning, expression, and purification of recombinants, were purchased from Bioss China and provided by Laboratory of Jilin Provincial Engineering Research Center of Animal Probiotics, in Jilin Agricultural University of China.

Mice
In   Electroporation of L. plantarum was carried out as follows: 10 μl of plasmid was gently added to 100 μl of L. plantarum NC8, and mixed gently for 5 min at 4˚C and were subjected to an electric pulse. The mixture of plasmids DNA and L. plantarum NC8 were then anaerobically without E.M incubated in MRS medium for 2 h at 37˚C. Recombinant NC8-pSIP409-pgsA-VP6-Dcpep and NC8-pSIP409-pgsA-VP7-Dcpep were picked on MRS agar medium including E.M.

Expression Plasmid Construction
The transformants of L. plantarum NC8 sequences were verified by plasmid DNA sequencing.

Immunization of Recombinant L. plantarum Strainsto BALB/c Mice
Mice were orally immunized with 109 CFU of L. plantaram strains and the immune protocol was administered on three consecutive days: the inoculation at days 0, 1, and 2; a booster at days 14, 15, and 16; a second booster at days 28, 29, and 30. Mice sera were collected on days 0, 9, 25, and 41 following the first immunization to evaluate the immunogenicity of the recombinant L. plantarum NC8. PBS was given in the similar approach to the control groups.

Sample Collection from Immunized Mice
To isolate mononuclear cells (MNCs) from spleen and peripheral blood; the blood, and fecal pellets were collected from mice 14 days after the first and second booosting immunization of NC8-pSIP409-pgsA-VP6-DCpep or NC8-pSIP409-pgsA-VP7-DCpep. On the same day of MNC isolation the isolated MNCs were stained with antibodies to mice cell markers and TLR antibodies straight without in vitro stimulation. was added for staining of TLR2, which is expressed on the cell surface, the TLR2 and its alike isotype match control tubes. Before flow cytometry analysis the cells were incubated, washed three times, fixed and resuspended in staining buffer and kept at 4˚C in dark. By using a 4 color FACSCalibur flow cytometer the analysis of the stained cells was performed and a minimum 22,000 cells were obtained. Data analysis was performing using FlowJo 7.6.1 software.

ELISA Analysis for Detection of Serum Cytokine Levels
Blood samples from mice were collected from the eye. Sera was processed and stored at −20˚C until tested. The ELISA was conducted using anti-mouse cytokine antibodies for detection of porcine IFN-γ, IL-4, IL-6, IL-10, IL-12, IFN-γ, TGF-β, and TNF-α as previously described [24].

Statistical Analysis
To evaluate frequencies of TLR-expressing cells in subpopulation of cells in spleen and blood among groups and the serum cytokine concentrations among groups the non-parametric Kruskal Wallis rank sum test was performed. The similar test was used when dissimilarities among these groups were detected, in a pairwise approach to make clear the nature of the dissimilarities. For the evaluation of association between frequencies of TLR-expressing macrophages/monocytes or cDCs and concentrations of cytokine in serum the Spearman's rank correlation test was used. At p < 0.05 was judged as statistical significance throughout.

Fecal Counts of Lactobacillus plantaram, PRV Infection and Clinical Signs
The   Within the treatment groups in both spleen and blood high unpredictability was observed for frequencies of TLR expressing APCs.

Induction of TLR9 Expressing CD14+ and CD14− APCs by Recombinants
The TLR9 expressing CD14+ and CD14− APCs patterns in the L. plantaram NC8-pSIP409-pgsA-Vp6-DCpep and L. plantaram NC8-pSIP409-pgsA-Vp7-DCpep groups were comparable to patterns of TLR3 with somewhat lesser frequencies as shown in (Figure 2), signifying that TLR9 is as well engaged in the PRV innate immune responses induction. There was minimal induction of TLR9 expressing   pSIP409-pgsA-Vp7-DCpep in blood contrasted to NC8-pSIP409-pgsA group and they were significantly higher than the PBS control group.

Concentrations of Serum IFN-α Significantly Correlated with TLR3-and TLR9-Expressing CD14− APCs Frequencies in Spleen
To recognize the associations among frequencies of TLR2, TLR3 and TLR9 expressing APCs in spleen or blood and serum concentrations of the cytokines between the four treatment groups the Spearman's rank correlation analysis was performed. Significant positive correlations were found between concentrations of IFN-α and frequencies of TLR3 expressing CD14− cDC and macrophages/monocytes and TLR9 expressing CD14− cDC and macrophages/monocytes in spleen. These associations recommend that the CD14− APCs in spleen may significantly contribute to the levels of IFN-α in serum.
The CD14+ and CD14− APCs expressing TLR2, TLR3 and TLR9 were both stimulated in spleen by PRV infection; on the other hand, the responses in blood of TLR expressing APC were incredibly dissimilar from that of spleen. In blood of the NC8-pSIP409-pgsA mice the frequencies of TLR2, TLR3 and TLR9 expressing APCs were comparable to the control PBS mice. In the NC8-pSIP409-pgsA mice group the lack of TLR responses in blood is the same with previous results showing that PRV infection did not augment total frequencies of APCs or CD14+ cDCs, and in blood reduced significantly the frequencies total of CD14+ macrophages/monocytes [17] [18] [19] [20] [21]. The significant dissimilarity among the APC response in blood and spleen the probable causes may be due to MNCs were only at one time point collected. Our study gives ad-World Journal of Vaccines ditional confirmation that TLR9 is involved in induction of the innate immune responses by both recombinant L. plantaram NC8-pSIP409-pgsA-Vp6-DCpep or L. plantaram NC8-pSIP409-pgsA-Vp7-DCpep because the highest frequencies induced of TLR9 expressing CD14+ macrophages/monocytes in blood and spleen, which is the same results with the effect of DCpep adjuvant vaccine B and T cell immune responses induced [27]. In spleen and blood the IFN-α level reduction associated significantly with the frequencies reduction of TLR3 and TLR9 expressing CD14− APCs, which perhaps recommend that the action of CD14− APCs significantly contributes to the response of IFN-α. The L. plantaram NC8 on IFN-γ, IL-4, IFN-α and TGF-β serum responses had significant influence.

Conclusion
This study after L. plantaram NC8 colonization elucidated the systemic responses of TLR2, TLR3, and TLR9 expressing macrophage/and monocyte cDC,

Declarations Ethics Approval and Consent to Participate
This study was carried out in agreement with the principles established by Jilin Agriculture University Changchun China and guide for the use of laboratory and care animals and all experimental protocols were approved by a Jilin Agriculture University (No. JLAU08201007).

Consent for Publication
Not applicable. Availability of data and materials: will be provided after publication when required.

Funding
This work was supported by The National Key Research and Development Program of China (2017YFD0501000).

Authors' Contributions
Author 1 SMS-searching data, wrote manuscript and acted as corresponding author. Author 2 WY-editing manuscript. Author 3 GY-editing the manuscript and supervision of the manuscript. Author 4 CW-editing the manuscript and supervision of the manuscript.