Heat shock increases hydrogen peroxide release from circulating hemocytes of the snail Biomphalaria glabrata.

Planorbid freshwater snails are important intermediate hosts for parasitic diseases caused by parasitic worms, most notably schistosomiasis. There are numerous reports of snails, specifically Biomphalaria glabrata, having compromised defences against schistosomes after being exposed to thermal stress. Environmental modifications to the defenses of schistosome transmitting snails could have negative ramifications for human disease risk in the context of climate change. Here the effects of heat shock on the production of hydrogen peroxide, a primary anti-microbial effector in many molluscs, were examined. The present findings show that heat shock increases NADPH oxidase 2 mRNA levels and hydrogen peroxide produced by snail hemocytes, and that both of these phenotypes could be reversed by an HSP-90 inhibitor. These findings indicate that snail defense systems are altered by heat shock at a molecular level in B. glabrata, and that snail immunity to many pathogens may be altered by the rapid variations in temperature that are associated with global climate change.

Acute exposure to physiological doses of triiodothyronine does not induce gonadal caspase 3 activity in goldfish in vitro.

Seasonally spawning fish rely on a dynamic and complex hormonal interplay to regulate cycles of gonadal development and the regression. Thyroid hormones have been shown to be a key player during gonadal development, and can regulate the activity of a number of essential reproductive hormones. Apoptosis is a vital cellular process that contributes to the hormonal control of gonadal development and regression, but the roles of thyroid hormones on gonadal apoptosis in goldfish have not been explored. The present study examines the role of acute T3 exposure on caspase 3-dependent apoptosis in dispersed goldfish gonadal tissue in vitro. We examined the levels of caspase 3 activity in early, mid, and late recrudescent gonadal tissue after exposure to physiological doses of T3 for up to 24 h. Acute treatment with T3 did not alter basal caspase 3 activity in goldfish gonads in vitro in these reproductive stages. This initial study suggests that transient increases in T3 levels are unlikely to directly contribute to basal caspase 3-dependent apoptosis in the gonadal tissue of goldfish, although we cannot rule out an interaction of T3 with other hormones involved in the control of apoptosis in the testis and ovary.

The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response.

The synthetic supercooling drug, icilin, and its primary receptor target, the cation channel transient receptor potential (TRP) melastatin-8 (TRPM8), have been described as potent negative regulators of inflammation in the colon. The aim of this study was to determine whether the anti-inflammatory action of icilin could potentially be used to treat autoimmune neuroinflammatory disorders, such as multiple sclerosis (MS). During experimental autoimmune encephalomyelitis (EAE)-a CD4+ T cell-driven murine model of MS-we found that both wild-type (WT) and TRPM8-deficient EAE mice were protected from disease progression during icilin treatment, as evidenced by delays in clinical onset and reductions in neuroinflammation. In vitro, icilin potently inhibited the proliferation of murine and human CD4+ T cells, with the peripheral expansion of autoantigen-restricted T cells similarly diminished by the administration of icilin in mice. Attenuation of both TRPM8-/- and TRP ankyrin-1-/- T-cell proliferation by icilin was consistent with the WT phenotype, which suggests a mechanism that is independent of these channels. In addition, icilin treatment altered the expressional profile of activated CD4+ T cells to one that was indicative of restricted effector function and limited neuroinflammatory potential. These findings identify a potent anti-inflammatory role for icilin in lymphocyte-mediated neuroinflammation and highlight clear pleiotropic effects of the compound beyond classic TRP channel activation.-Ewanchuk, B. W., Allan, E. R. O., Warren, A. L., Ramachandran, R., Yates, R. M. The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response.

Allelic variation in a single genomic region alters the hemolymph proteome in the snail Biomphalaria glabrata.

