REVIEW ISJ 10: 58-68, 2013 ISSN 1824-307X REVIEW Apolipophorins and insects immune response A Zdybicka-Barabas, M Cytryńska Department of Immunobiology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, 19 Akademicka St., 20-033 Lublin, Poland Accepted July 30, 2013 Abstract Insect lipoproteins, called lipophorins, are non-covalent assemblies of lipids and proteins serving as lipid transport vehicles. The protein moiety of lipophorin comprises two glycosylated apolipoproteins, apolipophorin I (apoLp-I) and apolipophorin II (apoLp-II), constantly present in a lipophorin particle, and an exchangeable protein, apolipophorin III (apoLp-III). ApoLp-III is an abundant protein occurring in hemolymph in lipid-free and lipid-bound state and playing an important role in lipid transport and insect innate immunity. In immune response apoLp-III serves as a pattern recognition molecule. It binds and detoxifies microbial cell wall components, i.e., lipopolysaccharide, lipoteichoic acid, and β-1,3-glucan. ApoLp-III activates expression of antimicrobial peptides and proteins, stimulates their antimicrobial activity, participates in regulation of the phenoloxidase system and in hemolymph clotting. In addition, the protein is involved in cellular immune response, influencing hemocyte adhesion, phagocytosis and nodule formation, and in gut immunity. Although apoLp-III is the best studied apolipophorin in insect immunity so far, a literature review suggests that all the three apolipoproteins, apoLp-I, apoLp-II and apoLp-III, function together in a coordinated defense against pathogens. Key Words: lipophorin; apolipophorin III; insect immunity; Galleria mellonella Introduction Insect lipoproteins, called lipophorins, are well- studied complexes of multifunctional molecules. These non-covalent assemblies of lipids and proteins serve as lipid transport vehicles. Lipophorins have a structure similar to mammalian lipoproteins. They possess a hydrophobic core composed of nonpolar lipid constituents, surrounded by a monolayer of amphiphilic phospholipids (PL) and apolipoproteins. The protein moiety of lipophorin comprises two glycosylated apolipoproteins, arising from a common precursor preproapolipophorin, i.e. apolipophorin I (apoLp-I, ca 240 kDa) and apolipophorin II (apoLp-II, ca 80kDa) which are present in a 1:1 molar ratio in lipophorin particles (Ryan et al., 1984; Weers et al., 1993; Blacklock and Ryan, 1994; Ryan and Van der Horst, 2000; Marinotti et al., 2006). The fat body, a functional analog of mammalian ___________________________________________________________________________ Corresponding author: Małgorzata Cytryńska Department of Immunobiology Institute of Biology and Biochemistry Faculty of Biology and Biotechnology Maria Curie-Skłodowska University 19 Akademicka St., 20-033 Lublin, Poland E-mail: cytryna@poczta.umcs.lublin.pl liver, is the site of apoLp-I, apoLp-II and lipid synthesis as well as lipid storage, and assembling of lipoprotein particles. Lipophorins are released as high or very high density lipoproteins into the hemolymph, depending on the insect species (Prasad et al., 1986; Venkatesh et al., 1987; De Capuro and De Bianchi, 1990; Weers et al., 1992; Van Heusden et al., 1998). One of the major roles of lipoprotein is lipid transport during larval development and long-distance flight of insects. Adipokinetic hormones (AKHs), which are stored in the secretory granules of neuroendocrine cells (corpus cardiaca), are released during flight activity and are involved in lipid mobilization (Beenakkers et al., 1985; Van der Horst, 2003). AKHs trigger conversion of the fat body triacylglycerol (TAG) stores into diacylglycerol (DAG) by a specific lipase. The insect lipids are then released as DAG and, after being assembled with apoLp-I and apoLp-II, form high-density lipophorins (HDLp). Two proteins, apoLp-I and apoLp-II, are constantly present in a lipophorin particle, whereas the third protein, apolipophorin III (apoLp-III), is an exchangeable molecule in these complexes. Association of DAG and apoLp-III with HDLp converts them to low- density lipophorins (LDLp), which serve as transport vehicles for lipids in hemolymph to a target tissue. 