Patrick C. Hanington

Comparison of select innate immune mechanisms of fish and mammals.

Abstract:  The study of innate immunity has become increasingly popular since the discovery of homologs of many of the innate immune system components and pathways in lower organisms including invertebrates. As fish occupy a key position in the evolution of the innate and adaptive immune responses, there has been a great deal of interest regarding similarities and differences between their defense mechanisms and those of higher vertebrates. This review focuses on describing select mechanisms of the innate immune responses of fish and the implications for evolution of immunity in higher vertebrates.

Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection

Invertebrates lack adaptive immune systems homologous to those of vertebrates, yet it is becoming increasingly clear that they can produce diversified antigen recognition molecules. We have previously noted that the snail Biomphalaria glabrata produces a secreted lectin, fibrinogen-related protein 3 (FREP3), unusual among invertebrate defense molecules because it is somatically diversified by gene conversion and point mutation. Here we implicate FREP3 in playing a central role in resistance to a major group of snail pathogens, digenetic trematodes. FREP3 is up-regulated in three models of resistance of B. glabrata to infection with Schistosoma mansoni or Echinostoma paraensei, and functions as an opsonin favoring phagocytosis by hemocytes. Knock-down of FREP3 in resistant snails using siRNA-mediated interference resulted in increased susceptibility to E. paraensei, providing a direct link between a gastropod immune molecule and resistance to trematodes. FREP3 up-regulation is also associated with heightened responsiveness following priming with attenuated digenetic trematodes (acquired resistance) in this model invertebrate immune system.

Two Macrophage Colony-Stimulating Factor Genes Exist in Fish That Differ in Gene Organization and Are Differentially Expressed

Mammalian macrophage CSF (MCSF; CSF-1) is the primary regulator of the mononuclear phagocyte lineage. We, for the first time, report the complete sequencing of five MCSF cDNAs from three fish species, rainbow trout, zebrafish, and goldfish. Despite the difference in the lengths of the MCSF transcripts, all of the fish MCSF molecules encode a signal peptide, a CSF-1 domain, a transmembrane domain, and an intracellular region. Each fish MCSF gene has a unique exon/intron structure. The primordial MCSF gene may have had a nine exon/eight intron structure. In this model, insertion of an intron in exon 6 in primitive fish created the fish type I MCSF, while the loss of this exon or part of the original exon 6 created the fish type II MCSF. Investigation of alternative splicing variants in trout suggests that no mammalian equivalent splice variants exist. The two trout MCSF genes are differentially expressed in vivo and contributed differently to the high-level expression of MCSF in spleen and head kidney. In contrast to the up-regulation of MCSF by PMA in mammals, in trout MCSF1 expression is down-regulated by PMA treatment. As in mammals, recombinant trout MCSF1 can promote the growth of head kidney leukocytes, and it up-regulates the expression of CXCR3 in head kidney macrophages, with the latter suggesting a role of MCSF in the trafficking of macrophages to sites of inflammation or injury where the CXCR3 ligands are expressed. Thus MCSF has an important role in the immune system of fish as in mammals.

A somatically diversified defense factor, FREP3, is a determinant of snail resistance to schistosome infection.

Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished.

Growth Factors of Lower Vertebrates CHARACTERIZATION OF GOLDFISH (CARASSIUS AURATUS L.) MACROPHAGE COLONY-STIMULATING FACTOR-1

Colony-stimulating factor-1 (CSF-1) regulates mononuclear cell proliferation, differentiation, and survival. The functions of CSF-1 are well documented in mammals; however, little is known about CSF-1 biology in lower vertebrates. This is the first report on the identification and functional characterization of a fish CSF-1 molecule expressed highly in the spleen and in phorbol 12-myristate 13-acetate-stimulated monocytes. Goldfish CSF-1 is a 199-amino acid protein that possesses the required cysteine residues to form important intra-chain and inter-chain disulfide bonds that allow CSF-1 to form a functional homodimer and to interact with its high affinity receptor, CSF-1R. Recombinant goldfish CSF-1 formed a homodimer and bound to the soluble goldfish CSF-1R. The addition of the recombinant CSF-1 to sorted goldfish progenitor cells, monocytes, and macrophages induced the differentiation of monocytes into macrophages and the proliferation of monocyte-like cells. The proliferation of these cells was abrogated by addition of an anti-CSF-1R antibody as well as the soluble CSF-1R. The ability of the soluble CSF-1R to inhibit CSF-1-induced proliferation represents a novel mechanism for the regulation of CSF-1 function.

Macrophage colony-stimulating factor (CSF-1) induces pro-inflammatory gene expression and enhances antimicrobial responses of goldfish (Carassius auratus L.) macrophages.

We report on the regulation of pro-inflammatory functions of goldfish macrophages and induction of gene expression by recombinant goldfish CSF-1 (rgCSF-1). Recombinant goldfish TNFalpha-2 (rg TNFalpha-2), rgIFNgamma but not rgTGFbeta induced time-dependent increase of CSF-1 expression in macrophages. Treatment of goldfish macrophages with rgCSF-1 increased expression of several immune genes including CXCL-8 (=IL-8), CCL-1, TNFalpha-1, TNFalpha-2, IL-1beta-1, IL-1beta-2, IL-12-p35, IL-12-p40, IFN, IL-10 and iNOS A and B. The rgCSF-1 treatment did not significantly alter the mRNA levels of TGFbeta and NRAMP in macrophages up to 48h post treatment. However, at 72h post treatment, the expression of TGFbeta increased whereas that of NRAMP decreased. The treatment of macrophages with rgCSF-1 enhanced their respiratory burst and nitric oxide responses that were abrogated after addition of soluble CSF-1 receptor (sCSF-1R) to cell cultures. Macrophages exhibited a concentration-dependent chemotactic response toward rgCSF-1 as well as an increase in phagocytic activity that was abrogated after addition of sCSF-1R to cell cultures. Our results indicate that in addition to being an important growth factor of goldfish macrophages, rgCSF-1 also plays a central role in the regulation of their pro-inflammatory responses.

A Novel Hematopoietic Granulin Induces Proliferation of Goldfish (Carassius auratus L.) Macrophages

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation.

A Novel Toll-Like Receptor (TLR) Influences Compatibility between the Gastropod Biomphalaria glabrata, and the Digenean Trematode Schistosoma mansoni.

Schistosomiasis, a devastating disease caused by parasitic flatworms of the genus Schistosoma, affects over 260 million people worldwide especially in tropical and sub-tropical regions. Schistosomes must undergo their larval development within specific species of snail intermediate hosts, a trait that is shared among almost all digenean trematodes. This unique and long-standing host-parasite relationship presents an opportunity to study both the importance of conserved immunological features in novel immunological roles, as well as new immunological adaptations that have arisen to combat a very specific type of immunological challenge. While it is well supported that the snail immune response is important for protecting against schistosome infection, very few specific snail immune factors have been identified and even fewer have been functionally characterized. Here, we provide the first functional report of a snail Toll-like receptor, which we demonstrate as playing an important role in the cellular immune response of the snail Biomphalaria glabrata following challenge with Schistosoma mansoni. This TLR (BgTLR) was identified as part of a peptide screen of snail immune cell surface proteins that differed in abundance between B. glabrata snails that differ in their compatibility phenotype to challenge by S. mansoni. The S. mansoni-resistant strain of B. glabrata (BS-90) displayed higher levels of BgTLR compared to the susceptible (M-line) strain. Transcript expression of BgTLR was found to be very responsive in BS-90 snails when challenged with S. mansoni, increasing 27 fold relative to β-actin (non-immune control gene); whereas expression in susceptible M-line snails was not significantly increased. Knockdown of BgTLR in BS-90 snails via targeted siRNA oligonucleotides was confirmed using a specific anti-BgTLR antibody and resulted in a significant alteration of the resistant phenotype, yielding patent infections in 43% of the normally resistant snails, which shed S. mansoni cercariae 1-week before the susceptible controls. Our results represent the first functional characterization of a gastropod TLR, and demonstrate that BgTLR is an important snail immune receptor that is capable of influencing infection outcome following S. mansoni challenge.

The role of fibrinogen-related proteins in the gastropod immune response.

Fibrinogen-related proteins or FREPs constitute a large family of molecules, defined by the presence of a fibrinogen-related domain (FReD). These molecules are found in all animals and are diverse in both form and function. Here, we review the current understanding of gastropod FREPs, which are characterized by the presence of a fibrinogen domain connected to one or two immunoglobulin superfamily domains by way of a short interceding region. We present a historical perspective on the discovery of FREPs in gastropods followed by a summary of advances made in the nearly two decades of research focused on the characterization of FREPs in Biomphalaria glabrata (BgFREPs). Topics covered include BgFREP genomic architecture, predicted structure and known functions, structural comparisons between BgFREPs, and evidence of somatic diversification. Also examined are the expression patterns of BgFREPs during snail development and immunological challenges. Recent functional characterization of the role BgFREPs play in the defence response against digenean trematodes is also presented, as well as new data investigating the nucleotide-level genomic conservation of FREPs among Pulmonate gastropods. Finally, we identify areas in need of further research. These include confirming and identifying the specific binding targets of BgFREPs and elucidating how they later engage snail haemocytes to elicit an immunological response, precise mechanisms and importance of BgFREP diversification, characterizing the tissue expression patterns of BgFREPs, as well as addressing whether gastropod FREPs retain immunological importance in alternative snail-trematode associations or more broadly in snail-pathogen interactions.

Macrophage colony stimulating factor (CSF-1) is a central growth factor of goldfish macrophages.

We recently characterized macrophage colony stimulating factor (CSF-1) of fish (the goldfish). Here, we report for the first time that goldfish CSF-1 acts through the CSF-1 receptor by showing loss of CSF-1 function in CSF-1R knockdown monocytes using RNAi, and demonstrate that goldfish CSF-1 administration in vivo increases the amount of circulating monocytes in blood. We also show that conditioned supernatants from goldfish fibroblast cultures induced the proliferation of goldfish monocytes indicating that, like in mammals, teleost fibroblasts are an important producer of CSF-1. The continuous addition of recombinant CSF-1 to primary goldfish macrophage cultures stabilized and extended their longevity and resulted in a long-term culture of functional macrophages capable of mounting a potent nitric oxide response upon activation with goldfish recombinant TNF-alpha.