Freshwater snails are obligate intermediate hosts for numerous parasitic trematodes, most notably schistosomes. Schistosomiasis is a devastating human and veterinary illness, which is primarily controlled by limiting the transmission of these parasites from their intermediate snail hosts. Understanding how this transmission occurs, as well as the basic immunobiology of these snails may be important for controlling this disease in the future. Allelic variation in the Guadeloupe resistance complex (GRC) of Biomphalaria glabrata partially determines their susceptibility to parasitic infection, and can influence the microbiome diversity and microbial defenses in the hemolymph of these snails. In the present study, we examine the most abundant proteins present in the hemolymph of snails that are resistant or susceptible to schistosomes, as determined by their GRC genotype. Using proteomic analysis, we found that snails with different GRC genotypes have differentially abundant hemolymph proteins that are not explained by differences in transcription. There are 13 revealed hemolymph proteins that differ significantly between resistant and susceptible genotypes, nearly 40% of which are involved in immune responses. These findings build on the mounting evidence that genes in the GRC region have multiple physiological roles, and likely contribute more extensively to the general immune response than previously believed. These data also raise the intriguing possibility that the GRC region controls resistance to schistosomes, not directly, but indirectly via its effects on the snail's proteome and potentially its microbiome.

Allelic variation partially regulates galactose-dependent hydrogen peroxide release from circulating hemocytes of the snail Biomphalaria glabrata.

Freshwater snails are the intermediate hosts for numerous parasitic worms that are detrimental to human and agricultural health. Understanding the immune responses of these snails could be vital for finding ways to block transmission of those parasites. Allelic variation in a recently discovered genomic region in the snail, Biomphalaria glabrata, influences their susceptibility to schistosomes. Here we tested whether genes in that region, termed the Guadeloupe Resistance Complex (GRC), are involved in recognition of common pathogen-associated molecules that have been shown to be stimulants of the hydrogen peroxide defense pathway. We show that hemocytes extracted from individuals with one of the three GRC genotypes released less hydrogen peroxide than the other two genotypes, after stimulation with galactose. This difference was not observed after stimulation with several other microbial-associated carbohydrates, despite those ligands sharing the same putative pathway for hydrogen peroxide release. Therefore, we conclude that allelic variation in the GRC region may influence the recognition of galactose, rather than the conserved downstream steps in the hydrogen peroxide pathway. These results thus are consistent with the hypothesis that proteins produced by this region are involved in pathogen recognition.

Allelic Variation in a Single Genomic Region Alters the Microbiome of the Snail Biomphalaria glabrata.

Freshwater snails are the intermediate hosts for numerous parasitic worms which can have negative consequences for human health and agriculture. Understanding the transmission of these diseases requires a more complete characterization of the immunobiology of snail hosts. This includes the characterization of its microbiome and genetic factors which may interact with this important commensal community. Allelic variation in the Guadeloupe resistance complex (GRC) genomic region of Guadeloupean Biomphalaria glabrata influences their susceptibility to schistosome infection and may have other roles in the snail immune response. In the present study, we examined whether a snail's GRC genotype has a role in shaping the bacterial diversity and composition present on or in whole snails. We show that the GRC haplotype, including the resistant genotype, has a significant effect on the diversity of bacterial species present in or on whole snails, including the relative abundances of Gemmatimonas aurantiaca and Micavibrio aeruginosavorus. These findings support the hypothesis that the GRC region is likely involved in pathways that can modify the microbial community of these snails and may have more immune roles in B. glabrata than originally believed. This is also one of few examples in which allelic variation at a particular locus has been shown to affect the microbiome in any species.

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

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-1ß 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-1ß 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-1ß, 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-1ß-driven neuroinflammation.

Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.

The level of proteolysis within phagosomes of dendritic cells (DCs) is thought to be tightly regulated, as it directly impacts the cell's efficiency to process antigen. Activity of the antimicrobial effector NADPH oxidase (NOX2) has been shown to reduce levels of proteolysis within phagosomes of both macrophages and DCs. However, the proposed mechanisms underlying these observations in these two myeloid cell lineages are dissimilar. Using real-time analysis of lumenal microenvironmental parameters within phagosomes in live bone marrow-derived DCs, we show that the levels of phagosomal proteolysis are diminished in the presence of NOX2 activity, but in contrast to previous reports, the acidification of the phagosome is largely unaffected. As found in macrophages, we show that NOX2 controls phagosomal proteolysis in DCs through redox modulation of local cysteine cathepsins. Aspartic cathepsins were unaffected by redox conditions, indicating that NOX2 skews the relative protease activities in these antigen processing compartments. The ability of DC phagosomes to reduce disulphides was also compromised by NOX2 activity, implicating this oxidase in the control of an additional antigen processing chemistry of DCs.

