Jonathan D. Stoltzfus

Early changes in interferon signaling define natural killer cell response and refractoriness to interferon‐based therapy of hepatitis C patients

Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN‐γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)‐induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN‐α‐based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN‐α‐based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor–apoptosis‐inducing ligand [TRAIL] expression and degranulation) and decreased IFN‐γ production. NK cells from patients with a greater than 2 log10 first‐phase HCV RNA decline to IFN‐α‐based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log10 first‐phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN‐α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN‐α during the second‐phase virological response. Conclusion: These data show that IFN‐α‐induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN‐γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN‐α‐based therapy. (Hepatology 2012)

The genomic basis of parasitism in the Strongyloides clade of nematodes

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families—families encoding astacin-like and SCP/TAPS proteins—is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

RNAseq Analysis of the Parasitic Nematode Strongyloides stercoralis Reveals Divergent Regulation of Canonical Dauer Pathways

The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode Strongyloides stercoralis differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode Caenorhabditis elegans has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor β (TGFβ) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in S. stercoralis, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for S. stercoralis homologs of C. elegans dauer genes identified by BLAST searches of the S. stercoralis genome as well as de novo assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to C. elegans, S. stercoralis has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven S. stercoralis genes encoding homologs of the single C. elegans dauer regulatory TGFβ ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in S. stercoralis and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.

Strongyloides stercoralis age-1: A Potential Regulator of Infective Larval Development in a Parasitic Nematode

Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The ‘dauer hypothesis’ predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 µM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases.

Ribavirin improves the IFN‐γ response of natural killer cells to IFN‐based therapy of hepatitis C virus infection

Ribavirin (RBV) is an important component of interferon (IFN)‐based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host IFN response, has been proposed as RBVs mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN‐α/β receptor signaling, as NK cell cytotoxicity and IFN‐γ production are regulated by signal transducer and activator of transcription (STAT)1‐ and STAT4‐phosphorylation, respectively. Specifically, pSTAT1‐dependent NK cell cytotoxicity increases and pSTAT4‐dependent IFN‐γ production decreases in response to endogenous, virus‐induced IFN‐α and during IFN‐α‐based therapy. To assess whether RBV has a direct effect on NK cells and/or improves the IFN‐γ response of NK cells in the presence of IFN‐α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of RBV pretreatment, who all received subsequent pegylated (Peg)IFN/ribavirin combination therapy. During RBV pretreatment, both the frequency of CD56dim NK cells with cytotoxic effector functions and the frequency of CD56bright NK cells with the capacity to produce IFN‐γ decreased (P = 0.049 and P = 0.001, respectively). In vitro or in vivo exposure of NK cells to RBV improved the pSTAT4 (P < 0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN‐α. This was associated with an increase in IFN‐γ production but not cytotoxicity of NK cells during subsequent IFN‐α‐based therapy. The frequency of IFN‐γ‐producing NK cells was greater in fast second‐phase virological responders than in slow responders. Conclusion: RBV enhances the pSTAT4 and IFN‐γ response of NK cells to IFN‐α‐stimulation. (Hepatology 2014;60:1160–1169)

cGMP and NHR Signaling Co-regulate Expression of Insulin-Like Peptides and Developmental Activation of Infective Larvae in Strongyloides stercoralis

The infectious form of the parasitic nematode Strongyloides stercoralis is a developmentally arrested third-stage larva (L3i), which is morphologically similar to the developmentally arrested dauer larva in the free-living nematode Caenorhabditis elegans. We hypothesize that the molecular pathways regulating C. elegans dauer development also control L3i arrest and activation in S. stercoralis. This study aimed to determine the factors that regulate L3i activation, with a focus on G protein-coupled receptor-mediated regulation of cyclic guanosine monophosphate (cGMP) pathway signaling, including its modulation of the insulin/IGF-1-like signaling (IIS) pathway. We found that application of the membrane-permeable cGMP analog 8-bromo-cGMP potently activated development of S. stercoralis L3i, as measured by resumption of feeding, with 85.1±2.2% of L3i feeding in 200 µM 8-bromo-cGMP in comparison to 0.6±0.3% in the buffer diluent. Utilizing RNAseq, we examined L3i stimulated with DMEM, 8-bromo-cGMP, or the DAF-12 nuclear hormone receptor (NHR) ligand Δ7-dafachronic acid (DA)—a signaling pathway downstream of IIS in C. elegans. L3i stimulated with 8-bromo-cGMP up-regulated transcripts of the putative agonistic insulin-like peptide (ILP) -encoding genes Ss-ilp-1 (20-fold) and Ss-ilp-6 (11-fold) in comparison to controls without stimulation. Surprisingly, we found that Δ7-DA similarly modulated transcript levels of ILP-encoding genes. Using the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002, we demonstrated that 400 nM Δ7-DA-mediated activation (93.3±1.1% L3i feeding) can be blocked using this IIS inhibitor at 100 µM (7.6±1.6% L3i feeding). To determine the tissues where promoters of ILP-encoding genes are active, we expressed promoter::egfp reporter constructs in transgenic S. stercoralis post-free-living larvae. Ss-ilp-1 and Ss-ilp-6 promoters are active in the hypodermis and neurons and the Ss-ilp-7 promoter is active in the intestine and a pair of head neurons. Together, these data provide evidence that cGMP and DAF-12 NHR signaling converge on IIS to regulate S. stercoralis L3i activation.