58 Fig. 1 Main functions of apolipophorin III in insects. (*) denotes processes in which lipophorin particles or apoLp- I/II are involved From 14 to 16 molecules of apoLp-III can be associated with the LDLp surface (Kawooya et al., 1984, 1986; Wells et al., 1987; Van der Horst et al., 1991). After release of a lipid load, apoLp-III dissociates from the complex and together with HDLp can be reused for another cycle of DAG transport (Weers et al., 1992; Blacklock and Ryan, 1994; Soulages et al., 1995; Ryan and Van der Horst, 2000; Niere et al., 2001). Among apolipophorins, especially apoLp-III is considered to be an important factor of insect immunity. The review summarizes available data on apoLp-III involvement in insect immune response (Fig. 1). Apolipophorin III and lipid interactions ApoLp-III was detected in different insect tissues, e.g., eggs, fat body, hemocytes, and hemolymph. This protein was found in hemolymph plasma of larvae, pupae, adults as well as in the molting fluid. It is a water-soluble and heat-stable protein of molecular mass 17-20 kDa depending on the insect species (Table 1). ApoLp-IIIs contain no cysteine, some of them are glycosylated in Orthoptera species, but other, e.g. in Lepidoptera species, lack this modification (Kawooya et al., 1984; Chino and Yazawa, 1986; Chung and Ourth, 2002). The studies on L. migratoria and G. mellonella have shown that the protein can occur as isoforms, differing in pI values (Chino and Yazawa, 1986; Van der Horst et al., 1991; Wiesner et al., 1997; Zdybicka-Barabas and Cytryńska, 2011). The apoLp-III molecule is formed by a bundle of five antiparallel amphipathic α-helices organized in an up-and-down topology, which are connected by short hinged loop regions (Breiter et al., 1991). This bundle motif is a stable arrangement of the protein, which allows it to exist in hemolymph. A majority of residues in the molecule interior are hydrophobic, while hydrophilic residues are exposed to the aqueous environment of hemolymph. Although the degree of amino acid sequence identity of apoLp- IIIs from evolutionally divergent species is low, the three-dimensional structure of these proteins in their lipid-free state shows a striking similarity in molecular architecture, which is connected with the physiological functions of apoLp-III. Physiologically, the protein occurs in a lipid-free or lipid-bound state that readily converts from one to the other depending on the metabolic setting. The low intrinsic stability of the helix bundle in the lipid-free state probably facilitates interaction with lipid surfaces. During the formation of the complex between apoLp-III and lipid, considerable conformational changes in the protein were observed, i.e., helix-helix interactions were replaced by helix-lipid interactions in the lipid-bound open conformation (Wientzek et al., 1994; Raussens et al., 1995). The lipid-bound state is the active form of 59 Table 1 Properties of apolipophorin III of selected insect species Molecular mass (a,b) (Da) Number of residues pI (a,b)/ isoforms Reference Lepidoptera Acherontia atropos 17247 b 20 kDaa 161 Surholt et al., 1988 Bombyx mori 18420 18378b 164 8.04 b Yamauchi et al., 2000 Diatraea grandiosella 17964b 165 6.8a Burks et al., 1992 Galleria mellonella 18075.5 a 18075b 163 6.38b 6.5a 5.9a 6.1a Weise et al., 1998 Zdybicka-Barabas and Cytryńska, 2011 Heliothis virescens 18 kDa a 17965.9a Chung and Ourth, 2002 Hyalophora cecropia 18 kDaa Telfer et al., 1991 Hyphantria cunea 18.3 kDa a 18344b 165 6.23 b Kim et al., 2004 Manduca sexta 18364 18380b 166 5.88 b Kawooya et al., 1984 Spodoptera exigua 16523b 149 6.25b Rizwan-Ul-Haq et al., 2011 Spodoptera litura 18.3 kDaa 166 Kim et al., 1998 Thitarodes pui 18606a 171 5.61b Sun et al., 2012 Orthoptera Acheta domesticus 17248 b 17.2 kDaa 161 Smith et al., 1994 Strobel et al., 1990 Locusta migratoria 20488a (Glc) 17583a (non-Glc) 17327b 162-164 5.35a /5.43a 5.10ba/5.11a 4.98a Van der Horst et al., 1984; 1991 Weers et al., 2000 Chino and Yazawa, 1986 Coleoptera Derobrachus geminatus 18039 b 18 kDaa 165 Smith et al., 1994 Diptera Anopheles gambiae 19247b 170 4.82b Gupta et al., 2010 a molecular mass and isoelectric point (pI) determined empirically; b theoretical molecular mass and pI; Glc – glycosylated; non-Glc – non-glycosylated the protein and occurs when apoLp-III associates with lipid-enriched lipophorins. It has been demonstrated that interaction of apoLp-III with model phospholipid vesicles, composed of dimyristoylphosphatidylcholine (DMPC), transforms them into discoidal particles surrounded by apoLp-III (Weers et al., 1999, 2000; Weers and Ryan, 2003; Vasques et al., 2009; Narayanaswami et al., 2010; Wan et al., 2011). The interaction of the protein with membrane lipid bilayers does not affect their permeability and occurs via polar and/or electrostatic