news and views dormancy and activation that has remained largely unexplored. Given the long-term natural temporal patterns of true relapse infections in P. cynomolgi and P. vivax, hypnozoite reactivation will probably involve a complex set of genetic, epigenetic and environmental cues that are still waiting to be dissected11. Given that in vitro hypnozoite activation seems to be continuous or frequent at a high rate after 8 days, whereas in vivo relapses are less frequent, stretching over greater time frames12, it will be important to compare the in vitro observations shown by the authors with the biology of

P. cynomolgi hypnozoites in livers from infected monkeys. Despite these current caveats and open questions, the work of Dembélé et al.8 will undoubtedly aid the development of similar long-term culture systems for P. vivax liver stage forms and contribute to the elimination efforts against this human parasite. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. White, N.J. & Imwong, M. Adv. Parasitol. 80, 113–150 (2012).

2. Wells, T.N., Burrows, J.N. & Baird, J.K. Trends Parasitol. 26, 145–151 (2010). 3. Baird, K.J., Maguire, J.D. & Price, R.N. Adv. Parasitol. 80, 203–270 (2012). 4. Noulin, F. et al. Trends Parasitol. 29, 286–294 (2013). 5. Mazier, D. et al. Nature 307, 367–369 (1984). 6. March, S. et al. Cell Host Microbe 14, 104–115 (2013). 7. Galinski, M.R., Meyer, E.V. & Barnwell, J.W. Adv. Parasitol. 81, 1–26 (2013). 8. Dembélé, L. et al. Nat. Med. 20, 307–312 (2014). 9. Mota, M.M. et al. Science 291, 141–144 (2001). 10. Dembélé, L. et al. PLoS ONE 6, e18162 (2011). 11. Krotoski, W.A. Prog. Clin. Parasitol. 1, 1–19 (1989). 12. Schmidt, L.H. Am. J. Trop. Med. Hyg. 35, 1077–1099 (1986).

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Regulating innate immunity with dopamine and electroacupuncture Sangeeta S Chavan & Kevin J Tracey Neural circuits are able to modulate immune responses by detecting inflammatory mediators and relaying signals back to the immune system. Here, in a mouse model of sepsis, the authors show that the immune responses can be modulated by electroacupuncture, which stimulates a neural circuit that results in the release of dopamine. The mechanism, like the inflammatory reflex, is neither sympathetic nor parasympathetic. Their results show a potential way forward in developing therapies for sepsis in dopamine agonists (pages 291–295). Recently mapped peripheral neural circuits sense the status of cytokines and other inflammatory mediators and relay that information to the central nervous system. Stimulation of the sensory neural signals mediates activation of the outgoing (efferent) neural signals that return to the immune system to control immunity in real time. These neural-immune reflexes have been implicated in modulating disease severity, as experimental activation of these neural immune reflex circuits attenuates inflammation. Now, in this issue, Torres-Rosas and colleagues1 report that activation of a neural-immune circuit using electroacupuncture attenuates inflammation. Using mouse models of sepsis, the investigators observed that activation of the sensory sciatic nerve by electroacupuncture in the leg inhibits cytokine release and improves survival. Further, the investigators mapped the reflex circuit to the vagus nerve (named for its wandering course to the visceral organs), a major sensory and motor conduit that innervates and controls the physiological functions of the reticuloenSangeeta S. Chavan and Kevin J. Tracey are at the Feinstein Institute for Medical Research, Laboratory of Biomedical Science, Manhasset, New York, USA. e-mail: [email protected] or [email protected]

