John F. Andersen

The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1)

Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition mouse dendritic cells (DCs) to promote Th2 lymphocyte differentiation. Using an in vitro bystander polarization assay as the readout, we purified and identified the major Th2-inducing component from soluble egg extract (SEA) as the secreted T2 ribonuclease, omega-1. The Th2-promoting activity of omega-1 was found to be sensitive to ribonuclease inhibition and did not require MyD88/TRIF signaling in DCs. In common with unfractioned SEA, the purified native protein suppresses lipopolysaccharide-induced DC activation, but unlike SEA, it fails to trigger interleukin 4 production from basophils. Importantly, omega-1–exposed DCs displayed pronounced cytoskeletal changes and exhibited decreased antigen-dependent conjugate formation with CD4+ T cells. Based on this evidence, we hypothesize that S. mansoni omega-1 acts by limiting the interaction of DCs with CD4+ T lymphocytes, thereby lowering the strength of the activation signal delivered.

Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells

Toxoplasma gondii releases factors that potently stimulate production of interleukin-12 (IL-12) from dendritic cells (DCs). Purification of this activity showed that cyclophilin-18 (C-18) was its principal component, and antibodies generated against recombinant C-18 inhibited tachyzoite extract–induced synthesis of IL-12. Recombinant C-18 showed high affinity for and triggered cell signaling through CCR5, a chemokine receptor important in parasite-induced IL-12 production by DCs. These findings suggest that the unusual potency of T. gondii in inducing IL-12 from DCs results from its synthesis of a unique chemokine mimic that signals through CCR5. The ability to generate this strong protective response may benefit parasite transmission by preventing the protozoan from overwhelming its intermediate hosts.

Function and Evolution of a Mosquito Salivary Protein Family

Saliva of blood-sucking arthropods contains a complex and diverse mixture of antihemostatic, antiinflammatory, and immunomodulatory compounds. The D7 salivary family of proteins is abundantly expressed in blood-feeding Diptera and is distantly related to the odorant-binding protein superfamily. In mosquitoes, two subfamilies exist, the long and short D7 proteins. Ticks and kissing bugs evolved salivary lipocalins that act as efficient scavengers of biogenic amines, and a similar function was postulated for the D7 proteins. Accordingly, we expressed the five members of the small D7 family of the African malaria vector Anopheles gambiae and a D7 long form from Aedes aegypti and showed by isothermal microcalorimetry, a modified and very sensitive non-equilibrium chromatography/spectrum distortion method, and by smooth muscle bioassay that four of these five short D7 proteins and the D7 long form bind serotonin with high affinity, as well as histamine and norepinephrine. The nonbinding D7 protein is poorly expressed in the salivary glands and appears to be on the path to becoming a pseudogene. Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties. It appears that counteracting biogenic amines is of strong adaptive value in the convergent evolution of arthropods to hematophagy. This adaptation has been solved independently in ticks, bugs, and mosquitoes by co-option of either member of the lipocalin or, as shown here, by the odorant-binding protein families.

Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis

Here we report the ability of the tick Ixodes scapularis, the main vector of Lyme disease in the United States, to actively and specifically affect the host proteolytic activity in the sites of infestation through the release of a cystatin constituent of its saliva. The cystatin presence in the saliva was verified both biochemically and immunologically. We named the protein sialostatin L because of its inhibitory action against cathepsin L. We also show that the proteases it targets, although limited in number, have a prominent role in the proteolytic cascades that take place in the extracellular and intracellular environment. As a result, sialostatin L displays an antiinflammatory role and inhibits proliferation of cytotoxic T lymphocytes. Beyond unraveling another component accounting for the properties of tick saliva, contributing to feeding success and pathogen transmission, we describe a novel tool for studying the role of papain-like proteases in diverse biologic phenomena and a protein with numerous potential pharmaceutical applications.

Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods.

Recent gene sequence and crystal structure determinations of salivary proteins from several blood-sucking arthropods have revealed an unusual evolutionary relationship: many such proteins derive their functions from lipocalin protein folds. Many blood-sucking arthropods have independently evolved the ability to overcome a host organisms means of preventing blood loss (called hemostasis). Most blood feeders have proteins that induce vasodilation, inhibit blood coagulation, and reduce inflammation, but do so by distinctly different mechanisms. Despite this diversity, in many cases the antihemostatic activities in such organisms reside in proteins with lipocalin folds. Thirteen such lipocalins are described in this review, with a particular focus on the heme-containing nitrophorins from Rhodnius prolixus, which transport nitric oxide, sequester histamine, and disrupt blood coagulation. Also described are the antiplatelet compounds RPAI, moubatin, and pallidipin from R. prolixus, Ornithodoros moubata, and Triatoma pallidipennis; the antithrombin protein triabin from T. pallidipennis; and the tick histamine binding proteins from Rhipicephalus appendiculatus.

