Increased hyaluronan by naked mole-rat Has2 improves healthspan in mice.

Abundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent-the naked mole-rat1,2. To study whether the benefits of HMM-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHas2). nmrHas2 mice showed an increase in hyaluronan levels in several tissues, and a lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHas2 mice shifted towards that of longer-lived species. The most notable change observed in nmrHas2 mice was attenuated inflammation across multiple tissues. HMM-HA reduced inflammation through several pathways, including a direct immunoregulatory effect on immune cells, protection from oxidative stress and improved gut barrier function during ageing. These beneficial effects were conferred by HMM-HA and were not specific to the nmrHas2 gene. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new paths for using HMM-HA to improve lifespan and healthspan.

DNA methylation clocks tick in naked mole rats but queens age more slowly than nonbreeders.

Naked mole rats (NMRs) live an exceptionally long life, appear not to exhibit age-related decline in physiological capacity and are resistant to age-related diseases. However, it has been unknown whether NMRs also evade aging according to a primary hallmark of aging: epigenetic changes. To address this question, we profiled n = 385 samples from 11 tissue types at loci that are highly conserved between mammalian species using a custom array (HorvathMammalMethylChip40). We observed strong epigenetic aging effects and developed seven highly accurate epigenetic clocks for several tissues (pan-tissue, blood, kidney, liver, skin clocks) and two dual-species (human-NMR) clocks. The skin clock correctly estimated induced pluripotent stem cells derived from NMR fibroblasts to be of prenatal age. The NMR epigenetic clocks revealed that breeding NMR queens age more slowly than nonbreeders, a feature that is also observed in some eusocial insects. Our results show that despite a phenotype of negligible senescence, the NMR ages epigenetically.

p16(Ink4a) and senescence-associated ß-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli.

Constitutive p16Ink4a expression, along with senescence-associated ß-galactosidase (SAßG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAßG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAßG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAßG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAßG-positive macrophages.

Murine mesenchymal cells that express elevated levels of the CDK inhibitor p16(Ink4a) in vivo are not necessarily senescent.

Age-related health decline has been attributed to the accumulation of senescent cells recognized in vivo by p16(Ink4a) expression. The pharmacological elimination of p16(Ink4a)-positive cells from the tissues of mice was shown to extend a healthy lifespan. Here, we describe a population of mesenchymal cells isolated from mice that are highly p16(INK4a)-positive are proficient in proliferation but lack other properties of cellular senescence. These data, along with earlier reports on p16(Ink4a)-positive macrophages, indicate that p16(Ink4a)-positive and senescent cell populations only partially intersect, therefore, extending the list of potential cellular targets for anti- aging therapies.

Genotoxicity of two new carbazole derivatives with antifungal activity.

The class of carbazoles includes compounds with high biological activities and broad spectra of action. PLX01107 and PLX01008 are xenomycins, a new subclass of antimicrobial carbazole derivatives demonstrating strong antifungal activity in vitro. We performed three tests, a bacterial reverse mutation assay (Ames test), in vitro cytokinesis-block micronucleus assay, and chromosome aberration test in mouse bone marrow cells, to investigate the possible genotoxicity of these compounds. Despite their structural similarity, the two compounds had different genotoxicity profiles. PLX01008 showed positive effects in all assays. PLX01107 showed no mutagenicity in the Ames test but demonstrated strong cytogenetic activity in vitro and in vivo. PLX01107 was also tested in the in vivo alkaline comet assay, where a weak but statistically significant increase in DNA damage was seen in liver cells 24h after treatment. Significantly increased levels of formamidopyrimidine DNA glycosylase (FPG)-sensitive sites were found in bone marrow cells of PLX01107-treated mice (FPG-modified comet assay), suggesting induction of oxidative or alkylation damage to DNA.

Aging of mice is associated with p16(Ink4a)- and ß-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells.

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and ß-galactosidase activity at pH6.0 (ß-gal(pH6)), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/ß-gal(pH6)-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/ß-gal(pH6)-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/ß-gal(pH6)-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/ß-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Small-molecule xenomycins inhibit all stages of the Plasmodium life cycle.

Widespread resistance to most antimalaria drugs in use has prompted the search for novel candidate compounds with activity against Plasmodium asexual blood stages to be developed for treatment. In addition, the current malaria eradication programs require the development of drugs that are effective against all stages of the parasite life cycle. We have analyzed the antimalarial properties of xenomycins, a novel subclass of small molecule compounds initially isolated for anticancer activity and similarity to quinacrine in biological effects on mammalian cells. In vitro studies show potent activity of Xenomycins against Plasmodium falciparum. Oral administration of xenomycins in mouse models result in effective clearance of liver and blood asexual and sexual stages, as well as effective inhibition of transmission to mosquitoes. These characteristics position xenomycins as antimalarial candidates with potential activity in prevention, treatment and elimination of this disease.

