FlgM is required to evade NLRC4-mediated host protection against flagellated Salmonella.

Salmonella enterica serovar Typhimurium is a leading cause of gastroenteritis worldwide and a deadly pathogen in children, immunocompromised patients, and the elderly. Salmonella induces innate immune responses through the NLRC4 inflammasome, which has been demonstrated to have distinct roles during systemic and mucosal detections of flagellin and non-flagellin molecules. We hypothesized that NLRC4 recognition of Salmonella flagellin is the dominant protective pathway during infection. To test this hypothesis, we used wild-type, flagellin-deficient, and flagellin-overproducing Salmonella to establish the role of flagellin in mediating NLRC4-dependent host resistance during systemic and mucosal infections in mice. We observed that during the systemic phase of infection, Salmonella efficiently evades NLRC4-mediated innate immunity. During mucosal Salmonella infection, flagellin recognition by the NLRC4 inflammasome pathway is the dominant mediator of protective innate immunity. Deletion of flgM results in constitutive expression of flagellin and severely limits systemic and mucosal Salmonella infections in an NLRC4 inflammasome-dependent manner. These data establish that recognition of Salmonella's flagellin by the NLRC4 inflammasome during mucosal infection is the dominant innate protective pathway for host resistance against the enteric pathogen and that FlgM-mediated evasion of the NLRC4 inflammasome enhances virulence and intestinal tissue destruction.

Polyphosphate Functions In Vivo as an Iron Chelator and Fenton Reaction Inhibitor.

Maintaining cellular iron homeostasis is critical for organismal survival. Whereas iron depletion negatively affects the many metabolic pathways that depend on the activity of iron-containing enzymes, any excess of iron can cause the rapid formation of highly toxic reactive oxygen species (ROS) through Fenton chemistry. Although several cellular iron chelators have been identified, little is known about if and how organisms can prevent the Fenton reaction. By studying the effects of cisplatin, a commonly used anticancer drug and effective antimicrobial, we discovered that cisplatin elicits severe iron stress and oxidative DNA damage in bacteria. We found that both of these effects are successfully prevented by polyphosphate (polyP), an abundant polymer consisting solely of covalently linked inorganic phosphates. Subsequent in vitro and in vivo studies revealed that polyP provides a crucial iron reservoir under nonstress conditions and effectively complexes free iron and blocks ROS formation during iron stress. These results demonstrate that polyP, a universally conserved biomolecule, plays a hitherto unrecognized role as an iron chelator and an inhibitor of the Fenton reaction.IMPORTANCE How do organisms deal with free iron? On the one hand, iron is an essential metal that plays crucial structural and functional roles in many organisms. On the other hand, free iron is extremely toxic, particularly under aerobic conditions, where iron rapidly undergoes the Fenton reaction and produces highly reactive hydroxyl radicals. Our study now demonstrates that we have discovered one of the first physiologically relevant nonproteinaceous iron chelators and Fenton inhibitors. We found that polyphosphate, a highly conserved and ubiquitous inorganic polyanion, chelates iron and, through its multivalency, prevents the interaction of iron with peroxide and therefore the formation of hydroxyl radicals. We show that polyP provides a crucial iron reservoir for metalloproteins under nonstress conditions and effectively chelates free iron during iron stress. Importantly, polyP is present in all cells and organisms and hence is likely to take on this crucial function in both prokaryotic and eukaryotic cells.

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice.

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

Automated phenotyping and lifespan assessment of a C. elegans model of Parkinson's disease.

