Clearance of schistosome parasites by resistant genotypes at a single genomic region in Biomphalaria glabrata snails involves cellular components of the hemolymph

Schistosomiasis is one of the most detrimental neglected tropical diseases. Controlling the spread of this parasitic illness requires effective sanitation, access to chemotherapeutic drugs, and control over populations of the freshwater snails, such as Biomphalaria glabrata, that are essential intermediate hosts for schistosomes. Effectively controlling this disease, while minimising ecological implications of such control, will require an extensive understanding of the immunological interactions between schistosomes and their molluscan intermediate hosts. Here we histologically characterise the clearance of schistosome larvae by snails that exhibit allelic variation at a single genomic region, the Guadeloupe resistance complex. We show that snails with a resistant Guadeloupe resistance complex genotype clear schistosomes within the first 24-48 h, and that this resistance can be transferred to susceptible snails via whole hemolymph but not cell-free plasma. These findings imply that Guadeloupe resistance complex-coded proteins help to coordinate hemocyte-mediated immune responses to schistosome infections in Guadeloupean snails

siRNA knockdown of Grctm6 increases the number of cercariae released by shedding snails but does not modify susceptibility.

<p>Resistant BgGUA snails were treated with <i>GFP</i> or <i>grctm6</i> oligos and exposed to 10 or 20 miracidia 3 days after injection of oligos. (A-B) Susceptibility over weeks 5–10 post challenge with (A) 10 (<i>n</i> = 65 <i>GFP</i>, 63 snails <i>grctm6</i>) or (B) 20 miracidia (<i>n</i> = 55 <i>GFP</i>, 37 <i>grctm6</i> snails). (C-D) The total number of cercariae released over a 15 h period (a 3 h period, every week, from weeks 5–10 post exposure) by shedding snails exposed to (C) 10 (<i>n</i> = 6 <i>GFP</i>, 5 <i>grctm6</i> snails) or (D) 20 miracidia (<i>n</i> = 12 <i>GFP</i>, 11 <i>grctm6</i> snails). Susceptibility data are presented as the proportion of infected snails +/- standard error of proportions. Cercariae counts are presented as mean +/- SD. Significant differences (Susceptibility: Z score of proportion; cercariae counts: ln transformed for equal variance, student t-test, <i>p</i><0.05) from the <i>GFP</i> control are denoted by asterisks (*). Note the different scales on the Y-axes in figures for 10-miracidial versus 20-miracidial challenges.</p

siRNA mediated knockdown of Grctm6.

<p>(A) Mortality of resistant BgGUA snails injected with siRNA taken on 4 separate days post injection (<i>n</i> = 3–6 independent experiments per day). (B) The kinetics of siRNA mediated knockdown of <i>grctm6</i> mRNA in whole resistant snails after injection compared to <i>GFP</i> siRNA oligos during the first 4 days post-injection <b>(</b><i>n</i> = 8–10 snails/treatment/day). A reduction of 60% below control was evident by day 3. (C) Western blot analysis of Grctm6 protein levels in resistant hemolymph 2–4 days after injecting snails with <i>grctm6</i> or <i>GFP</i> siRNA. Equivalent total protein was loaded into each well (750 ng). The band shown in these representative blots is at the ~68 kDa size (~42 for BgActin loading control). Densitometry was preformed using Image J software (Days 2 and 4: <i>n</i> = 3 samples/treatment/day, <i>n</i> = hemolymph pooled from 4–5 individuals; Day 3: <i>n</i> = 5 samples/treatment, <i>n</i> = hemolymph pooled from 6–10 individuals, averaged from three independent Western blots and presented relative to the BgActin loading control for each individual day). A ~30% reduction, below control relative to BgActin, was found in samples taken on Day 3. mRNA levels are normalized to β-actin and relative to <i>GFP</i> oligo injected individuals taken on each individual day (no comparison between days was made). All data are presented as mean +/- SD; significant differences (student t-test, <i>p</i><0.05) from the <i>GFP</i> control are denoted by asterisks (*).</p

<i>grctm6</i> mRNA expression and protein detection.

<p>(A) Constitutive <i>grctm6</i> mRNA levels in resistant whole snail (WS), head-foot (HF), albumen gland (ABG), and hemolymph (HL) lysates (<i>n</i> = 4 samples). HL has significantly elevated levels compared to HF only. (B) Western blot analysis of constitutive Grctm6 protein levels in resistant whole snail after hemolymph was removed (WS- HL), head-foot (HF), albumen gland (ABG), hemolymph with cells removed (HL—cells), whole hemolymph (HL), hemocyte lysates, sample buffer containing no snail tissue, negative control peptide, and Grctm6 peptide (provided by genscript). Equivalent total protein was loaded into each well for experimental samples (500 ng/sample). The band shown is at the ~68 kDa size for Grctm6 (~42 for BgActin loading control), Grctm6 peptides appear lower down on the gel as they are not the full length protein. HL- cells shows lower levels of BgActin protein because most of the actin producing cells were removed by centrifugation. Additionally, hemolymph samples with differing total protein concentrations are shown below. mRNA levels are normalized to β-actin and presented as mean +/- SD relative to WS samples. Significant differences (ln transformed for equal variance, ANOVA, <i>p</i><0.05) are denoted by asterisks (*).</p

<i>grctm6</i> encodes a single-pass transmembrane protein with an extraordinarily variable extracellular domain.