forces at the bilayer surface without penetration of the hydrophobic core of the bilayer (Zhang et al., 1993; Sahoo et al., 1998). The packing defects in the phospholipid bilayer generate sites of apoLp-III binding, which was reported for various DMPC and sphingomyelin (SM) vesicles (Chiu et al., 2009). It is known that lipid binding involves the hydrophobic surface of the helix bundle. Upon lipid binding, apoLp-III molecule undergoes conformational changes and its hydrophobic interior is exposed to the lipid environment, which allows hydrophobic amino acid side-chains to gain direct access to the lipid surface (Wientzek et al., 1994; Garda et al., 2002; Sahoo et al., 2002). A plausible model of the interactions of apoLp-III with model phospholipid particles is based on reorientation in the bundle of α-helices, i.e., three 60 of them move away from the two others (Breiter et al., 1991; Ryan and Van der Horst, 2000; Van der Horst et al., 2001, 2002). It has been suggested that the lipid binding is initiated at one end of the helix bundle. Different models have been proposed for description of the initial binding steps. One model has been suggested for L. migratoria apoLp-III by Breiter et al. (1991), where directed helix-lipid contact is made possible by conformational opening of the bundle involving ‘hinged’ loops connecting helices 2 and 3 and helices 4 and 5. The resulting exposure of its interior permits formation of a stable interaction with hydrophobic patches on lipophorin particles which appear as a function of DAG enrichment. In turn, Kawooya et al. (1986) have proposed that M. sexta apoLp-III recognizes potential lipid surface binding sites via one of its end and then Breiter et al. (1991) have shown that loop A connecting helices 1 and 2 and loop C between helices 3 and 4 play this role. Furthermore, in the structure of apoLp-III of L. migratoria, M. sexta, and Thitarodes pui short additional helices (called 4’ and 3’) located at one end of the α-helix bundle are present. They adapt an orientation nearly perpendicular to the long axis of the bundle (Wang et al., 1997; Narayanaswami et al., 1999; Fan et al., 2003; Sun et al., 2012). Because they are exposed to the solvent, it has been suggested that these short helices play a critical role in initiating the lipid binding with apoLp-III. It is believed that repositioning of the first and last helices in the molecule is an essential step in the binding process of apoLp-III, facilitating further opening of the hydrophobic helix bundle interior and separation of all helices from each other, which allows the protein to spread out on the lipid surface (Narayanaswami et al., 1996; Garda et al., 2002; Sahoo et al., 2002). It has been demonstrated that apoLp-III of M. sexta, L. migratoria and G. mellonella were unable to bind lipid surfaces when helix 1 and helix 5 were tethered by a disulfide bond (Garda et al., 2002; Sahoo et al., 2002; Leon et al., 2006a). Based on spectroscopic analyses, Raussens et al. (1995) inferred that the helices of M. sexta apoLp-III in the lipid-bound state are oriented perpendicularly to fatty acyl chains. ApoLp-III shares similarities in the structure and in the mechanism of lipid binding with the N-terminal domain of human apolipoprotein E (apoE) and apolipoprotein A-I (apoA-I). The structure of the 22 kDa N-terminal domain in apoE3 in the lipid-free state comprises four amphipathic α-helices with buried hydrophobic residues and exposed hydrophilic residues (Aggerbeck et al., 1988; Wilson et al., 1991). This domain can alter its conformation in a manner similar to apoLp-III on the lipoprotein particle. Like the human apolipoproteins, apoLp-III forms discoidal complexes with phospholipid liposomes in which extended helices of the protein are wrapped around nanodiscs (Saito et al., 2004; Hatters et al., 2006; Davidson et al., 2007; Narayanaswami et al., 2010). Involvement of apolipophorin III in insects immunity The level of apoLp-III in hemolymph of immune- challenged insects, e.g., G. mellonella, Heliothis virescens, Plutella xylostella, undergoes changes, depending on the insect species and the pathogen/parasite, which indicates participation of apoLp-III in immune response against microbial infections (Chung and Ourth, 2002; Song et al., 2008; Zdybicka-Barabas and Cytryńska, 2011). It has been postulated that during an early step of insect immune response an interaction of apoLp-III with lipids occurs and that apoLp-III in the lipid- bound state is involved in insect