Phenotypic analysis of Caenorhabditis elegans has greatly advanced our understanding of the molecular mechanisms implicated in the aging process as well as in age-related pathologies. However, conventional high-resolution imaging methods and survival assays are labor-intensive and subject to operator-based variations and decreased reproducibility. Recent advances in microfluidics and automated flatbed scanner technologies have significantly improved experimentation by eliminating handling errors and increasing the sensitivity in measurements. Here, we introduce a medium-throughput microfluidic platform, which efficiently positions and immobilizes single worms through pressurization for high resolution imaging. Worms are sorted based on select imaging criteria, and subsequently transferred into multi-well plates for automated lifespan assessment. To illustrate the applicability of this method, we imaged α-synuclein deposits in a C. elegans model of Parkinson's Disease (PD). We found that age synchronized individuals expressing human α-synuclein vary greatly in the quantity and size of intracellular α-synuclein foci at early stages in life. Subsequent lifespan analysis of the individuals, however, did not reveal any correlation between the number or extent of α-synuclein deposits and subsequent lifespan. These studies suggest that the observed natural variations in α-synuclein deposits found in C. elegans models of PD do not originate from inherent differences in the fitness of the organism or contribute to alterations in lifespan.

Rapamycin persistently improves cardiac function in aged, male and female mice, even following cessation of treatment.

Even in healthy aging, cardiac morbidity and mortality increase with age in both mice and humans. These effects include a decline in diastolic function, left ventricular hypertrophy, metabolic substrate shifts, and alterations in the cardiac proteome. Previous work from our laboratory indicated that short-term (10-week) treatment with rapamycin, an mTORC1 inhibitor, improved measures of these age-related changes. In this report, we demonstrate that the rapamycin-dependent improvement of diastolic function is highly persistent, while decreases in both cardiac hypertrophy and passive stiffness are substantially persistent 8 weeks after cessation of an 8-week treatment of rapamycin in both male and female 22- to 24-month-old C57BL/6NIA mice. The proteomic and metabolomic abundance changes that occur after 8 weeks of rapamycin treatment have varying persistence after 8 further weeks without the drug. However, rapamycin did lead to a persistent increase in abundance of electron transport chain (ETC) complex components, most of which belonged to Complex I. Although ETC protein abundance and Complex I activity were each differentially affected in males and females, the ratio of Complex I activity to Complex I protein abundance was equally and persistently reduced after rapamycin treatment in both sexes. Thus, rapamycin treatment in the aged mice persistently improved diastolic function and myocardial stiffness, persistently altered the cardiac proteome in the absence of persistent metabolic changes, and led to persistent alterations in mitochondrial respiratory chain activity. These observations suggest that an optimal translational regimen for rapamycin therapy that promotes enhancement of healthspan may involve intermittent short-term treatments.

Measuring biological age in mice using differential mass spectrometry.

Aging is an ill-defined process that increases the risk of morbidity and mortality. Aging is also heterogeneous meaning that biological and chronological age can differ. Here, we used unbiased differential mass spectrometry to quantify thousands of proteins in mouse liver and select those that that consistently change in expression as mice age. A panel of 14 proteins from inbred C57BL/6 mice was used to equate chronological and biological age in this reference population, against which other mice could be compared. This "biological age calculator" identified two strains of f1 hybrid mice as biologically younger than inbred mice and progeroid mice as being biologically older. In an independent validation experiment, the calculator identified mice treated with rapamycin, known to extend lifespan of mice, as 18% younger than mice fed a placebo diet. This demonstrates that it is possible to measure subtle changes in biologic age in mammals using a proteomics approach.

Accumulation of Nucleolar Inorganic Polyphosphate Is a Cellular Response to Cisplatin-Induced Apoptosis.

The chemotherapeutic drug cisplatin, which targets DNA, serves as one of the main staples in cancer treatment. Yet, the therapeutic application of cisplatin is limited by two major challenges: the occurrence of reversible and irreversible side effects due to non-specific toxicity, and the intrinsic or developing resistance of tumor cells toward cisplatin. Here we demonstrate that cancer cells respond to cisplatin treatment with the nucleolar accumulation of inorganic polyphosphate (polyP), a universally conserved high-energy compound. PolyP accumulation positively correlates with the levels of activated caspase-3, suggesting a novel role of polyP in cisplatin-mediated apoptosis. In support of this finding, we discovered that administration of exogenous polyP increases cisplatin-induced toxicity in select cancer cell lines, raising the exciting possibility that enhancing endogenous polyP levels might be a novel mechanism to sensitize cancer cells to cisplatin treatment.

