We validated the bioinformatic predictions for N. brasiliensis by quantitative real-time PCR and mass spectrometry and showed that N. brasiliensis produces endocannabinoids at all life cycle stages. Taken together, these studies show for the first time the production of endocannabinoids by parasitic helminths and suggest that helminth infection-induced endocannabinoids functionally influence the host immune response and parasite burden. These findings support a new area of investigation into the function of the endocannabinoid system in infectious diseases.We examined if N. brasiliensis-induced endocannabinoids were associated with health outcomes for the host or parasite by correlative analyses between endocannabinoid levels and infection-induced weight loss or parasite egg burdens. N. brasiliensis infectious larval stage 3 helminths migrate to the lung, where they develop into L4, followed by infection of the small intestine, where they develop into adults and produce eggs . At an infectious dose of 500 L3 helminths, N. brasiliensis infection of the lung, which occurs between days 1 and 3 postinfection, causes lung hemorrhaging and inflammation likely due to the physical damage of the worm burrowing through the lung tissue. During this acute infection of the lung, we observed significant weight loss that was remarkably resolved once the N. brasiliensis parasites were established in the intestine, at day 6 . Consistent with this weight loss, analysis of feeding patterns in naive and infected mice revealed reduced food intake and motor activity at day 1 postinfection,indoor grow shelves when the N. brasiliensis parasites had reached the lung, that was also resolved by day 6 postinfection .
We also observed a significant positive correlation between N. brasiliensis-induced acute weight loss at day 3 and N. brasiliensis egg burdens at day 7 post infection . This correlation suggests that acute infection-induced weight loss at day 3 may be a good predictor of subsequent parasite establishment in the intestine. The effect of N. brasiliensis infection on mouse weight changes may be due to lung tissue inflammation or changes in mouse feeding behavior. Given that endocannabinoids can regulate both these processes, we investigated correlations between endocannabinoids and N. brasiliensis parasite burdens. We observed that 2-AG intestinal levels from day 7 infected mice were negatively correlated with early infection-induced weight loss and day 7 parasite egg burden . To comprehensively define the relationship between endocannabinoids versus host and parasite fitness, we performed Spearman correlation analyses across all experiments . We observed a negative correlation between day 7 infected jejunum 2-AG and OEA and early infection-induced weight changes and day 7 parasite egg burden. In contrast, we observed a positive correlation between plasma 2-AG and OEA, weight loss, and parasite egg burden. These data indicate that high endocannabinoid levels locally in the intestine are associated with reduced early infection-induced weight loss and decreased parasite egg burden.In this study, we investigated the endocannabinoid signaling system following infection with N. brasiliensis. While recognized for its critical function in the central and enteric nervous systems, the endocannabinoid system is also activated by and can influence inflammatory immune responses . For example, 2-AG and AEA are anti-inflammatory, which has provided the basis for the potential therapeutic use of synthetic cannabinoids, or cannabis, in autoimmune or inflammatory diseases . Oral treatment of mice with AEA promoted a tolerogenic immune response and regulatory macrophages in the intestine that were protective in a nonobese diabetic model .
Despite an immune function in the intestine, the functional significance of endocannabinoids in intestinal parasite infection has not been examined. Our finding that helminth infection triggers significant endocannabinoid synthesis that is correlated with both host health outcomes and parasite fecundity suggests that endocannabinoids may be an important player in host-helminth dynamics. Inhibition of CB1R led to decreased expression of the Th2 cytokine IL-5 in the intestine and IL-4, IL-5, and IL-10 in the spleen but no difference in the Th1 cytokine IFN- . This was associated with increased parasite egg and worm burdens, suggesting that CB1R signaling may be important for the optimal host immune response to keep helminth burdens in check. Endocannabinoids also signal through CB2R ; however, CB2R antagonist treatment did not significantly change cytokine responses or helminth burdens. CB1R and CB2R inhibition was conducted during a short time frame, days 4 to 6 postinfection, and we cannot confirm complete abrogation of CB1R or CB2R signaling in the intestine with this treatment regime. Future studies with earlier treatment regimes or CB1R/CB2R-deficient mice may delineate functional differences between these signaling pathways in helminth infection. Cannabinoid CB1Rs are expressed in cholecystokinin -positive enteroendocrine cells in the duodenum of mice and ghrelin-expressing cells of the stomach of rats , and N. brasiliensis infection in ratsis associated with elevations in circulating levels of CCK . Thus, it is possible that helminth infection may induce changes in feeding behavior, as seen in Fig. 2, by a mechanism that includes endocannabinoid-mediated changes in the production and/or release of peptides important for feeding behavior, including CCK. We observed significant correlations between endocannabinoid levels, infection-induced weight loss, and parasite burdens.
