The photopigment in the human eye that transduces light for circadian and neuroendocrine regulation, is unknown. The aim of this study was to establish an action spectrum for lightinduced melatonin suppression that could help elucidate the ocular photoreceptor system for regulating the human pineal gland. Subjects (37 females, 35 males, mean age of 24.5 Ϯ 0.3 years) were healthy and had normal color vision. Full-field, monochromatic light exposures took place between 2:00 and 3:30 A.M. while subjects' pupils were dilated. Blood samples collected before and after light exposures were quantified for melatonin. Each subject was tested with at least seven different irradiances of one wavelength with a minimum of 1 week between each nighttime exposure. Nighttime melatonin suppression tests (n ϭ 627) were completed with wavelengths from 420 to 600 nm. The data were fit to eight univariant, sigmoidal fluence-response curves (R 2 ϭ 0.81-0.95). The action spectrum constructed from these data fit an opsin template (R 2 ϭ 0.91), which identifies 446-477 nm as the most potent wavelength region providing circadian input for regulating melatonin secretion. The results suggest that, in humans, a single photopigment may be primarily responsible for melatonin suppression, and its peak absorbance appears to be distinct from that of rod and cone cell photopigments for vision. The data also suggest that this new photopigment is retinaldehyde based. These findings suggest that there is a novel opsin photopigment in the human eye that mediates circadian photoreception.

To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1β secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

In nature, Legionella pneumophila replicates exclusively as an intracellular parasite of amoebae, but it also persists in the environment as a free-living microbe. Studies of how this opportunistic pathogen recognizes and responds to distinct extracellular and intracellular environments identified a link between the growth phase and expression of traits previously correlated with virulence. When cultured in broth, only post-exponential-phase L. pneumophila was sodium sensitive, cytotoxic, osmotically resistant, competent to evade macrophage lysosomes, infectious, and motile. Likewise, the L. pneumophila phenotype changed during growth in macrophages. During the intracellular replication period, this bacterium was sodium resistant and lacked flagella; concomitant with macrophage lysis, L. pneumophilabecame sodium sensitive and flagellated. Expression of the virulent phenotype was a response to starvation, since exponential-phaseL. pneumophila became cytotoxic, sodium sensitive, and motile after incubation in broth from stationary-phase cultures, except when it was supplemented with amino acids. Together, these data indicate that while nutrients are plentiful, intracellularL. pneumophila organisms are dedicated to replication; when amino acids become limiting, the progeny express virulence factors to escape the spent host, to disperse and survive in the aquatic environment, and to reestablish a protected intracellular niche favorable for growth.

The circadian and neurobehavioral effects of light are primarily mediated by a retinal ganglion cell photoreceptor in the mammalian eye containing the photopigment melanopsin. Nine action spectrum studies using rodents, monkeys, and humans for these responses indicate peak sensitivities in the blue region of the visible spectrum ranging from 459 to 484 nm, with some disagreement in short-wavelength sensitivity of the spectrum. The aim of this work was to quantify the sensitivity of human volunteers to monochromatic 420-nm light for plasma melatonin suppression. Adult female (n = 14) and male (n = 12) subjects participated in 2 studies, each employing a within-subjects design. In a fluence-response study, subjects (n = 8) were tested with 8 light irradiances at 420 nm ranging over a 4-log unit photon density range of 1010 to 1014 photons/cm 2/sec and 1 dark exposure control night. In the other study, subjects (n = 18) completed an experiment comparing melatonin suppression with equal photon doses (1.21 × 1013 photons/cm2/sec) of 420 nm and 460 nm monochromatic light and a dark exposure control night. The first study demonstrated a clear fluence-response relationship between 420-nm light and melatonin suppression (p < 0.001) with a half-saturation constant of 2.74 × 1011 photons/cm2/sec. The second study showed that 460-nm light is significantly stronger than 420-nm light for suppressing melatonin (p < 0.04). Together, the results clarify the visible short-wavelength sensitivity of the human melatonin suppression action spectrum. This basic physiological finding may be useful for optimizing lighting for therapeutic and other applications.