Circulating levels of monocyte chemoattractant protein-1 as a potential measure of biological age in mice and frailty in humans.

A serum biomarker of biological versus chronological age would have significant impact on clinical care. It could be used to identify individuals at risk of early-onset frailty or the multimorbidities associated with old age. It may also serve as a surrogate endpoint in clinical trials targeting mechanisms of aging. Here, we identified MCP-1/CCL2, a chemokine responsible for recruiting monocytes, as a potential biomarker of biological age. Circulating monocyte chemoattractant protein-1 (MCP-1) levels increased in an age-dependent manner in wild-type (WT) mice. That age-dependent increase was accelerated in Ercc1-/Δ and Bubr1H/H mouse models of progeria. Genetic and pharmacologic interventions that slow aging of Ercc1-/Δ and WT mice lowered serum MCP-1 levels significantly. Finally, in elderly humans with aortic stenosis, MCP-1 levels were significantly higher in frail individuals compared to nonfrail. These data support the conclusion that MCP-1 can be used as a measure of mammalian biological age that is responsive to interventions that extend healthy aging.

Rapamycin treatment attenuates age-associated periodontitis in mice.

Interventions that target biological mechanisms of aging have great potential to enhance quality of life by delaying morbidity and mortality. The FDA-approved drug rapamycin is a compelling candidate for such an intervention. In a previous study, it was reported that 3 months of rapamycin treatment is sufficient to increase life expectancy and remodel the gut microbiome in aged mice. Transient treatment with rapamycin or a rapamycin derivative has also been shown to delay immune stem cell senescence and rejuvenate immune function in aged mice and elderly people. Periodontal disease is an important age-related disease involving altered immune function, pathological changes to the oral microbiome, and systemic inflammation. Periodontal disease is defined clinically by loss of alveolar bone and by connective tissue degeneration. Here, we describe significant alveolar bone loss during aging in two different mouse strain backgrounds and report that rapamycin treatment is sufficient to reverse age-associated periodontal disease in mice. Partial restoration of youthful levels of alveolar bone is observed in 22-month-old rapamycin-treated mice as rapidly as 8 weeks after initiation of treatment. To the best of our knowledge, this represents the first intervention shown to substantially prevent or reverse age-associated alveolar bone loss. These findings suggest the possibility that inhibition of mTOR with rapamycin or other pharmacological agents may be useful to treat a clinically relevant condition for which there is currently no effective treatment.

A New Preclinical Paradigm for Testing Anti-Aging Therapeutics.

Testing drugs for anti-aging effects has historically been conducted in mouse life-span studies, but are costly and time consuming, and more importantly, difficult to recapitulate in humans. In addition, life-span studies in mice are not well suited to testing drug combinations that target multiple factors involved in aging. Additional paradigms for testing therapeutics aimed at slowing aging are needed. A new paradigm, designated as the Geropathology Grading Platform (GGP), is based on a standardized set of guidelines developed to detect the presence or absence of low-impact histopathological lesions and to determine the level of severity of high-impact lesions in organs from aged mice. The GGP generates a numerical score for each age-related lesion in an organ, summed for total lesions, and averaged over multiple mice to obtain a composite lesion score (CLS). Preliminary studies show that the platform generates CLSs that increase with the age of mice in an organ-dependent manner. The CLSs are sensitive enough to detect changes elicited by interventions that extend mouse life span, and thus help validate the GGP as a novel tool to measure biological aging. While currently optimized for mice, the GGP could be adapted to any preclinical animal model.

Mitochondrial dysfunction in cardiac aging.

Cardiovascular diseases are the leading cause of death in most developed nations. While it has received the least public attention, aging is the dominant risk factor for developing cardiovascular diseases, as the prevalence of cardiovascular diseases increases dramatically with increasing age. Cardiac aging is an intrinsic process that results in impaired cardiac function, along with cellular and molecular changes. Mitochondria play a great role in these processes, as cardiac function is an energetically demanding process. In this review, we examine mitochondrial dysfunction in cardiac aging. Recent research has demonstrated that mitochondrial dysfunction can disrupt morphology, signaling pathways, and protein interactions; conversely, mitochondrial homeostasis is maintained by mechanisms that include fission/fusion, autophagy, and unfolded protein responses. Finally, we describe some of the recent findings in mitochondrial targeted treatments to help meet the challenges of mitochondrial dysfunction in aging.

Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.

Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10-week RP or 40% CR. Protein abundance and turnover were measured in vivo using (2) H3 -leucine heavy isotope labeling followed by LC-MS/MS, and translation was assessed by polysome profiling. We observed 35-60% increased protein half-lives after CR and 15% increased half-lives after RP compared to age-matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions.

Quality control systems in cardiac aging.

Cardiac aging is an intrinsic process that results in impaired cardiac function, along with cellular and molecular changes. These degenerative changes are intimately associated with quality control mechanisms. This review provides a general overview of the clinical and cellular changes which manifest in cardiac aging, and the quality control mechanisms involved in maintaining homeostasis and retarding aging. These mechanisms include autophagy, ubiquitin-mediated turnover, apoptosis, mitochondrial quality control and cardiac matrix homeostasis. Finally, we discuss aging interventions that have been observed to impact cardiac health outcomes. These include caloric restriction, rapamycin, resveratrol, GDF11, mitochondrial antioxidants and cardiolipin-targeted therapeutics. A greater understanding of the quality control mechanisms that promote cardiac homeostasis will help to understand the benefits of these interventions, and hopefully lead to further improved therapeutic modalities.

Flagellin induces antibody responses through a TLR5- and inflammasome-independent pathway.

Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1Rs and IL-18Rs, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. In this study, we define roles of known flagellin receptors and MyD88 in Ab responses generated toward flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype-specific Ab responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote Ab responses toward flagellin and coadministered OVA in C57BL/6 mice. We demonstrate IgG2c responses toward flagellin were TLR5 and inflammasome dependent; IgG1 was the dominant isotype and partially TLR5 and inflammasome dependent. Our data indicate a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5 and MyD88 dependent and caspase-1 independent. Unlike C57BL/6 mice, flagellin-immunized A/J mice induced codominant IgG1 and IgG2a responses. Furthermore, MyD88-independent, flagellin-induced Ab responses were even more pronounced in A/J MyD88(-/-) mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward coadministered OVA, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to Ab production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.

Innate immune detection of flagellin positively and negatively regulates salmonella infection.

Salmonella enterica serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type Salmonella infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression in vivo permits Salmonella to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used Salmonella deficient in the anti-sigma factor flgM, which overproduce flagella and are attenuated in vivo. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of flgM(-) S. Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of flgM(-) S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.

The innate immune response to Salmonella enterica serovar Typhimurium by macrophages is dependent on TREM2-DAP12.

Macrophage recognition of Salmonella enterica serovar Typhimurium leads to a cascade of signaling events, including the activation of Src family and Syk kinases and the production of reactive oxygen species (ROS), which are critical for host innate defense during early stages of bacterial infection. ROS production depends on the NADPH oxidase, but little is known about the innate immune receptors and proximal adapters that regulate Salmonella-induced ROS. Herein, we demonstrate that serovar Typhimurium induces ROS through a pathway that requires both triggering receptor expressed on myeloid cells 2 (TREM2) and DAP12. This pathway is highly analogous to the pathways utilized by Fc receptors and integrins to regulate ROS production. Oral infection of mice with serovar Typhimurium demonstrates that the DAP12-dependent pathway regulates cecal colonization during early stages of Salmonella infection. Thus, DAP12 is an important regulator of Salmonella-induced ROS production in macrophages, and TREM2 is essential for linking DAP12 to the innate response to serovar Typhimurium.

Melorheostosis of the index finger: a case report.

We describe a case of melorheostosis involving multiple bones of the left index finger. This patient presented with a many-year history of a finger mass, and demonstrated the typical radiographic findings of melorheostosis on radiographs and computed tomography (CT) of the hand. Following excisional biopsy and bone grafting, this patient is doing well and undergoing physical therapy.