However, further functional studies are necessary to determine causal relationships between these multiple parameters and the contribution, if any, of infection-induced endocannabinoids to feeding behavior. In addition to host endocannabinoid expression, we show for the first time that N. brasiliensis produces endocannabinoids and that genes encoding endocannabinoid biosynthetic and degradative enzymes are present in the genomes of multiple parasitic nematodes, including some of the most common helminth parasites of humans. One proposed strategy by which parasites modulate host immunity is by releasing molecules that are already native within the host, or at least native-like molecules . For example, A. suum and T. canis have been shown to synthesize morphine or morphinelike substrates, and morphine is a known immunomodulator . It is well recognized that the endocannabinoid system is conserved in a diverse variety of vertebrates, including pythons and goldfish ; however, whether it evolved earlier and is functional in more primitive eukaryotic organisms is less well understood . We observed that N. brasiliensis has predicted genes encoding the NAPE-PLD and FAAH enzymes that catalyze endocannabinoid synthesis and breakdown. Moreover, mass spectrometry analysis revealed that infectious L3 N. brasiliensis produced extremely high levels of AEA, reaching 100 to 1,000 times that found in tissue or blood of mice. A recent study showed that truffles, the fruiting bodies of fungi, produce AEA potentially as an attractant and feeding stimulant for animals to ensure its dissemination . Given that AEA is anti-inflammatory,indoor garden table high-level synthesis at the infectious stage may also function to dampen the host immune response. It is possible that in addition to endocannabinoids, N. brasiliensis may produce and release other signaling molecules endogenous to the host, such as morphine, and that there is overlap in the biological effects of these molecules on host immunity or behavior. Given the difference in mass between N. brasiliensis and the host, the endocannabinoids detected in the infected mice are likely host derived. However, it is possible that N. brasiliensisderived endocannabinoids may functionally impact the host at the cellular level. Future studies are necessary to test these hypotheses. Inhibition of CB1R signaling significantly increased N. brasiliensis egg burden; however, whether the functional effect was through influencing the host or alteration of the endocannabinoid system in N. brasiliensis is unclear. In the host, the increased N. brasiliensis burdens when CB1R signaling is inhibited could be due to the altered immune response or to reduced intestinal motility. Indeed, immune cells express both CB1 and CB2 receptors and intestinal epithelial cells express CB1R . Whether the CB1R inhibitor-mediated increase in N. brasiliensis burden was through direct effects on immune cells, effects on the intestinal epithelial cells, or a combination of such effects is unclear and would require further studies. In addition to regulating immune responses, the endocannabinoid system throughout the gastrointestinal tract plays a variety of physiological roles, including the control of motility, immune function, mucosal barrier function, and feeding behavior . Although outside the scope of the present study, future studies should include an evaluation of the impact that endocannabinoid-induced changes in intestinal motility have on egg burden. Furthermore, it is possible that endocannabinoid metabolism and/or release may be secondarily affected by perturbations in physiological responses governed by endocannabinoids themselves.
It is also possible that CB1R inhibition could directly affect N. brasiliensis. Although the N. brasiliensis genome does not have canonical cannabinoid receptor genes, genes encoding endocannabinoid degradative enzymes are present in the N. brasiliensis genome, suggesting that N. brasiliensis may also respond to the endocannabinoids it produces through an as-yetun identified receptor. Consistent with this, C. elegans produces and responds to cannabinoids through NPR-19 . Inhibiting CB1R signaling was ultimately bene- ficial to N. brasiliensis, leading to improved parasite fertility. Why N. brasiliensis would produce endocannabinoids that adversely affect its fertility is unclear at present. Our data, however, support a functional impact of the host and parasite endocannabinoid system and suggest that further studies delineating the beneficial or detrimental function of endocannabinoids in the host versus the helminth are warranted. For example, the timing of cannabinoid receptor signaling inhibition may be critical. In our studies, we inhibited CB1R signaling after adult parasite establishment in the intestine; however, N. brasiliensis produces the most AEA during the initial infection, possibly as an anti-inflammatory mechanism to downregulate the host immune response. N. brasiliensis-derived AEA may also prevent excessive host inflammation that could lead to host mortality, which would be an equally adverse outcome for the helminth. Notwithstanding this, our discovery of parasite-derived endocannabinoids implicates the endocannabinoid system as a primitive pathway that contributes to host-pathogen interaction and suggests that investigation of the existence of the endocannabinoid system in other pathogens is warranted. The complexity of host-helminth interaction and the numerous factors that influence the health outcomes for the parasite and the host are increasingly recognized. In addition to an optimized Th2 response for parasite expulsion, parasitic helminths trigger a multitude of non-immune pathways that affect physiological processes, such as feeding and metabolism, but can also influence the immune response . Our findings suggest that the endocannabinoid system is a previously unrecognized contributor to this dynamic process and may therefore have a significant impact on the host’s health outcome beyond parasite expulsion.Heavy, chronic methamphetamine exposure is associated with central nervous system injury and neurocognitive deficits . Human studies in abstinent users have described deficits in executive function, attention, learning and memory, information processing speed, and motor skills . Functions localized to the prefrontal cortex and frontostriatal connections may be especially vulnerable to METH effects . The PFC plays a critical role in decision-making and inhibitory control , with DA as the major neurotransmitter implicated in the evaluation of rewards, maintenance of addictive behaviors, and differences cognitive function . Although METH-associated CNS injury is evident, METH exposure parameters often do not inform the degree of impairment seen among people with a history of METH dependence . This suggests individual differences invulnerability to the effects of METH, which may result from a combination of environmental and genetic factors. Examining genetic variability may offer insight as to how individual differences contribute to risk for cognitive dysfunction in chronic METH use. Mechanisms of METH-related injury include alterations in dopamine , serotonin, GABA and glutamate systems . METH principally modulates DA neurotransmission and increases extracellular DA concentrations by a number of means, which include stimulating DA release and inhibiting reuptake via the DA transporter . In addition to dopaminergic activity in the synapse, an important mechanism of DA-related METH neurotoxicity may occur at the receptor level; for example, a recent study shows that phasic METH-indueed DA release impacts D1 DA receptor availability which is negatively associated with cortical thickness . While DA is critical for cognitive function, overexposure to DA in the synapse caused by stimulant exposure likely plays a role in neural compromise, including damage to DA terminals, microvascular injury, and structural and functional abnormalities on neuroimaging . Thus, regulatory mechanisms that assist in removing DA from the synapse, play an important role in DA homeostasis in the brain . Catechol-O-methyltransferase , COMT accounts for more than 60% of the metabolic degradation of released DA in the PFC . A single nucleotide polymorphism of COMT involves a Val to Met amino acid substitution at codon 158 in the membrane-bound COMT .