immunity (Wiesner et al., 1997; Dettloff et al., 2001a,b). A relationship between the lipid transport and immune function of apoLp-III has been shown in crickets. It appeared that the two processes compete for the protein, as after flight, the crickets became less able to fight bacterial infection (Adamo et al., 2008). Recognition of non-self Proper recognition of invading pathogens by the immune system is a necessary prerequisite for activation and mounting of effective humoral as well as cellular immune response. It has been documented that apoLp-III binds microbial cell wall components, such as Gram-negative bacteria lipopolysaccharide (LPS), Gram-positive bacteria lipoteichoic acids (LTA), and fungal β-1,3-glucan (Dunphy and Halwani, 1997; Halwani et al., 2000; Pratt and Weers, 2004; Whitten et al., 2004; Leon et al., 2006a, b; Ma et al., 2006). Due to this property, apoLp-III is considered as a pathogen recognition receptor (PRR). In their work, Halwani et al. (2000) reported on interaction of G. mellonella apoLp-III with LTAs of Bacillus subtilis, Enterococcus hirae, and Streptococcus pyogenes. They also demonstrated that E. hirae LTA promoted binding of apoLp-III to E. hirae cells. In addition, binding of LTAs by apoLp-III prevented loss of plasmatocytes caused by B. subtilis LTAs in G. mellonella larvae. Our recent study has demonstrated binding of G. mellonella apoLp-III to different Gram-positive and Gram- negative bacteria. The results suggested that, in addition to LTAs, apoLp-III interacted with other cell surface components of Gram-positive bacteria, since it bound to some of the tested bacteria lacking LTAs in their cell walls, e.g., B. circulans (Zdybicka- Barabas and Cytryńska, 2011). Lipid A and the carbohydrate part of the LPS molecule are involved in interaction with apoLp-III. The binding causes conformational changes in the apoLp-III molecule, leading to rearranging and opening of the bundle of α-helices, similar to binding to lipid surfaces upon interaction with lipoprotein complexes (Leon et al., 2006a, b). Recently, a model of interaction between G. mellonella apoLp-III and E. coli LPS aggregates has been proposed. ApoLp-III disaggregated LPS by forming protein- LPS complexes. According to this model, the initial step is binding of apoLp-III to the LPS carbohydrate moiety through ionic interactions. The second step, leading to strong lipid A binding, is likely to be driven by hydrophobic interaction. It has been calculated that four apoLp-III molecules can interact with 24 molecules of E. coli LPS. Analysis of similar complexes formed between apoLp-III and K. pneumoniae LPS revealed that the complexes 61 contained three apoLp-III and nine LPS molecules (Oztug et al., 2012). Whitten et al. (2004) demonstrated binding of apoLp-III to β-1,3-glucan, a fungal cell wall component. Moreover, they showed that the survival rate of G. mellonella larvae injected with conidia of entomopathogenic fungus Metarhizium anisopliae coated by apoLp-III was higher in comparison with that one in larvae challenged by non-coated conidia, indicating a protective role of apoLp-III against fungal infection. Binding of G. mellonella apoLp-III to the cell surface of different yeasts and conidia of filamentous fungi has also been demonstrated (Zdybicka-Barabas et al., 2012). An analysis of in vitro treatment of Candida albicans, Zygosaccharomyces marxianus, and Fusarium oxysporum with apoLp-III revealed morphological and metabolic activity changes, suggesting a role of this protein not only in fungi recognition but also in antifungal activity of hemolymph. ApoLp-III as a signaling molecule and its role in antimicrobial activity induction As reported by Dettloff et al. (2001a), shortly after injection into G. mellonella hemocoel, biotinylated apoLp-III was detected in the immune- competent hemocytes, suggesting functioning of apoLp-III as a signaling molecule in insect hemolymph. In accordance with this finding, G. mellonella bacterial challenge led to formation of apoLp-III-lipid complexes, assembled into LDLp which were taken up by granulocytes (Dettloff et al., 2001b; Niere et al., 2001). It was reported that apoLp-III potentiated antimicrobial activity in insect hemolymph. An injection of apoLp-III into the hemocoel of G. mellonella larvae led to an increase in hemolymph lysozyme and anti-E. coli activity, similar to bacterial challenge (Wiesner et al., 1997; Halwani and Dunphy, 1999; Niere et al., 1999). The constitutive presence of apoLp-III in hemolymph and