Strongyloides stercoralis daf-2 encodes a divergent ortholog of Caenorhabditis elegans DAF-2

We hypothesise that developmental arrest in infectious larvae of parasitic nematodes is regulated by signalling pathways homologous to Caenorhabditis elegans DAF (dauer formation) pathways. Alignment of Strongyloides stercoralis (Ss) DAF-2 with DAF-2 of C. elegans and homologs of other species shows that most structural motifs in these insulin-like receptors are conserved. However, the catalytic domain of Ss-DAF-2 contains two substitutions (Q1242 and Q1256), that would result in constitutive dauer formation in C. elegans or diabetes in vertebrate animals. Ss-daf-2 also shows two alternately spliced isoforms, the constitutively expressed Ss-daf-2a, and Ss-daf-2b, which is only expressed in stages leading to parasitism.

Regulation of Life Cycle Checkpoints and Developmental Activation of Infective Larvae in Strongyloides stercoralis by Dafachronic Acid

The complex life cycle of the parasitic nematode Strongyloides stercoralis leads to either developmental arrest of infectious third-stage larvae (iL3) or growth to reproductive adults. In the free-living nematode Caenorhabditis elegans, analogous determination between dauer arrest and reproductive growth is governed by dafachronic acids (DAs), a class of steroid hormones that are ligands for the nuclear hormone receptor DAF-12. Biosynthesis of DAs requires the cytochrome P450 (CYP) DAF-9. We tested the hypothesis that DAs also regulate S. stercoralis development via DAF-12 signaling at three points. First, we found that 1 μM Δ7-DA stimulated 100% of post-parasitic first-stage larvae (L1s) to develop to free-living adults instead of iL3 at 37°C, while 69.4±12.0% (SD) of post-parasitic L1s developed to iL3 in controls. Second, we found that 1 μM Δ7-DA prevented post-free-living iL3 arrest and stimulated 85.2±16.9% of larvae to develop to free-living rhabditiform third- and fourth-stages, compared to 0% in the control. This induction required 24–48 hours of Δ7-DA exposure. Third, we found that the CYP inhibitor ketoconazole prevented iL3 feeding in host-like conditions, with only 5.6±2.9% of iL3 feeding in 40 μM ketoconazole, compared to 98.8±0.4% in the positive control. This inhibition was partially rescued by Δ7-DA, with 71.2±16.4% of iL3 feeding in 400 nM Δ7-DA and 35 μM ketoconazole, providing the first evidence of endogenous DA production in S. stercoralis. We then characterized the 26 CYP-encoding genes in S. stercoralis and identified a homolog with sequence and developmental regulation similar to DAF-9. Overall, these data demonstrate that DAF-12 signaling regulates S. stercoralis development, showing that in the post-parasitic generation, loss of DAF-12 signaling favors iL3 arrest, while increased DAF-12 signaling favors reproductive development; that in the post-free-living generation, absence of DAF-12 signaling is crucial for iL3 arrest; and that endogenous DA production regulates iL3 activation.

The Nuclear Receptor DAF-12 Regulates Nutrient Metabolism and Reproductive Growth in Nematodes

Appropriate nutrient response is essential for growth and reproduction. Under favorable nutrient conditions, the C. elegans nuclear receptor DAF-12 is activated by dafachronic acids, hormones that commit larvae to reproductive growth. Here, we report that in addition to its well-studied role in controlling developmental gene expression, the DAF-12 endocrine system governs expression of a gene network that stimulates the aerobic catabolism of fatty acids. Thus, activation of the DAF-12 transcriptome coordinately mobilizes energy stores to permit reproductive growth. DAF-12 regulation of this metabolic gene network is conserved in the human parasite, Strongyloides stercoralis, and inhibition of specific steps in this network blocks reproductive growth in both of the nematodes. Our study provides a molecular understanding for metabolic adaptation of nematodes to their environment, and suggests a new therapeutic strategy for treating parasitic diseases.

Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis.

Background Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs. Methodology/Principal Findings The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5′-UTR, a 17 bp 3′-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis. Conclusions/Significance The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.