Attachment and phagocytosis by salmon macrophages of agarose beads coated with human C3b and C3bi (2023)

Complement plays an important role in the innate immune system, and it comprises about 35 individual proteins. In mammals, complement is activated via three different pathways, the classical pathway, the alternative pathway, and the lectin pathway. All three activation pathways produce C3-convertase in different forms. C3-convertase cleaves C3 to C3a and C3b and initiates a cascade of cleavage and activation, eventually resulting in the formation of the membrane attack complex. Complement activation results in the generation of activated fragments that are involved in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production. Although the complement system has been studied extensively in mammals, complement is less well understood in teleosts. This review summarizes the current knowledge of the teleost complement components involved in phagocytosis, chemotaxis, and cell lysis. We report the characterized complement components in various teleost species. In addition, we provide a comprehensive compilation of complement regulators, and this information is used to analyze the role of complement regulators in pathogen infection. The influence of complement receptors on the immune responses of teleosts is reviewed. Finally, we propose directions for future study of the molecular evolution, structure, and function of complement components in teleosts. This review provides new insights into the complement system of recognition and defense, and such knowledge is essential for the development of new immune strategies in aquaculture.

Recently, our laboratory had produced five families of transgenic rainbow trout harboring cecropin P1 transgene, and via repeated challenge studies these fish exhibited a significant elevation of resistance to infection by microbial pathogens. By cDNA microarray and mRNA deep sequencing (mRNA-seq) analyses on two of the five families of cecropin P1 transgenic fish, differentially expressed genes (DEGs) relevant to the innate and adaptive immune pathways in three different immune-related tissues, (i.e. spleen, kidney and liver) were profiled. These results supported our hypothesis that in addition to its direct microbicidal activity, the transgene product of cecropin P1 induces immunomodulatory activity in the transgenic host. Here, we have adapted the technique of quantitative reverse transcription real time PCR (RT-qPCR) array to analyze the expression of genes relevant to the innate and adaptive immune pathways in the rest three families. A RT-qPCR array was constructed with oligonucleotide primers of fifty-two innate/adaptive immune relevant DEGs shown to be the most perturbed by cecropin P1 transgene product in previous studies. Messenger RNA isolated from the spleen, kidney and liver of transgenic fish and non-transgenic fish control were studied on this array. Results of RT-qPCR array revealed that statistically significant perturbations of gene expression were detected in pathways of cytokine/chemokine signaling, Toll-like receptor signaling, complement cascade, antigen processing/presentation, lysosomal phagocytosis and leukocyte trans-endothelial migration in the transgenic spleen; extracellular matrix (ECM) organization and leukocyte trans-endothelial migration pathways in the transgenic kidney; lysosomal activity pathway in the transgenic liver. Furthermore, genes related to the pathways of the peroxisome proliferator-activated receptors (PPAR) signaling, lipid metabolism process and arachidonic acid metabolism were also impacted in the transgenic liver. Findings of the current study are in good agreement with those discoveries in previous two transgenic families by cDNA microarray and mRNA-seq analyses.

Fungal infections are a major cause of animal and plant morbidity and mortality worldwide. Effective biological therapeutics could complement current antifungal drugs, but understanding of their invivo mechanisms has been hampered by technical barriers to intravital imaging of host-pathogen interactions. Here we characterize the fungal infection of zebrafish as a model to understand the mechanism-of-action for biological antifungal therapeutics through intravital imaging of these transparent animals. We find that non-specific human IgG enhances phagocytosis by zebrafish phagocytes invivo. Polyclonal anti-Candida antibodies enhance containment of fungi invivo and promote survival. Analysis suggests that early phagocytic containment is a strong prognostic indicator for overall survival. Although polyclonal anti-Candida antibodies protect against disease, this is not necessarily the case for individual monoclonal anti-Candida antibodies. Thus, the zebrafish appears to provide a useful model host for testing if a biological therapeutic promotes phagocytosis invivo and enhances protection against candidemia.

Immune complexes activate the classical pathway of complement resulting in the covalent deposition of fragments of the third (C3b) and fourth (C4b) components of complement, thus opsonizing the complexes for uptake by CD35 found on human erythrocytes. The complexes are then transported to and cleared from the circulation by the reticuloendothelial system. It has been shown that rainbow trout can remove immune complexes from the circulation in a complement-dependent manner similar to that found in the human. However, the cell or cell types involved have not been identified. The purpose of this study was to investigate whether a complement-dependent immune adherence receptor is expressed on erythrocytes from the rainbow trout (Oncorhynchus mykiss) and the channel catfish (Ictalarus punctatus). Coating fluorescent microparticles with BSA, and then binding them to anti-BSA created an artificial immune complex that was incubated with normal fish serum, normal human serum or EDTA-treated serum. The complement-coated immune complexes were then incubated with either fish or human erythrocytes and analyzed for binding by flow cytometry and further visualized by fluorescence microscopy. Our results indicate that erythrocytes from rainbow trout are capable of binding immune complexes when pretreated with serum from either the trout or human, but not when pretreated with serum containing EDTA. By contrast, erythrocytes from the channel catfish did not bind immune complexes pretreated with autologous or human serum. These data suggest that differences exist in receptor distribution between two closely related species of fish, and a potentially homologous relationship in receptor expression, and possibility function, exist between two highly divergent species.