<p>Alignment of the protein products of the three alleles of <i>grctm6</i> found in BgGUA. Residues identical to those in the <i>R</i> allele are indicated by dots. The putative signal peptide is shown with a dark blue outlined box. The putative transmembrane domain is shown with an orange box, while the extracellular and intracellular domains are flanked by green and purple bars, respectively. Highly variable regions, defined by 20aa windows containing at least 10 sequence differences, have a yellow background. Cysteines are shown in red. Asparagines predicted to be glycosylated are shown in blue/green.</p

Susceptibility of BgGUA lines is not explained by <i>grctm6</i> mRNA levels.

<p>(A) The susceptibility of the three different homozygous BgGUA genotypes (<i>S1S1</i>, <i>S2S2</i>, <i>RR</i>) after challenge with 20 miracidia (<i>n</i> = 64, 68, 86 snails respectively). Susceptibility data are presented as the proportion of infected snails +/- standard error of proportions. (B) Constitutive <i>grctm6</i> mRNA levels (whole snail) of these same homozygous lines (<i>n</i> = 17, 21, 17 snails respectively). Note that the susceptibility differences among the three genotypes does not correlate with expression levels of <i>grctm6</i>. mRNA levels are normalized to β-actin and presented as mean +/- SD relative to levels in <i>S2S2</i>. Significant differences among lines (Susceptibility: Z score of proportion; mRNA levels: ln transformed for equal variance, ANOVA, <i>p</i><0.05) are denoted by asterisks (*).</p

siRNA knockdown of Grctm6 increases the number of cercariae released by shedding snails but does not modify susceptibility.

<p>Resistant BgGUA snails were treated with <i>GFP</i> or <i>grctm6</i> oligos and exposed to 10 or 20 miracidia 3 days after injection of oligos. (A-B) Susceptibility over weeks 5–10 post challenge with (A) 10 (<i>n</i> = 65 <i>GFP</i>, 63 snails <i>grctm6</i>) or (B) 20 miracidia (<i>n</i> = 55 <i>GFP</i>, 37 <i>grctm6</i> snails). (C-D) The total number of cercariae released over a 15 h period (a 3 h period, every week, from weeks 5–10 post exposure) by shedding snails exposed to (C) 10 (<i>n</i> = 6 <i>GFP</i>, 5 <i>grctm6</i> snails) or (D) 20 miracidia (<i>n</i> = 12 <i>GFP</i>, 11 <i>grctm6</i> snails). Susceptibility data are presented as the proportion of infected snails +/- standard error of proportions. Cercariae counts are presented as mean +/- SD. Significant differences (Susceptibility: Z score of proportion; cercariae counts: ln transformed for equal variance, student t-test, <i>p</i><0.05) from the <i>GFP</i> control are denoted by asterisks (*). Note the different scales on the Y-axes in figures for 10-miracidial versus 20-miracidial challenges.</p

A role for cathepsin Z in neuroinflammation provides mechanistic support for an epigenetic risk factor in multiple sclerosis

Abstract Background Hypomethylation of the cathepsin Z locus has been proposed as an epigenetic risk factor for multiple sclerosis (MS). Cathepsin Z is a unique lysosomal cysteine cathepsin expressed primarily by antigen presenting cells. While cathepsin Z expression has been associated with neuroinflammatory disorders, a role for cathepsin Z in mediating neuroinflammation has not been previously established. Methods Experimental autoimmune encephalomyelitis (EAE) was induced in both wildtype mice and mice deficient in cathepsin Z. The effects of cathepsin Z-deficiency on the processing and presentation of the autoantigen myelin oligodendrocyte glycoprotein, and on the production of IL-1β and IL-18 were determined in vitro from cells derived from wildtype and cathepsin Z-deficient mice. The effects of cathepsin Z-deficiency on CD4+ T cell activation, migration, and infiltration to the CNS were determined in vivo. Statistical analyses of parametric data were performed by one-way ANOVA followed by Tukey post-hoc tests, or by an unpaired Student’s t test. EAE clinical scoring was analyzed using the Mann–Whitney U test. Results We showed that mice deficient in cathepsin Z have reduced neuroinflammation and dramatically lowered circulating levels of IL-1β during EAE. Deficiency in cathepsin Z did not impact either the processing or the presentation of MOG, or MOG- specific CD4+ T cell activation and trafficking. Consistently, we found that cathepsin Z-deficiency reduced the efficiency of antigen presenting cells to secrete IL-1β, which in turn reduced the ability of mice to generate Th17 responses—critical steps in the pathogenesis of EAE and MS. Conclusion Together, these data support a novel role for cathepsin Z in the propagation of IL-1β-driven neuroinflammation