Chicken cathelicidin-3 protected mice from avian pathogenic Escherichia coli infection mediated by triggering chemokine CCL3 pathway

Abstract Host defense peptides (HDPs) possess both direct bacteriocidal and immunomodulatory activities. In this study, chicken cathelicidin-1 (cCATH-1) and chicken cathelicidin-3 (cCATH-3) were selected based on their markedly different minimum inhibitory concentrations (MICs) against avian pathogenic Escherichia coli (APEC) strains, with MICs of cCATH-1 being 4- to 16-fold lower than those of cCATH-3. However, following intraperitoneal challenge with APEC strain E058 (4 × 10⁶ CFU/mouse), 80% of cCATH-3 treated mice survived while 20% of cCATH-1 treated mice survived. Flow cytometry (FC) revealed significantly lower peripheral blood Ly6C high monocytes but higher peritoneal Ly6C high macrophages in cCATH-3-treated mice at 4 and 8 h post-treatment. Peritoneal CCL3 secretion was significantly upregulated in cCATH-3 treated mice at 8 h post-treatment. Upon knockout of macrophages, the protection of cCATH-3 in mice against APEC E058 challenge was markedly abolished. In vitro, peritoneal macrophages treated with cCATH-3 showed stronger bacteriocidal activity than those treated with cCATH-1. Mouse CCL-3 (mCCL3)-specific antibody neutralize the effect of mCCL3 on macrophages chemotaxis in vitro. mCCL3-specific antibody administration resulted in 0% survival in cCATH-3-treated mice versus 60% survival in cCATH-3-treated mice administered with mock antibody, while cCATH-1-treated mice showed 0% survival administered with either mCCL3-specific antibody or mock antibody after APEC E058 strain challenge. In summary, cCATH-3 provides robust protection against APEC infection compared with cCATH-1 in mice, partially through CCL3-mediated macrophage chemotaxis and enhanced bacteriocidal activity in activated macrophages, revealing a novel mechanism of avian cathelicidins and supporting HDPs as alternatives to antibiotics for controlling avian colibacillosis.