A terminal degradation product (C3d) of mammalian complement component C3 plays an important role in modulation of the adaptive immune response through the interaction with complement receptor type 2 (CR2) on B cells. The present study is aimed at determining whether this is a functional bridge between the innate and adaptive immune systems in bony fish. The fragmentation of the complement component C3 in carp (Cyprinus carpio) serum, activated with zymosan, was analysed to ascertain if carp C3 also generates a mammalian C3d-like fragment under physiological conditions. A 35 kDa peptide reactive to the anti-carp C3 α-chain was detected on the zymosan particles and in the activated serum. Its N-terminal amino acid sequence identified it as carp C3d derived from the C3-H1 isoform. Another C3 isoform, C3-S, of carp was found to yield a C3d fragment at lower efficiency than C3-H1. Recombinant C3d fragments derived from C3-H1 and C3-S were produced in Escherichia coli as fusion proteins with glutathione-S-transferase (GST), and used for ligands to examine the presence of a possible CR2-like C3d receptor on carp lymphocytes. An enzyme-linked immunoadsorbent assay system, using the recombinant C3d proteins and anti-GST on a microplate to which was attached carp peripheral lymphocytes, detected a significant binding of carp C3d to the lymphocyte. The degree of binding of C3-H1-derived C3d was higher than that of C3-S-derived C3d. In addition, the binding of both ligands was inhibited by anti-C3 α-chain, but not by EDTA or EGTA, indicating that the putative C3d receptor does not require divalent cation. These properties agree well with those reported for mammalian CR2.

We have developed a method for isolating goldfish microglia. Cells were identified as microglia immunohistochemically with NN-2, a monoclonal antibody (MAb) raised against teleost retinal microglial cells, and by their phagocytic abilities. Morphological characterization of the cells identified round, phase-bright cells as well as flattened macrophage-like cells. Ramified cells were also seen but they were rare. Fusion of macrophage-like cells occurred in high density cultures and resulted in the formation of giant cells that disintegrated a few days later. Immunohistochemical studies demonstrated that virtually all of the cells in our cultures were NN-2+ and did not label with either antiGFAP (an astrocyte marker) or MAb 6D2 (an oligodendrocyte marker). Cells identified as microglia were intensely phagocytic and ingested latex microspheres, DiIAcLDL and goldfish myelin in vitro. In addition, we labelled microglial cells in vivo with intracranial injections of fluorescent dextran and found that microglia isolated from these animals contained the dextran and phagocytosed microspheres. We also studied the effect of myelin on microsphere uptake and compared the effect of myelin and opsonized myelin on the phagocytic activity of the cells. Our results showed a clear increase in the phagocytic activity of microglia when incubated with myelin, with an enhanced effect of opsonized myelin.

Experimental Cell Research, Volume 319, Issue 18, 2013, pp. 2759-2769

Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates.

Otolaryngologic Clinics of North America, Volume 54, Issue 2, 2021, pp. 247-257

Vaccine, Volume 35, Issue 13, 2017, pp. 1712-1720

Trends in Parasitology, Volume 30, Issue 9, 2014, pp. 436-444

The past 10 years have generated new insights into the complex interaction between CD36 (cluster of differentiation 36) and malaria. These range from the crystallization of the CD36 homolog, LIMPII (lysosomal integral membrane protein II), permitting modeling of CD36 and its binding to diverse ligands, to cell biology-based studies of CD36 and large population genetic studies assessing the association of CD36 polymorphisms and malarial disease severity. Collectively these lines of evidence indicate that a receptor other than CD36 is associated with severity. CD36 plays an important role in innate immunity and in the phagocytic uptake of multiple pathogens including malaria. CD36 polymorphisms lack association with severity, and isolates that cause severe disease primarily bind to endothelial protein C receptor (EPCR) rather than to CD36.

Microbes and Infection, Volume 16, Issue 12, 2014, pp. 1002-1012

Recent years have seen a great advance in knowledge of how a host senses infection. Nucleic acids, as a common denominator to all pathogens, are at the centre of several of the sensing pathways, especially those involved with the recognition of viruses. In this review we discuss the current knowledge on how intracellular DNA is sensed by the mammalian host.

Experimental Parasitology, Volume 197, 2019, pp. 57-64

Leishmaniasis is a vector borne parasitic disease affecting millions of people worldwide and is spreading into further areas because of global warming. The development of new active substances against these single-cell eukaryotic parasites is of great importance. Leishmania tarentolae promastigotes (LtP) are non-pathogenic for mammals and serve as model organisms for pathogenic Leishmania in basic research. However, it is important to refine methods to study the process of the infection of mammalian macrophages by LtP and pathogenic Leishmania. Important stages of the infection are phagocytosis by macrophages and multiplication of Leishmania amastigotes in the phagolysosome of macrophages. In this study, advanced methods using electron spin resonance (ESR) spectroscopy and genetically manipulated LtP were used to monitor the infection of adherent J774 macrophages with LtP. An ESR method was established to detect the formation of superoxide radicals directly in adherent J774 cells and to investigate the effect of LtP on this activity. J774 cells responded with a burst of superoxide radicals in the presence of phorbol myristate acetate as positive control. In contrast, challenging J774 cells with LtP resulted in a much lower burst of superoxide radicals. To facilitate LtP detection in the phagolysosome of J774 macrophages, LtP expressing enhanced green fluorescent protein (EGFP-LtP) were constructed. After different infection times with EGFP-LtP, the J774 cells were visualized by phase contrast microscopy and the cell number was determined. The intramacrophage Leishmania tarentolae amastigotes (LtA) expressing EGFP were detected by fluorescence microscopy and then counted with ImageJ. These experiments showed that LtP are taken up by J774 cells and form intraphagolysosomal amastigotes. LtA under our conditions multiplied intracellularly and were able to persist about 48 h in J774 cells. These experiments showed that ESR spectroscopy of attached macrophages and the use of the EGFP-LtP are suitable methods to study the initial phase of Leishmania infection in vitro.