NADPH oxidase modifies patterns of MHC class II-restricted epitopic repertoires through redox control of antigen processing.

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow-derived macrophages, but not dendritic cells, to process and present the I-A(b)-immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-A(b) binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-A(b) mice that were deficient in the p47(phox) or gp91(phox) subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4(+) T cells in the CNS, despite eliciting a normal primary CD4(+) T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4(+) T cell-driven autoimmune disease processes.

γ-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.

Although it is known that lysosomal cysteine cathepsins require a reducing environment for optimal activity, it is not firmly established how these enzymes are maintained in their reduced-active state in the acidic and occasionally oxidative environment within phagosomes and lysosomes. γ-Interferon-inducible lysosomal thiol reductase (GILT) has been the only enzyme described in the endosomes, lysosomes, and phagosomes with the potential to catalyze the reduction of cysteine cathepsins. Our goal in the current study was to assess the effect of GILT on major phagosomal functions with an emphasis on proteolytic efficiency in murine bone marrow-derived macrophages. Assessment of phagosomal disulfide reduction upon internalization of IgG-opsonized experimental particles confirmed a major role for GILT in phagosomal disulfide reduction in both resting and interferon-γ-activated macrophages. Furthermore we observed a decrease in early phagosomal proteolytic efficiency in GILT-deficient macrophages, specifically in the absence of an NADPH oxidase-mediated respiratory burst. This deficiency was more prominent in IL-4-activated macrophages that inherently possess lower levels of NADPH oxidase activity. Finally, we provide evidence that GILT is required for optimal activity of the lysosomal cysteine protease, cathepsin S. In summary, our results suggest a role for GILT in maintaining cysteine cathepsin proteolytic efficiency in phagosomes, particularly in the absence of high NADPH oxidase activity, which is characteristic of alternatively activated macrophages.

Alternative activation of macrophages by IL-4 enhances the proteolytic capacity of their phagosomes through synergistic mechanisms.

Alternatively activated macrophages, generated in a T-helper 2 environment, have demonstrated roles in wound repair and tissue remodeling in addition to being charged with immune tasks. Because the hydrolytic chemistries of the phagosomal lumen are central to many of these functions, we investigated their modification after alternative activation with IL-4 and IL-13. Most significantly, we found striking up-regulation of the proteolytic levels within the phagosome of IL-4-activated macrophages. Two synergistic mechanisms were determined to underlie this up-regulation. First, IL-4-activated macrophages displayed increased expression of cathepsin S and L, providing greater proteolytic machinery to the phagosome despite unchanged rates of lysosomal contribution. Secondly, decreased phagosomal NADPH oxidase (NOX2) activity, at least partially resulting from decreased expression of the NOX2 subunit gp91(phox), resulted in a more reductive lumenal microenvironment, which in turn, enhanced activities of local cysteine cathepsins. Decreased NOX2 activity additionally increased the phagosome's ability to reduce disulfides, further enhancing the efficiency of the macrophage to degrade proteins containing disulfide bonds. Together, these changes initiated by IL-4 act synergistically to rapidly and dramatically enhance the macrophage's ability to degrade phagocytosed protein, which, we reason, better equips this cell for its roles in wound repair and tissue remodeling.

Structural variants identified using non-Mendelian inheritance patterns advance the mechanistic understanding of autism spectrum disorder.