Macrophages are the guardians of the innate immune system, recognizing a broad array of pathogen-associated molecular patterns (PAMPs) to initiate immediate defenses and to recruit the adaptive branch of the immune system. Toll-like receptors (TLRs) detect extracellular microbial products, such as lipopolysaccharide, peptidoglycan, lipotechoic acid, and fl agellin (1), whereas surveillance of the cytosol is the task of nucleotide-binding oligomerization domain (NOD) leucine-rich repeat (LRR) proteins. The best-characterized members of the NOD-LRR family are NOD1 and NOD2, which recognize distinct elements of bacterial cell wall peptidoglycan in the cytosol to mount or modulate a proinfl ammatory immune response or to promote apoptosis (2). In mouse macrophages, the NOD-LRR protein Naip5 (Birc1e) restricts intracellular replication of the opportunistic human pathogen Legionella pneumophila (3-5). Naip5 is comprised of three modules: NH 2-terminal baculoviral inhibitor of apoptosis repeats, a central NOD domain, and COOH-terminal LRRs (2). By analogy to other NOD-LRR proteins, the LRR region is thought to recognize microbial products, triggering oligomerization via the NOD domain and activation of a cellular response that is governed by various NH 2terminal eff ector-binding domains (2). Whereas virtually all mice are resistant to L. pneumophila, the A/J strain encodes a naip5 allele that confers susceptibility to infection (3). Whether the

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When microbes contaminate the macrophage cytoplasm, leukocytes undergo a proinflammatory death that is initiated by nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) that bind and activate caspase-1. We report that these inflammasome components also regulate autophagy, a vesicular pathway to eliminate cytosolic debris. In response to infection with flagellate Legionella pneumophila, C57BL/6J mouse macrophages equipped with caspase-1 and the NLR proteins NAIP5 and NLRC4 stimulated autophagosome turnover. A second trigger of inflammasome assembly, K+ efflux, also rapidly activated autophagy in macrophages that produced caspase-1. Autophagy protects infected macrophages from pyroptosis, since caspase-1-dependent cell death occurred more frequently when autophagy was dampened pharmacologically by either 3-methyladenine or an inhibitor of the Atg4 protease. Accordingly, in addition to coordinating pyroptosis, both (pro-) caspase-1 protein and NLR components of inflammasomes equip macrophages to recruit autophagy, a disposal pathway that raises the threshold of contaminants necessary to trigger proinflammatory leukocyte death.IMPORTANCE An exciting development in the innate-immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Nutrient deprivation has long been known to induce autophagy; how infection triggers this disposal pathway is an active area of research. Autophagy is encountered by many of the intracellular pathogens that are known to trigger pyroptosis, an inflammatory cell death initiated when nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) activate caspase-1 within inflammasome complexes. Therefore, we tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. By exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, we demonstrate for the first time that, when confronted with cytosolic contamination, primary mouse macrophages rely not only on the NLR proteins NAIP5 and NLRC4 but also on (pro-)caspase-1 protein to mount a rapid autophagic response that wards off proinflammatory cell death.

The basic goal of this research is to determine the best combination of light wavelengths for use as a lighting countermeasure for circadian and sleep disruption during space exploration, as well as for individuals living on Earth. Action spectra employing monochromatic light and selected monochromatic wavelength comparisons have shown that short‐wavelength visible light in the blue‐appearing portion of the spectrum is most potent for neuroendocrine, circadian, and neurobehavioral regulation. The studies presented here tested the hypothesis that broad spectrum, polychromatic fluorescent light enriched in the short‐wavelength portion of the visible spectrum is more potent for pineal melatonin suppression in healthy men and women. A total of 24 subjects were tested across three separate experiments. Each experiment used a within‐subjects study design that tested eight volunteers to establish the full‐range fluence–response relationship between corneal light irradiance and nocturnal plasma melatonin suppression. Each experiment tested one of the three types of fluorescent lamps that differed in their relative emission of light in the short‐wavelength end of the visible spectrum between 400 and 500 nm. A hazard analysis, based on national and international eye safety criteria, determined that all light exposures used in this study were safe. Each fluence–response curve demonstrated that increasing corneal irradiances of light evoked progressively increasing suppression of nocturnal melatonin. Comparison of these fluence–response curves supports the hypothesis that polychromatic fluorescent light is more potent for melatonin regulation when enriched in the short‐wavelength spectrum.