Molecular Mechanism Of SLC15A4 Regulation Of Immune Response In Bovine Rumen Epithelial Cells

The immune function of the rumen epithelium is important for maintaining the physiological health of the cow.Feeding cows high concentrate diets for long periods of time can cause significant bacterial mortality in the rumen and the production of bacterial small peptides,which can induce ruminal inflammation as well as cause systemic inflammatory responses,leading to significant losses.Among the oligopeptide transporter POTs family,SLC15A4（PHT1）is the most abundantly expressed in the rumen,which can mediate the cellular transport of a series of short-chain peptides and peptidomimetics using the potential difference between the inner and outer membrane.SLC15A4 is mainly located in the cell membrane and is responsible for the transport of di-/tripeptides and peptidomimetics from the extracellular to the cytoplasm,and plays an important role in maintaining the balance of short-chain peptides in the rumen.Bacterial cytosolic acyl dipeptide（MDP）is a biologically active minimal peptidoglycan motif common to all bacteria,which can be recognized by NOD2 receptors in the cytoplasm and activate signaling pathways such as NF-κB to promote the release of cellular inflammatory factors.However,it has not been investigated whether SLC15A4 can mediate MDP-regulated immune responses in bovine rumen epithelial cells（BREC）.Therefore,this experiment was conducted to investigate the molecular mechanism of SLC15A4 gene regulation in the immune response of bovine rumen epithelial cells by CRISPR/Cas9 gene editing technology to provide a theoretical basis for studying the rumen immune response in ruminants.The experimental results were as follows:1.CRISPR/Cas9 system to establish SLC15A4 knockout cell line in bovine rumen epithelial cells.The results showed that（1）using the wild-type BREC genome as a template,the PCR product was gel electrophoresed to obtain a 386 bp target band,which was consistent with the sequencing results.This indicates that SLC15A4 is present in the wild-type BREC genome.（2）LentiCRISPRv2-sgRNA vector was successfully constructed.sgRNA1,sgRNA2,sgRNA3 and sgRNA4 sequences were correctly inserted into lentiCRISPRv2,and the position,direction and sequence of the inserted sequences were as expected.（3）The sgRNA2 has cleavage activity on the second exonic region of BREC SLC15A4.Sequencing results of SLC15A4 CX upstream primers and downstream primers showed that both set peaks started from the target site sgRNA-2.This indicates that the LentiCRISPRv2-sgRNA2 vector was successfully transferred into BREC and caused insertion or deletion of SLC15A4 gene bases.（4）The monoclonal BREC inserted a base T at the target sgRNA2.Sequencing and TA cloning sequencing peak plots showed that this knockout was a pure knockout.Since the base insertion was not a multiple of 3 and caused a shift mutation downstream of the insertion,resulting in an early appearance of the stop codon TAG at base 816,causing a change in protein sequence or spatial conformation,the cell was determined to be a successful knockout cell line.2.Transcriptome analysis of the effects of SLC15A4 on differential mRNA expression and signaling pathways in BREC.The results showed that（1）SLC15A4 knockdown affected multiple signaling pathways in BREC and had an impact on the transport function of BREC.（2）The expression of IL-6 and IL-8 in BREC was highly significantly downregulated after knockdown of SLC15A4（P<0.001）.The expression of IL-1β,IL-32 and TNF-α was also downregulated.（3）The overall trend of chemokines was down-regulated after knockdown of SLC15A4,with CCL2,CCL5 and CXCL8 being highly significantly down-regulated（P<0.001）.While CCL28,CXCL2,CXCL3,CXCL5,CXCL9 and CXCL12 were significantly decreased in expression compared to wild-type BREC（P<0.05）.（4）No significant changes in NF-κB repressor kinases（IKKB,IKKE）and NF-κB repressors（IKBA,IKBB,IKBD,IKBE and IKBZ）in the NF-κB signaling pathway were observed after knockdown of SLC15A4.However,four of the five members of NF-κB transcription factor family（NF-κB1,NF-κ B2,RELA and RELB）were significantly down-regulated（P<0.05）.（5）After knockdown of SLC15A4,both NOD1 and NOD2 were highly significantly down-regulated（P<0.001）.Also key kinases RIPK1,RIPK2 and RIPK3 downstream of the NOD receptor signaling pathway were significantly down-regulated（P<0.05）.（6）Knockdown of SLC15A4 significantly upregulated TLR2,TLR3,TLR4 and MYD88 in the BREC Toll receptor signaling pathway（P<0.05）.Among the TNF receptor related factors,TRAF3 was significantly up-regulated,but TRAF6 had no effect.IRAK1 and IRAK2 in IL-1 receptor-associated kinase were significantly up-regulated（P<0.05）.（7）ERK2 was significantly upregulated and ERK1 and ERK8 were significantly downregulated in the MAPK signaling pathway in BREC after knockdown of SLC15A4（P<0.05）.JNK1 and JNK3 in JNK kinase were significantly up-regulated.The p38 β,p38 γ and p38 α in p38 kinase were significantly up-regulated（P<0.05）.（8）The qRT-PCR validation results showed that IL-6,CXCL2,CXCL3,CXCL9,CCL2,CCL5,NOD1,NOD2 and RIPK2 expressions were significantly down-regulated（P<0.05）in the SLC15A4 KO group compared with the WT group,and there was no difference in TNF-α expression,which was consistent with the results of transcriptome analysis above.The above results imply that SLC15 A4 knockdown affects the expression of MAPK,Toll,NOD-RIPK,and NF-κB signaling pathways as well as pro-inflammatory and chemokines in the BREC immune response.3.SLC15A4 mediates bacterial dipeptide MDP to regulate immune responses in bovine rumen epithelial cells.The experiment was divided into three groups:wild-type BREC（WT）;wild-type BREC with 10 μg/mL MDP added（WT+MDP）;and BREC with 10 μg/mL MDP knockout added（SLC15A4 KO+MDP）.The results showed that（1）MDP could significantly increase the expression of IL-6,TNF-α,CXCL2,CXCL3,CXCL9,CCL2 and CCL5 in BREC.The expression of IL-6,TNF-α,CXCL2,CXCL3,CXCL9,CCL2 and CCL5 induced by MDP was significantly decreased after SLC15A4 knockdown（P<0.05）.（2）MDP significantly increased the expression of NOD1,NOD2 and RIPK2 in BREC（P<0.05）.Expression of ERK1/2,p65,p-ERK1/2 and p-p65 kinases was significantly upregulated upon MDP addition.Knockdown of SLC15A4 decreased the expression of NOD1,NOD2 receptor and RIPK2 kinases,while expression of ERK1/2,p65,p-ERK1/2 and p-p65 kinases was significantly suppressed.This suggests that the NOD-RIPK pathway,NF-κB pathway and MAPK pathway activated by MDP were significantly inhibited after SLC15A4 knockdown.CONCLUSIONS:SLC15A4 can mediate MDP regulation of the NOD-RIPK pathway in BREC to modulate the NF-κB pathway and MAPK pathway,which in turn affects the expression of inflammatory factors and chemokines,thereby regulating the immune response in BREC.