The heritability of autism spectrum disorder (ASD), based on 680,000 families and five countries, is estimated to be nearly 80%, yet heritability reported from SNP-based studies are consistently lower, and few significant loci have been identified with genome-wide association studies. This gap in genomic information may reside in rare variants, interaction among variants (epistasis), or cryptic structural variation (SV) and may provide mechanisms that underlie ASD. Here we use a method to identify potential SVs based on non-Mendelian inheritance patterns in pedigrees using parent-child genotypes from ASD families and demonstrate that they are enriched in ASD-risk genes. Most are in non-coding genic space and are over-represented in expression quantitative trait loci, suggesting that they affect gene regulation, which we confirm with their overlap of differentially expressed genes in postmortem brain tissue of ASD individuals. We then identify an SV in the GRIK2 gene that alters RNA splicing and a regulatory region of the ACMSD gene in the kynurenine pathway as significantly associated with a non-verbal ASD phenotype, supporting our hypothesis that these currently excluded loci can provide a clearer mechanistic understanding of ASD. Finally, we use an explainable artificial intelligence approach to define subgroups demonstrating their use in the context of precision medicine.

Direct effects of triiodothyronine on production of anterior pituitary hormones and gonadal steroids in goldfish.

The present study investigated the effects of triiodothyronine (T3) on pituitary gonadotropin (GTH) subunits, thyroid stimulating hormone (TSH) ß subunit, and growth hormone (GH) mRNA levels, as well as gonadal steroid secretion during different stages of reproduction in goldfish. Goldfish pituitary cells cultured with T3 exhibited lower tshß mRNA levels in all reproductive stages and lower luteinising hormone ß (lhß) mRNA levels in early recrudescence, whereas gh and fshß mRNA levels were not altered. T3 injections significantly reduced circulating oestrogen (OE2) concentrations in early and mid recrudescent male goldfish, but were without effect on the circulating level of OE2 in female fish. T3 injections also reduced circulating levels of testosterone in both male and female goldfish during the mid stage of gonadal recrudescence. In vitro culture of goldfish ovarian follicles at the late stage of gonadal recrudescence, in the presence of T3, resulted in reduced OE2 secretion; no consistent effect of T3 on testosterone secretion was observed in cultured goldfish ovarian follicles and testis. These findings support the hypothesis that T3 impairs reproduction by inhibiting production of gonadal steroids and pituitary luteinising hormone production in goldfish.

TBBPA downregulates thyroid receptor and estrogen receptor mRNA levels in goldfish gonadal tissue.

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant that has been shown to be a potential thyroid disrupting chemical. Currently, TBBPA is not included in the UN's list of endocrine disruptors and adverse effects of TBBPA in mammals has not been fully investigated. However, there is clear evidence that TBBPA exerts adverse health effects on reproduction of aquatic species. Therefore, it is important to provide more information on potential endocrine disruptive effects of TBBPA in vertebrate species. In this study we investigated the effect of TBBPA on transcript levels of estrogen receptors (ERs) and thyroid receptors (TRs) in the gonadal tissue of goldfish in vivo and in vitro. ERß mRNA levels were significantly lower in testis and ovary following exposure to TBBPA. TRα mRNA levels were also downregulated in testis tissue. Importantly, these phenotypic effects occurred at lower, environmentally relevant, concentrations in vivo. Furthermore, exposure to TBBPA also reduced ERß and TRα mRNA abundance in goldfish testes and ovaries in vivo, which is similar to previously observed T3 responses in these tissues. These findings suggest that TBBPA may be a thyroid hormone mimic, capable of disrupting reproduction by affecting steroid hormone receptors. Our findings suggest that it is important to study TBBPA as an endocrine disruptor in aquatic organisms as it may have implications for both conservation and aquaculture.

Auto-regulation of thyroid hormone receptors in the goldfish ovary and testis.