the fact that intrahemocoelic injection of apoLp-III (recombinant or purified from insects) resulted in a negligible increase in the soluble apoLp-III fraction in hemolymph, ruled out hormone- or cytokine- related activity of the protein. Evidence provided by Dettloff et al. (2001b) for LDLp formation after bacterial challenge and uptake of lipid-bound apoLp-III by the hemocytes, indicated that activation of the immune system by apoLp-III might be rather connected with conformational changes and an increase in the lipid-bound fraction of the protein in hemolymph. Intensification of hemolymph antimicrobial activity after apoLp-III injection could result from activation of antimicrobial gene expression, which was demonstrated in apoLp-III- challenged Hyphantria cunea. In this insect, apoLp- III injection induced the expression of lysozyme and cecropin-like peptides (hyphancins). In addition, apoLp-III was detected in H. cunea granulocytes which underwent degranulation and degradation upon E. coli immune challenge. The authors postulated a relationship between a local discharge of apoLp-III from the granulocytes in response to bacterial challenge and activation of immune response (Kim et al., 2004). The existence of immune signals upstream of cell-bound receptors has been postulated by Rahman et al. (2006). They found that lipophorin particles mediated recognition and inactivation of LPS and bacteria in immune-challenged flour moth Ephestia kuehniella larvae. Moreover, an association of pattern recognition receptors, lectins as well as regulatory proteins activating prophenoloxidase with sub-population of lipophorin particles has been demonstrated (Rahman et al., 2006). Detoxification of non-self components The ability of apoLp-III to bind microbial cell wall components, e.g., LPS, implies participation of the protein in detoxification processes. Dunphy and Halwani (1997) demonstrated that G. mellonella apoLp-III bound to LPS isolated from the outer membrane of insect pathogenic bacteria Xenorhabdus nematophilus, which reduced the LPS toxicity and prevented G. mellonella hemocyte damage. The role of lipophorins, in which apoLp-III is an exchangeable component, in LPS detoxification was suggested by Kato et al. (1994a, b) in their study on Bombyx mori. The results indicated that formation of the lipophorin-LPS complex in B. mori hemolymph, similar to the lipoprotein-LPS complex in mammalian serum, caused a striking decrease in LPS biological activity, reflected by significant reduction in cecropin gene inducibility. As demonstrated by Ma et al. (2006), antibodies against LPS-binding proteins, such as immulectin-2, cross-reacted with proteins associated with purified lipophorin particles formed in G. mellonella hemolymph in vitro upon LPS addition. The results also indicated that lipophorin particles responded to LPS by forming insoluble aggregates sequestering LPS into non-toxic complexes. ApoLp-III can also be considered as an LTA- neutralizing protein, since binding of G. mellonella apoLp-III to B. subtilis LTAs prevented loss of plasmatocytes caused by LTA, indicating protection of the insect against the toxin (Halwani et al., 2000). Antimicrobial activity of apoLp-III and synergistic action with defense peptides Our study has revealed that G. mellonella apoLp-III exhibits antibacterial activity against certain Gram-positive and Gram-negative bacteria. Among the most susceptible bacteria were Salmonella typhimurium, K. pneumoniae, B. circulans, and Listeria monocytogenes (Zdybicka- Barabas and Cytryńska, 2011). Interestingly, apoLp-III also inhibited significantly growth of Legionella dumoffii cultured in a medium supplemented with choline (Palusinska-Szysz et al., 2012). AFM imaging and analysis of apoLp-III- treated bacteria revealed considerable alterations of the structure and nanomechanical properties of the cell surface, e.g. roughness, elasticity, and adhesion (Fig. 2) (Zdybicka-Barabas et al., 2011). The results underline the important role of apoLp- III in insect antibacterial defense, similarly to the role demonstrated for mammalian apoE in immune 62 Fig. 2 AFM analysis of K. pneumoniae cell surface alterations after treatment with G. mellonella apoLp-III. Three dimensional (a), amplitude (b), and topography (c) images are presented. Section profiles corresponding to lines in (c) are demonstrated in (d). 63 response against K. pneumoniae and L. monocytogenes (Roselaar and Daugherty, 1998; Bont et al., 1999). In G. mellonella, it was demonstrated that apoLp-III acted synergistically with other defense proteins and peptides against bacteria. Synergistic action of apoLp-III and lysozyme against M. lysodeikticus was suggested by Halwani and Dunphy (1999) on the basis of experiments on apoLp-III and EWL. In addition, they demonstrated an increase in hydrophobicity and the negative charge of the bacterial cells treated with apoLp-III, which could, to some extent, explain why apoLp-III enhances the antibacterial activity of cationic defense proteins and peptides. Recently, we have presented evidence for increasing G. mellonella lysozyme muramidase activity in the presence of apoLp-III, leading to an increase in lysozyme perforating activity of the E. coli cell membrane. Our research indicated that three defense factors present constitutively in G. mellonella hemolymph, namely apoLp-III, lysozyme, and anionic peptide 2, act in synergy against bacteria. ApoLp-III increases the enzymatic (muramidase) activity of lysozyme, whereas anionic peptide 2 seems to stimulate the non-enzymatic lysozyme activity (Zdybicka-Barabas et al., 2012, 2013). Moreover, an increase in cecropin A anti-E. coli activity in the presence of apoLp-III has been demonstrated (Park et al., 2005). In addition to enhancing antibacterial activity of other defense proteins and peptides, involvement of apoLp-III in regulation of phenoloxidase activity in G. mellonella has been reported; however a possible mechanism of this phenomenon has not been explained yet (Halwani et al., 2000; Park et al., 2005). Recently, Gupta et al. (2010) have reported on apoLp-III participation in midgut immune defense of Anopheles gambiae against Plasmodium berghei. In A. gambiae G3 females, invasion of P. berghei ookinetes triggered a strong transcriptional activation of apoLp-III in the midgut epithelial cells. Expression of apoLp-III in these cells stimulated antiplasmodial response, while silencing of apoLp-III by systemic injection of dsRNA greatly increased Plasmodium infection. Contribution of apoLp-I and apoLp-II, in addition to apoLp-III, in insect defense mechanisms against pathogens has been recently suggested (Hanada et al., 2011). It has been found that ApoLp, consisting of apoLp-I and apoLp-II, of the B. mori silkworms’ hemolymph is involved in resistance against Staphylococcus aureus infection by suppressing the expression of virulence genes encoding α- and β- hemolysin. Moreover, ApoLp also decreased expression of saeRS and RNAIII, important for activation of these hemolysin genes. It is possible that apoLp-I and apoLp-II, together with apoLp-III, function in coordinated antimicrobial defense in insects. Role of apoLp-III in cellular immune response Analysis of the properties of G. mellonella hemocytes after treatment with apoLp-III in vitro and after injection of apoLp-III into larval hemocoel revealed impaired adhesion of plasmatocytes and subpopulation of granulocytes to glass slides (Zakarian et al., 2002; Whitten et al., 2004). Since the ability to adhere to and spread on non-self surfaces is essential for hemocytes involved in cellular immune response, the finding suggested a role of apoLp-III in this arm of insect immunity. In the same study, delayed removal of apoLp-III-coated B. subtilis cells from the hemolymph was reported. Given the reduced hemocyte adhesion upon apoLp- III treatment, the authors postulated that apoLp-III may down-regulate nodule formation and/or phagocytosis (Zakarian et al., 2002). On the other hand, binding of apoLp-III to yeast cells (S. cerevisiae) enhanced the phagocytic activity of G. mellonella hemocytes in vitro, suggesting importance of apoLp-III opsonizing activity for effective clearance of the invaders (Wiesner et al., 1997). Similarly, the findings described by Whitten et al. (2004) presenting more effective in vivo nodule formation in larvae injected with apoLp-III could point towards a stimulating role of apoLp-III in cellular response. In support of this idea are the results presented by Son and Kim (2011) on the role of apoLp-III in activation of cellular response in diamondback moth, P. xylostella. Knockdown of apoLp-III expression by RNA interference caused a significant decrease in the apoLp-III level and resulted in considerable suppression of hemocyte nodule formation in response to bacterial challenge. Injection of recombinant apoLp-III to P. xylostella larvae parasitized by an endoparasitic wasp Cotesia plutellae restored the hemocyte activity. In addition, apoLp-III reduced pathogenicity of