Regulation of inducible heme oxygenase and cyclooxygenase isozymes in a mouse model of spotted fever group rickettsiosis.

Vascular endothelial cells (ECs) lining the blood vessels are the preferred primary targets of pathogenic Rickettsia species in the host. In response to oxidative stress triggered by infection, ECs launch defense mechanisms such as expression of heme oxygenase-1 (HO-1). Previous evidence from an established animal model of Rocky Mountain spotted fever also suggests selective modulation of anti-oxidant enzyme activities in the target host tissues. In this study, we have examined the expression profiles of HO-1 and COX-2 in different tissues during Rickettsia conorii infection of susceptible C3H/HeN mice. RNA hybridization with murine HO-1 and COX-2-specific complementary DNA probes revealed increased HO-1 expression in the liver and brain of mice infected with three different doses of R. conorii ranging from 2.25×10(3) to 2.25×10(5) pfu, relatively non-remarkable changes in the lungs, and a trend for down-regulation in the spleen. The most prominent HO-1 response was evident in the liver with ∼4-fold increase on day 4 post-infection, followed by a decline on day 7. HO-1 expression in the brain, however, peaked with significantly higher levels on day 7. Following infection with both sub-lethal as well as lethal doses of infection, the transcript encoding COX-2 also displayed a pattern of increased expression in the liver and brain. Although immunohistochemical staining revealed increased abundance of HO-1 protein in the liver of infected mice, adjoining serial sections did not exhibit positive staining for COX-2 in infected tissues. The levels of monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived cytokine (KC) were significantly higher in the sera of infected mice and corresponded with the onset and severity of the disease. Treatment of infected animals with anti-oxidants α-lipoic acid and N-acetylcysteine and HO inhibitor stannous protoporphyrin (SnPPIX) showed only selective beneficial effects on HO-1 and COX-2 expression in the liver and spleen and serum levels of KC and MCP-1. R. conorii infection of susceptible mice, therefore, results in selective regulation of the expression of HO-1 and COX-2 in a manner dependent on the target host tissue's cellular environment and the propensity of infection with rickettsiae.

Rickettsia rickettsii infection of human macrovascular and microvascular endothelial cells reveals activation of both common and cell type-specific host response mechanisms.

Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-kappaB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-kappaB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E(2) after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

Rickettsia rickettsii infection protects human microvascular endothelial cells against staurosporine-induced apoptosis by a cIAP(2)-independent mechanism.

BACKGROUND: Manipulation of host cell death is an important determinant of the outcome of an infection. Here, we investigate whether Rickettsia rickettsii-infected host endothelial cells resist the effects of staurosporine, a potent inducer of apoptosis, and we explore the mechanisms underlying the anti-apoptotic effect of infection. METHODS: Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine. The extent of apoptosis was evaluated with flow cytometry. mRNA and protein expression levels were determined by use of microarray or polymerase chain reaction and immunoblotting, respectively. RESULTS: Staurosporine-induced apoptosis in endothelial cells infected for 24 and 48 h was significantly reduced, compared with simultaneously treated uninfected cells. A microarray of human genes involved in apoptosis and polymerase chain reaction analyses revealed increased steady-state mRNA expression of cIAP2 (a member of the inhibitor-of-apoptosis family of proteins) at 24 h after infection. The levels of cIAP2 protein (+/-SD) in infected cells were 3.5 +/- 1.7 -fold and 2.3 +/- 1.2 -fold higher than that in uninfected control cells at 24 and 48 h after infection. Nucleofection of human-specific cIAP2-targeted siRNA resulted in inhibition of protein expression by > or = 50% but had no effect on infection-induced protection against apoptosis. CONCLUSIONS: R. rickettsii-induced expression of cIAP2 in host endothelial cells is likely not a major contributor to protection against staurosporine-induced cell death.

Host-cell interactions with pathogenic Rickettsia species.

Pathogenic Rickettsia species are Gram-negative, obligate intracellular bacteria responsible for the spotted fever and typhus groups of diseases around the world. It is now well established that a majority of sequelae associated with human rickettsioses are the outcome of the pathogen's affinity for endothelium lining the blood vessels, the consequences of which are vascular inflammation, insult to vascular integrity and compromised vascular permeability, collectively termed 'Rickettsial vasculitis'. Signaling mechanisms leading to transcriptional activation of target cells in response to Rickettsial adhesion and/or invasion, differential activation of host-cell signaling due to infection with spotted fever versus typhus subgroups of Rickettsiae, and their contributions to the host's immune responses and determination of cell fate are the major subtopics of this review. Also included is a succinct analysis of established in vivo models and their use for understanding Rickettsial interactions with host cells and pathogenesis of vasculotropic rickettsioses. Continued progress in these important but relatively under-explored areas of bacterial pathogenesis research should further highlight unique aspects of Rickettsial interactions with host cells, elucidate the biological basis of endothelial tropism and reveal novel chemotherapeutic and vaccination strategies for debilitating Rickettsial diseases.