Thyroid hormones, acting via their cognate thyroid receptors (TRs) act as mediators and modulators of several physiological processes and homeostasis. A clear role for the TRs in reproduction has not yet been established although several lines of recent evidence suggest that they are involved in the regulation of reproduction. To further study the role of TRs in control of reproduction, we investigated homologous regulation of TR subtypes in the gonads of goldfish, in vivo and in vitro. It was found that tri-iodothyronine (T(3)) down-regulates the traditional TRs (TRα-1 and TRß) and up-regulates a dominant-negative form, TRα-t. This indicates a 'feedback' mechanism whereby an acute treatment with T(3) down regulates further T(3) mediated response. The results provide novel information on auto-regulation of TRs in the goldfish ovary and testis, and support the hypothesis that thyroid hormones are involved in the control of reproduction.

Thyroid hormone and reproduction: regulation of estrogen receptors in goldfish gonads.

There is increasing evidence that thyroid hormones influence reproduction in vertebrates. However, little information is available on the mechanisms by which this happens. As a first step in determining these mechanisms, we test the hypothesis that the estrogen receptor subtypes (ERalpha, ERbeta-1, and ERbeta-2) are regulated by the thyroid hormone, (T(3)), in the gonads of goldfish. All three subtypes were down-regulated by T(3) in the testis or ovary. We also found evidence that T(3) decreased pituitary gonadotropin expression and decreased transcript for gonadal aromatase. Collectively, it appears that T(3) acts to diminish estrogen signaling by (1) decreasing pituitary LH expression and thus steroidogenesis, (2) down-regulating gonadal aromatase expression and thus decreasing estrogen synthesis from androgens, and (3) decreasing sensitivity to estrogen by down-regulating the ER subtypes. Goldfish are seasonal breeders, spawning once a year, and thus have two distinct periods of growth: somatic and reproductive. Circulating thyroid hormone levels have been found to increase just after spawning. Therefore, we propose that this may be an endocrine mechanism that goldfish use to switch their energy expenditure from reproductive to growth efforts in the goldfish.

Neither heat pulse, nor multigenerational exposure to a modest increase in water temperature, alters the susceptibility of Guadeloupean Biomphalaria glabrata to Schistosoma mansoni infection.

There are increasing concerns regarding the role global climate change will have on many vector-borne diseases. Both mathematical models and laboratory experiments suggest that schistosomiasis risk may change as a result of the effects of increasing temperatures on the planorbid snails that host schistosomes. Heat pulse/heat shock of the BS90 strain of Biomphalaria glabrata was shown to increase the rate of infection by Schistosoma mansoni, but the result was not replicable in a follow up experiment by a different lab. We characterised the susceptibility and cercarial shedding of Guadeloupean B. glabrata after infection with S. mansoni under two temperature regimes: multigenerational exposure to small increases in temperature, and extreme heat pulse events. Neither long-term, multigenerational rearing at elevated temperatures, nor transient heat pulse modified the susceptibility of Guadeloupean B. glabrata to infection (prevalence) or shedding of schistosome cercaria (intensity of infection). These findings suggest that heat pulse-induced susceptibility in snail hosts may be dependent on the strain of the snail and/or schistosome, or on some as-yet unidentified environmental co-factor.

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.

The behavioral effects of antibiotic treatment on the snail Biomphalaria glabrata.

Schistosomiasis is a detrimental neglected tropical disease that is transmitted by Planorbid snails. Understanding the transmission and control of this disease requires an extensive understanding of these intermediate hosts, which is only achieved by the effective rearing and study of species such as Biomphalaria glabrata. This species is the intermediate host for Schistosoma mansoni in the New World, and is also the main model for studying schistosomes in mollusks. Antibiotics are used routinely in B. glabrata tissue culture, and occasionally on live snails. Here we show that standard doses of three common antibiotics (penicillin, streptomycin and gentamicin) drastically diminish the activity of healthy B. glabrata, but that treated snails recover rapidly when placed in fresh water. Ampicillin treated snails did not show altered activity. We suggest that researchers keep these apparent toxicities in mind if a need for antibiotic treatment of live Planorbid snails arises.

Schistosome infectivity in the snail, Biomphalaria glabrata, is partially dependent on the expression of Grctm6, a Guadeloupe Resistance Complex protein.

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis.