The pathogenic mechanisms of APEC are complex and diverse, causing huge economic losses worldwide. Studies have shown that T6SS secretes effectors that not only facilitate bacterial nutrient acquisition, organelle synthesis, or defense against host immunity but also assist in the precise input of virulence factors. Previous laboratory experiments confirmed that APEC infection led to pathological changes in the chicken trachea and caused the infiltration of heterophilic granulocytes(4). In another study, infection with Salmonella enteritidis resulted in differential protein expression in heterophilic granulocytes and the release of granule proteins that capture and kill invading microorganisms, in vitro and in vivo(11). Another study showed that important heterosexual-mediated defense mechanisms in chickens are achieved by chicken heterophil extracellular traps (HETs)(12).
We further investigated how APEC infection caused the infiltration of heterophilic granulocytes. The pathological sections showed that heterophilic granulocyte infiltration occurred after AE17 infection of chicken trachea. Therefore, we sequenced the tracheal tissues of the PBS group and the AE17 experimental group by TMT proteomics and inferred the cause of heterophilic granulocyte infiltration from the sequencing results. Our results showed that differentially expressed proteins appeared after infection by AE17 to varying degrees. The GO results suggest that heterophilic granulocyte infiltration is involved in a variety of biological processes. Of interest to us, serine-type endopeptidase inhibitor activity and regulation of complement activation were particularly significant in the upregulated proteins. In the downregulated proteins, the structural constituent of ribosome and protein localization to endoplasmic reticulum processes were significant. Bioinformatics analysis showed that these differentially expressed proteins function in biological processes, cellular components, molecular functions, subcellular locations, and signaling pathways. Subcellular localization analysis showed that these differential proteins were mainly distributed in the cytoplasm, nucleus, and mitochondria. Serine protease inhibitors are widely found in animals, plants, and microorganisms. They are natural inhibitors of serine proteases and involved in many important life processes, such as coagulation, complement activation, inflammatory response, cell migration, cell matrix reconstitution, and tumor suppression; upregulation of serine protease inhibitors implies thrombosis, hemostasis, and fibrinolysis and causes fibrin production(13). Serine protease inhibitors can also act as potent inhibitors of thrombolytic serine proteases to modulate the inflammatory response(14). Thus, upregulation of the complement system with serine protease inhibitors is a manifestation of the involvement in the inflammatory response, indirectly causing the infiltration of heterophilic granulocytes. KEGG pathway analysis showed that the three pathways Complement and coagulation cascades, Protein export and Phagosome upregulated proteins differed significantly and Ribosome pathway downregulated proteins differed significantly, these results are consistent with the GO analysis results, we think that the phenomenon causing heterophilic granulocyte infiltration is related to These results are consistent with the results of GO analysis, and we believe that the phenomenon of heterophilic granulocyte infiltration is related to differential proteins in these pathways. The extraordinary ability of pathogens to use the host hemostatic system to support microbial survival and dissemination following infection by Streptococcus pyogenes has been previously reported(15), the coagulation cascade includes both the contact system and the tissue factor pathway, both of which lead to fibrin formation. Analysis of the Complement and coagulation cascades pathway showed high fold differences between the C3 and C4 proteins, which have a high degree of homology(16). The C3 protein is a key complement protein in the alternative pathway(17), maintaining T-cell homeostasis in vivo and inducing the production of autocrine pro-inflammatory cytokines(18). The complement system is a mediator of the protein hydrolysis cascade reaction and innate immunity in plasma, a non-specific defense mechanism against pathogens(19). The main consequences of complement activation are pathogenic conditioning, inflammation, and recruitment of immunoreactive cells, as well as direct killing by pathogens. Coagulation is the conversion of another series of zymogens to serine proteases, culminating in the formation of thrombin, the enzyme responsible for the conversion of soluble fibrinogen into insoluble fibrin clots. Based on the GO analysis, upregulation of serine protease inhibitor expression. Protease-activated receptors, such as those activated by thrombin, are members of the G protein-coupled receptors and function as mediators of innate immunity(20). The kinin-releasing enzyme-kinin system is an endogenous metabolic cascade reaction whose triggering leads to the release of vasoactive kinins (bradykinin-associated peptides)(21), which are associated with inflammatory processes. In addition, the complement body enhances the function of strong antibodies and phagocytes(22).
We therefore infer that the alternative pathway is mainly involved in the inflammatory response causing heterophil infiltration after AE17 infection; the Complement and coagulation cascades pathway also showed high levels of FGA, FGB, and FGG differential ploidy; as a fibronectin class, it can be enhanced in this process by binding to interleukins (IL), thereby increasing resistance to infection(23). These three proteins are also present in the Neutrophil extracellular trap formation pathway, and heterophilic granulocytes are functionally similar to neutrophils, We therefore infer that the upregulated expression of these three proteins indirectly causes the infiltration of heterophilic granulocytes. Integrins as surface receptors of phagocytes can also mediate phagocytosis(24), and the upregulation of heterophilic granulocytes, such as avian phagocytes integrin protein, implies infiltration of heterophilic granulocytes. Interestingly, the differential multiplicity of iCb was higher in the Phagosome pathway, which is involved in the inflammatory response in synergy with the complement pathway. To enable B lymphocytes to change the expression of igM to igG for an optimal immune response, IGH expression is upregulated(25). Pathogen phagocytosis is ultimately accomplished through combinatorial interactions between phagocytic receptors(26). Through the Protein export pathway, we found that the up-regulated expression of the transport proteins SEC61α and SEC61β formed the Sec61 complex, which constitutes the protein conduction channel. The Sec61 complex plays an important role in the transmembrane transport of proteins and provides a new pathway for the transport of newly synthesized peptides to the ER lumen(27). In addition, Sec61 is also a co-transporter with the lumenal protein Bip, which is an upstream protein of the antigen presentation pathway and completes the immune response with phagocytes(28). In summary, when AE17 infects the chicken tracheal mucosa, it causes an increase in protein secretion, causing antigen presentation to stimulate the body's defense response, mainly through the complement and coagulation system of C3 protein, involved in the alternative pathway, to participate in the inflammatory response, causing downstream integrin protein-mediated phagocytosis. Consequently, heterophilic granulocytes appear to infiltrate. Further, the ability of the complement system to respond rapidly and extensively to microbial invaders, apoptotic cells, and other threats by inducing a robust elimination response is essential for its role as a host defense and surveillance system. The identification of novel complement-mediated indications and the clinical availability of the first therapeutic complement inhibitors have also sparked new interest in the development of complement-targeted drugs, and recent developments hold great promise for both diagnosis and treatment(29). It is therefore our hope that the present study provides a foundation for future research on the complement system.