dothelial system, liver, lung, spleen, kidneys and gut. This gives direct evidence for another vagus nerve circuit among the growing list of recently discovered circuits that mediate antiinflammatory effects. Physiological systems in mammals are regulated by the autonomic nervous system that has two principal divisions, the sympathetic pathway and the parasympathetic pathway. Unique anatomical and functional features distinguish the sympathetic and parasympathetic pathways. In general, sympathetic pathways mediate physiological responses, such as increased heart rate and blood pressure and mobilization of energy stores via catecholaminergic neurotransmitters. In contrast, parasympathetic pathways produce effects that oppose sympathetic actions, such as decreased heart rate and cardiac contractility and enhanced digestive functions, via cholinergic neurotransmitters. Today, the convergence of advanced imaging technologies, neural and cellular identification methods, molecular biology and optogenetics has revealed neural circuits that regulate immunity but cannot be neatly classified as either sympathetic or parasympathetic. The activity of these reflex circuits is initiated by sensory neural signals in response to bacterial products, cytokines or other inflammatory mediators. For example, seminal

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observations by Linda Watkins and her colleagues2 first demonstrated that fever in rodents after intra-abdominal administration of the cytokine IL1 required an intact vagus nerve (Fig. 1a). Stimulation of the sensory arc by these inflammatory mediators activates motor neural signals that return to the immune system to control its function by signaling through efferent neurons or the hypothalamic-pituitary adrenal (HPA) axis. Activation of the HPA via neuronal input results in glucocorticoid release, which controls peripheral immune responses. These neural reflexes are essential for maintaining balanced immune responses during infection and injury, termed ‘immunological homeostasis’ (Fig. 1). A prototypical neural circuit, the ‘inflammatory reflex’ is defined by cholinergic and adrenergic neurons, splenic T cells and splenic macrophages (Fig. 1e). Accordingly, as we and others have noted, this circuit is neither sympathetic nor parasympathetic4. The efferent action potentials traveling down the vagus nerve culminate in the celiac ganglion, the site of origin of adrenergic splenic nerve. This nerve regulates acetylcholine release by a subset of splenic T cells5,6. Bacterial pathogens can directly activate sensory neurons independently of innate immune cells. These sensory neurons can activate axon-axon reflexes that 239

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news and views Figure 1 Neural reflex immunity circuits. (a–d) Sensory (afferent) neural signals are activated by a stimulus (in this case either by inflammatory mediators (a) or bacteria (b) or acupuncture (c) or pressure (d), respectively). Sensory signals project to the interneurons in the brainstem that lead to outgoing (efferent) neural signals. The precise nature of this interaction is unknown (black box). The outgoing signals generated in the brainstem suppress innate immune responses and inflammation either via the vagus nerve to the adrenal medulla, resulting in the release of dopamine, as shown in this study by Torres-Rosas and colleagues1 (c); via the adrenergic nerve to the blood vessel near the fifth lumbar cord (gateway reflex, d); or via the vagus nerve to the celiac ganglion to the spleen to acetylcholine-producing (ChAT+) T cells (inflammatory reflex, e) or they induce the local axon-axon reflex (b). Outgoing signals are also relayed to the nuclei controlling the function of the HPA axis, resulting in increased glucocorticoid hormone release by the adrenal gland, which suppresses innate immune responses. Acupuncture stimulation, as shown by Torres-Rosas et al.1 and represented in c, could represent a possible means for modulating these circuits, in particular in the modulation of sepsis.

locally modulate the onset of innate immune cell activation, recruitment, cytokine release and inflammation and hence bypass the above circuit (Fig. 1b). Now, Torres-Rosas and colleagues1 have mapped another discrete immunoregulatory neural circuit, which we note is neither fully sympathetic nor parasympathetic (Fig. 1c). Studying a mouse model of sepsis, they observed that electroacupuncture at the sciatic nerve inhibited cytokine production in a voltagedependent manner. By surgical sectioning of the sciatic and vagus nerves, they discerned a new sciatic-to-vagus nerve circuit that regulates the innate immune response. Signals arising in the sciatic nerve culminate on efferent vagus nerve signals. The efferent vagus nerve signals terminate on the release of dopamine in the adrenal medulla. Dopamine induced by sciatic nerve stimulation targets dopaminergic type 1 (D1) receptors and suppresses systemic inflammation and improves survival in septic animals. This demonstrates that vagus nerve–mediated dopamine release contributes to the control of immunological homeostasis. The precise route by which sensory signals elicited from electroacupuncture transits the brainstem to activate outgoing vagus signals remains unknown, although there may be analogy to a third neural immune reflex circuit, termed the ‘gateway reflex’, that regulates T-cell recruitment into the central nervous system7 (Fig. 1d). In this circuit, sensory signals arise in the hind limb, are transmitted to the spinal cord and brain stem and then descend in the sympathetic chain to be relayed via adrenergic neurons 240

