Mannose-Binding Lectin Possesses Agglutination Activity and Promotes Opsonophagocytosis of Macrophages with Calreticulin Interaction in an Early Vertebrate

Abstract

The innate immune system is an ancient defense system in the process of biological evolution, which can quickly and efficiently resist pathogen infection. In mammals, mannose-binding lectin (MBL) is a key molecule in the innate immune and plays an essential role in the first line of host defense against pathogenic bacteria. However, the evolutionary origins and ancient roles of immune defense of MBL and its mechanism in clearance of microbial pathogens are still unclear, especially in early vertebrates. In this study, Oreochromis niloticus MBL (OnMBL) was successfully isolated and purified from the serum of Nile tilapia (O. niloticus). The OnMBL was able to bind and agglutinate with two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila. Interestingly, the OnMBL was able to significantly inhibit the proliferation of pathogenic bacteria and reduce the inflammatory response. Upon bacterial challenge, the downregulation of OnMBL expression by RNA interference could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by reinjection of the OnMBL, which restored the healthy status of the knockdown tilapia. Moreover, a mechanistic analysis revealed that the OnMBL could clear pathogenic bacteria by collaborating with cell-surface calreticulin to facilitate phagocytosis in a complement activation-independent manner. To our knowledge, these results provide the first evidence on the antibacterial response mechanism of MBL performing evolutionary conserved function to promote opsonophagocytosis of macrophages in early vertebrates and reveals new insights into the understanding of the evolutionary origins and ancient roles basis of the C-type lectins in the innate immune defense.

Chitosan/Calcium-Coated Ginsenoside Rb1 Phosphate Flower-like Microparticles as an Adjuvant to Enhance Immune Responses

“…In the process of the immune response, dendritic cells play an important role in the interaction of surface MHC molecules with T and B lymphocytes. Cytokine secretion exerts important functions in initiating and regulating immune responses by immune cells, especially macrophages [ 32 , 33 ]. Taken together, GRb1/IL-4@CS/CaP could significantly improve the humoral and cellular immune response in chicken organisms.…”

Functional Characterization of Serum Amyloid P Component (SAP) in Host Defense against Bacterial Infection in a Primary Vertebrate

“…The fish were immunized with 100 μL (1 × 10 6 CFU/fish, 1 × 10 7 CFU/mL concentration) live bacteria liquid such as S. agalactiae (ZQ1901) or A. hydrophila (BYK00810) dissolved in PBS (10 mM phosphate, 150 mM NaCl, pH 7.4), and grouped in stimulation groups, respectively [ 35 ]. As for the control group, PBS of equal volume was immunized [ 25 , 36 , 37 , 38 ].…”

“…After a successful acquisition of total RNA, the template was synthesized with the HiScript ® II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Similarly, the templates of every sample collected before were synthesized using the same protocol and then they were stored at −80 °C for further analysis [ 36 ].…”

“…The constitutive OnSAP expressions including healthy and bacterial injection situations were determined by qRT-PCR whose total volume was 20 μL containing 10 μL 2 × TaKaRa Ex Taq™SYBR premix, 3 μL of diluted cDNA (100 ng/μL), 2 μL of each primer shown in Supplementary Table S1 (2 μM), 2.6 μL DEPC treated water (Vazyme, Nanjing, China), and 0.4 μL Rox Reference Dye II (TaKaRa, Kyoto, Japan). β -actin was employed as an internal control to normalize the relative OnSAP expression level, whose fold changes was calculated using the 2 −ΔΔCt method [ 33 , 34 , 36 , 39 ]. The qRT-PCR program follows the description of the published works [ 40 ].…”

“…After adding 1 μL of trypan blue (0.4%) 10,000 individual cells of each sample were analyzed in flow cytometer FACS Aria III (BD Biosciences, Franklin Lakes, NJ, USA). Defined as the percentage of the macrophages with one or more engulfed bacteria within the total cell population, the phagocytosis rate was analyzed by Flowjo V10 software [ 34 , 36 ].…”