Prostaglandin E2 Is a Major Inhibitor of Dendritic Cell Maturation and Function in Ixodes scapularis Saliva

Tick saliva is thought to contain a number of molecules that prevent host immune and inflammatory responses. In this study, the effects of Ixodes scapularis saliva on cytokine production by bone marrow-derived dendritic cells (DCs) from C57BL/6 mice stimulated by TLR-2, TLR-4, and TLR-9 ligands were studied. Saliva at remarkably diluted concentrations (<1/2000) promotes a dose-dependent inhibition of IL-12 and TNF-α production induced by all TLR ligands used. Using a combination of fractionation techniques (microcon filtration, molecular sieving, and reversed-phase chromatography), we unambiguously identified PGE2 as the salivary inhibitor of IL-12 and TNF-α production by DCs. Moreover, we have found that I. scapularis saliva (dilution 1/200; ∼10 nM PGE2) marginally inhibited LPS-induced CD40, but not CD80, CD86, or MHC class II expression. In addition, saliva significantly suppressed the ability of DCs to stimulate Ag-specific CD4+ T cell proliferation and IL-2 production. Notably, the effect of saliva on DC maturation and function was reproduced by comparable concentrations of standard PGE2. These findings indicate that PGE2 accounts for most inhibition of DC function observed with saliva in vitro. The role of salivary PGE2 in vector-host interaction and host immune modulation and inflammation in vivo is also discussed. This study is the first to identify molecularly a DC inhibitor from blood-sucking arthropods.

HDP—A Novel Heme Detoxification Protein from the Malaria Parasite

When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous “Outbound–Inbound” trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target.

Human CD300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells.

CD300a is an immunoreceptor tyrosine-based inhibitory motif (ITIM) containing molecule that belongs to the CD300 family of paired activating/inhibitory receptors. It has been shown that its ligation inhibits activation signals on cells of both myeloid and lymphoid lineages. The ligands for CD300a have not been identified. Here, we show that a CD300a-Ig fusion protein specifically binds to apoptotic cells that are evolutionary apart, such as human and insect cells, suggesting that the ligand has to be conserved. Using surface plasmon resonance, ultracentrifugation, ELISA, and reporter cell assays, we identified phosphatidylethanolamine (PE) and phosphatidylserine (PS), 2 phospholipids that translocate to the outer leaflet of the plasma membrane of dead cells, as the ligands for CD300a. Mutational and structural modeling studies identified residues that are involved in the binding of CD300a to PE and PS and that form a cavity where the hydrophilic heads of PE and PS, can penetrate. CD300a down-regulates the uptake of apoptotic cells by macrophages and its ectopic expression in CD300a-negative cell lines also decreased the engulfment of dead cells. Collectively, our results indicate that PE and PS are ligands for CD300a, and that this interaction plays an important role in regulating the removal of dead cells.

Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial

The nitrophorins comprise an unusual family of proteins that use ferric (Fe(III)) heme to transport highly reactive nitric oxide (NO) from the salivary gland of a blood sucking bug to the victim, resulting in vasodilation and reduced blood coagulation. We have determined structures of nitrophorin 4 in complexes with H2O, cyanide and nitric oxide. These structures reveal a remarkable feature: the nitrophorins have a broadly open distal pocket in the absence of NO, but upon NO binding, three or more water molecules are expelled and two loops fold into the distal pocket, resulting in the packing of hydrophobic groups around the NO molecule and increased distortion of the heme. In this way, the protein apparently forms a ‘hydrophobic trap’ for the NO molecule. The structures are very accurate, ranging between 1.6 and 1.4 Å resolutions.

Inhibition of hemostasis by a high-affinity biogenic amine-binding protein from the saliva of a blood-feeding insect

The saliva of the blood-feeding insectRhodnius prolixus contains numerous pharmacologically active substances. Included among these are a number of lipocalin proteins that bind various ligands important in hemostasis and inflammation. One such protein is a biogenic amine-binding protein (ABP) that binds serotonin, epinephrine, and norepinephrine. Based on amino acid alignments, it is most similar to the nitrophorin group of lipocalins found in the same insect species. Physiologically, this protein appears to act as both a vasodilator and platelet aggregation inhibitor. This protein inhibits smooth muscle contraction of the rat uterus in response to serotonin and of the rabbit aorta in response to norepinephrine. Platelet aggregation induced by a combination of low concentrations of ADP and either serotonin or epinephrine is inhibited because of the binding of serotonin and epinephrine. Potentiation of aggregation induced by low concentrations of collagen along with serotonin or epinephrine is also inhibited. Dissociation constants for biogenic amines were measured using isothermal titration calorimetry and the Hummel-Dreyer method of equilibrium gel filtration. In this manner, K d values of 102, 24, and 345 nm were found for serotonin, norepinephrine, and epinephrine, respectively. Molecular modeling of ABP suggests that ligand binding is mediated by interaction with the side chains of aromatic amino acids and charged residues that line the binding pocket.