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that terminate on endothelial cells to regulate expression of chemokine receptors. This study by Torres-Rosas et al.1 and other recent studies focus attention on the possibility of using advances in neurophysiology, molecular biology and cellular biology to provide refined maps of the neural-immune reflex circuits that regulate immunity. These modern tools and techniques add precision beyond the generalized or simplistic characterizations of sympathetic or parasympathetic. For example, in the present study, the sensory sciatic nerve signals activate efferent vagus nerve signals, but these findings do not exclude the possibility that other central nervous system signals to the immune system are also activated, including signals to other organs, descending signals in the sympathetic chain and humoral mediators via the HPA axis. Henry Dale, who, early in the twentieth century, isolated the first neurotransmitter, acetylcholine, from spleen, cautioned against using nomenclature such as sympathetic or parasympathetic to describe neural circuits. Indeed, the prototypical

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neural-immune reflex identified more than a decade ago is dependent upon the vagus nerve (a classic parasympathetic nerve), the splenic nerve (a classic sympathetic nerve) and acetylcholine released by splenic T cells3,6. As Dale predicted, generic terms such as sympathetic and parasympathetic are at best imprecise and at worst incorrect to describe these immunoregulatory circuits. The identification of another neuralimmune reflex circuit by Torres-Rosas and colleagues1 is timely. From a pharmacological perspective, their findings suggest that it may be possible to target dopaminergic receptors as anti-inflammatory therapeutics. In addition, it may be possible to stimulate the sensory sciatic nerve to mediate

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news and views anti-inflammatory efferent vagus nerve signals. In all likelihood, Henry Dale would be pleased to learn about the specificity of these neural circuits as uniquely defined by their physiology and function.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Torres-Rosas et al. Nat. Med. 20, 291–295 (2014). 2. Watkins, L.R. et al. Neurosci. Lett. 183, 27–31 (1995).

3. Tracey, K.J. Nature 420, 853–859 (2002). 4. Andersson, U. & Tracey, K.J. Annu. Rev. Immunol. 30, 313–335 (2012). 5. Rosas-Ballina, M. et al. Proc. Natl. Acad. Sci. USA 105, 11008–11013 (2008). 6. Rosas-Ballina, M. et al. Science 334, 98–101 (2011). 7. Arima, Y. et al. Cell 148, 447–457 (2012).

One shot forward for HIV prevention Lawrence Corey & M Juliana McElrath

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The expression of antibodies to protect against an infectious disease can be achieved by the injection into the host of vectors carrying the gene to the relevant antibodies. Here the authors demonstrate the applicability of this technique to protection from HIV in a humanized mouse model, showing this to be a valid route to pursue in vaccine development for humans (pages 296–300). Persistent levels of functional antibodies have been the mainstay for preventing the acquisition of viral infections. This is most economically achieved by administration of an immunogen that induces neutralizing antibodies (active vaccination); however, direct infusion of antibodies themselves (passive immunoprophylaxis) has also proven to be effective, especially for rapid protection from a defined exposure to an infectious agent such as hepatitis A or B or respiratory syncytial virus infection in infants. Technological advances over the last five years have markedly increased the identification and characterization of epitope specificities of naturally occurring, broadly neutralizing antibodies to HIV-1 (ref. 1). Molecular procedures to insert the genes of neutralizing antibodies into a persistent viral vector such as recombinant adeno-associated virus (AAV) have been developed2,3. Upon injection into the muscle of animals, the vectors transduce cells to continuously secrete the expressed antibodies into serum (vectored immunoprophylaxis). The ability of the AAV vector to persist episomally in muscle cells provides the potential for prolonged secretion of antibodies. One important question for the HIV-1 vaccine field is whether the expressed neutralizing antibodies will diffuse to relevant mucosal epithelial surfaces in concentrations that are sufficiently high and durable enough to prevent against sexual acquisition of HIV to mediate protection from infection. The article by Balazs et al.4 in this issue illustrates the potential of this approach in protecting against mucosal HIV-1 infection. Although these

Lawrence Corey and M. Juliana McElrath are at the Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. e-mail: [email protected] or [email protected]

findings provide a critical step forward, they also raise concerns that even vectored immunoprophylaxis may, by itself, be an imperfect modality for HIV prevention. The investigators used a humanized boneliver-thymus mouse model of female immunodeficient NOD/SCID/γc mice implanted with human liver and thymus tissue and transplanted with autologous human fetal liver CD34+ stem cells (BLT mice). These were each administered a single dose of an AAV vector expressing one of a wide variety of currently available broadly targeted neutralizing antibodies with known epitope specificities4. The majority of antibodies protected against intravenous HIV-1 challenge in the humanized mouse model at concentrations as low as 350 ng/ml. More importantly, given that HIV is sexually transmitted, one potent, broadly neutralizing antibody to the CD4 binding region, called VRC-07, was consistently detected in the vaginal fluid of the mice at a thousandfold lower concentration than in serum. To see whether this concentration of antibody was high enough to protect against infection, a repeated low-dose intravaginal viral challenge designed to mimic HIV-1 exposure and acquisition in humans was given to the mice (Fig. 1). The VRC-07 anti–HIV-1 neutralizing IgG in the mucosa seemed high enough to reduce experimental HIV-1 challenge rates among vector-immunized animals by 62.5%. One of the vagaries of this mouse model is that despite homogeneity in genetics of the animals and the HIV-1 inoculation source, there was great variability in the number of HIV-1 inoculations required to infect the control animals. Whether variations in the extent and persistence of engraftment of the BLThumanized mice contribute to the variability in exposures needed for infection is unclear. More sobering data showed that even among

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mice from whom protection from challenge was achieved, as measured by serial measurements of plasma HIV, localized replication in the genital tract post challenge was detected, suggesting that sterilizing immunity was not universally achieved. Furthermore, there was detectable depletion of CD4+ T cells in the spleen and gut, indicating that systematic spread of HIV-1 still occurred in animals, despite the presence of neutralizing antibodies in the mucosa at the time of challenge. In addition, escape HIV-1 envelope variants were detected soon after infection, despite challenge with a more homogeneous virus inoculum than likely to occur in humans susceptible to mucosal exposures. For nearly two decades, great interest has developed in the use of AAV vectors for gene therapy and more recently for immunoprophylaxis against infectious diseases5. The appeal of these vectors is their ability to transduce cells, both dividing and nondividing, and their apparent lack of pathogenicity in humans6. However, pre-existing immunity from natural AAV infections with various AAV serotypes is prevalent, and the resulting extent of AAV antibody cross-reactivity may have an impact on the efficacy of HIV-1 antibody delivery and distribution following AAV-vectored immunoprophylaxis in humans, particularly in developing countries. Moreover, the high AAV vector particle doses administered (109–1011 genome copies) in the mice reported in Balazs et al.4 to achieve in vivo protection against HIV-1 may be challenging in humans if even higher doses are needed to achieve protective antibody levels. In time, these barriers may be overcome with use of less common AAV serotypes and improvements in transduction efficiencies. Although the development of a vaccine regimen that induces immune responses including potent, broadly reactive neutralizing antibodies 241