entomopathogenic bacteria X. nematophila toward P. xylostella larvae (Son and Kim, 2011). Although the exact role of apoLp-III in cellular immune response is difficult to define clearly on the basis of available data, it seems that the effect of the protein activity depends on the pathogen. Involvement in clot formation Clot formation can be considered as an integral part of insect immune response, because in addition to sealing wounds and limiting loss of body fluids, a clot entraps microbes at the wound site, thereby preventing invading the hemocoel. Moreover, upon activation of the PO system, the entrapped pathogens can be more easily killed and eliminated. Lipophorins have been identified as a common clotting factor in several insect species, e.g. G. mellonella, Tenebrio molitor, L. migratoria, Periplaneta americana, and Leucophaea maderae (Duvic and Brehélin, 1998; Altincicek et al., 2008; Dushay, 2009). The presence of apoLp-I and apoLp-I/II in the Anopheles gambiae and Drosophila melanogaster clots, respectively, has also been reported (Scherfer et al., 2004; Agianian et al., 2007). Proteomic analysis revealed that apoLp-III, together with other apolipoproteins, was a component of G. mellonella net-like coagulation structures containing endogenous extracellular nucleic acids. Moreover, apoLp-III was detected among specific RNA-binding proteins, suggesting its role in extracellular RNA-mediated immune response (Altincicek et al., 2008). 64 Apolipophorin III and entomopathogens One of the strategies developed by entomopathogenic organisms to cope with the host immune system is decreasing of the apoLp-III level. Reduction of the protein level is achieved by suppression of apoLp-III expression, which was described in P. xylostella parasitized by the entomopathogenic wasp C. plutellae (Son and Kim, 2011). Another way involves proteolytic degradation of apoLp-III by extracellular proteinases produced by entomopathogenic bacteria during infection, e.g. Pseudomonas aeruginosa elastase B and protease IV in infected G. mellonella larvae (Andrejko et al., 2005, 2008, 2013; Andrejko and Mizerska-Dudka, 2012). A very interesting strategy developed by entomopathogenic nematodes Steinernema feltiae infecting G. mellonella larvae was reported by Brivio et al. (2005, 2010). Hemolymph of the infected larvae was depleted of humoral immune factors which were attracted by and adsorbed to specific nematode surface molecules. Among proteins removed in this way from the insect hemolymph, a lipopolysaccharide binding protein (LBP), a peptidoglycan recognition protein LB (PGRP-LB), gloverin-like peptide and apoLp-III were identified. Removal of the immune factors, including apoLp-III, from the insect hemolymph by S. feltiae seems to protect nematode symbiotic bacteria X. nematophila, which kill the insect host and establish suitable conditions for reproduction of the nematodes (Brivio et al., 2005, 2010). Summary Apolipophorin III is a multifunctional insect protein involved in lipid transport and immune response. In addition, its role during programmed cell death has been described in M. sexta skeletal muscles and neurons (Sun et al., 1995). In insect immune response apoLp-III serves as a pattern recognition molecule. It binds and detoxifies microbial cell wall components, i.e. LPS, LTA, and β-1,3-glucan. ApoLp-III activates expression of antimicrobial peptides and proteins, stimulates their antimicrobial activity, and participates in regulation of phenoloxidase activity in insect hemolymph. In addition, the protein is involved in cellular immune response, influencing hemocyte adhesion, phagocytosis and nodule formation, and in gut immunity. Reduction of the apoLp-III level by entomopathogens through e.g., suppression of apoLp-III expression and/or degradation of the protein by entomopathogen proteases seems to be a common strategy to avoid host immune response and indicates that apoLp-III is an important component of insect immunity. Although apoLp-III is the best studied apolipophorin in insect immunity so far, a literature review suggests that all the three apolipoproteins, apoLp-I, apoLp-II and apoLp-III, function together in a coordinated defense against pathogens. Acknowledgements The authors would like to thank Prof. T Jakubowicz (Department of Immunobiology, Maria Curie-Skłodowska University, Lublin, Poland) for critical reading of and comments to the manuscript. References Adamo SA, Roberts JL, Easy RH, Ross NW. 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