Rickettsia raoultii sp. nov., a spotted fever group rickettsia associated with Dermacentor ticks in Europe and Russia.

We describe the characterization of a novel Rickettsia species cultivated from Dermacentor ticks collected in Russia and France, for which we propose the name Rickettsia raoultii sp. nov. Using multigene sequencing, we demonstrated that five rickettsial isolates from Dermacentor silvarum, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor nuttalli ticks were classified within this novel spotted fever rickettsia species. This rickettsia also exhibited a serotype distinct from previously described Rickettsia species. The type strain of Rickettsia raoultii sp. nov. is strain Khabarovsk(T) (=CSUR R3(T) =ATCC VR-1596(T)).

Comparative analysis of host-cell signalling mechanisms activated in response to infection with Rickettsia conorii and Rickettsia typhi.

The Gram-negative intracellular bacteria Rickettsia conorii and Rickettsia typhi are the aetiological agents of Mediterranean spotted fever and endemic typhus, respectively, in humans. Infection of endothelial cells (ECs) lining vessel walls, and the resultant vascular inflammation and haemostatic alterations are salient pathogenetic features of both of these rickettsial diseases. An important consideration, however, is that dramatic differences in the intracellular motility and accumulation patterns for spotted fever versus typhus group rickettsiae have been documented, suggesting the possibility of unique and potentially different interactions with host cells. This study characterized and compared R. conorii- and R. typhi-mediated effects on cultured human ECs. The DNA-binding activity of nuclear transcription factor-kappaB (NF-kappaB) and the phosphorylation status of stress-activated p38 kinase were determined as indicators of NF-kappaB and p38 activation. R. conorii infection resulted in a biphasic activation of NF-kappaB, with an early increase in DNA-binding activity at 3 h, followed by a later peak at 24 h. The activated NF-kappaB species were composed mainly of RelA p65-p50 heterodimers and p50 homodimers. R. typhi infection of ECs resulted in only early activation of NF-kappaB at 3 h, composed primarily of p65-p50 heterodimers. Whilst R. conorii infection induced increased phosphorylation of p38 kinase (threefold mean induction) with the maximal response at 3 h, a considerably less-intense response peaking at about 6 h post-infection was found with R. typhi. Furthermore, mRNA expression of the chemokines interleukin (IL)-8 and monocyte chemoattractant protein-1 in ECs infected with either Rickettsia species was higher than the corresponding controls, but there were distinct differences in the secretion patterns for IL-8, suggesting the possibility of involvement of post-transcriptional control mechanisms or differences in the release from intracellular storage sites. Thus, the intensity and kinetics of host-cell responses triggered by spotted fever and typhus species exhibit distinct variations that could subsequently lead to differences in the extent of endothelial activation and inflammation and serve as important determinants of pathogenesis.

Infection of human endothelial cells with spotted Fever group rickettsiae stimulates cyclooxygenase 2 expression and release of vasoactive prostaglandins.

Rickettsiae, a diverse group of obligately intracellular gram-negative bacteria, include etiologic agents of the spotted fever and typhus groups of diseases. Rocky Mountain spotted fever and boutonneuse fever, due to Rickettsia rickettsii and R. conorii, respectively, are characterized by widespread infection of the vascular endothelium, microvascular injury, and vasculitis. Cultured human endothelial cells (EC) are highly susceptible to infection and respond by altering the expression of adhesion molecules, regulatory cytokines, and the antioxidant enzyme heme oxygenase (HO). In the vasculature, HO regulates the cyclooxygenase (COX) enzymes, among which the inducible isozyme COX-2 facilitates the synthesis of prostaglandins (PGs). Using in vitro and ex vivo models of infection, we demonstrate here that R. rickettsii infection of human EC causes robust induction of COX-2 mRNA and protein expression but has no apparent effect on the constitutive COX-1 isoform. Cells infected with viable rickettsiae consistently displayed significantly increased secretion of 6-keto-PGF(1alpha) and PGE(2). R. rickettsii-induced COX-2 was sensitive to inhibitors of de novo transcription and the pyridinylimidazole-based compound SB 203580, suggesting that this transcriptional host cell response involves signaling through p38 mitogen-activated protein kinase. PG production by infected cells was abrogated by NS 398 (a selective COX-2 inhibitor) and indomethacin (a pan-COX inhibitor). Immunohistochemical staining of sections of infected umbilical cords and corresponding uninfected controls revealed comparatively more intense and abundant staining for COX-2 in infected endothelia. Induction of the endothelial COX-2 system and the resultant enhanced release of vasoactive PGs may contribute to the regulation of inflammatory responses and vascular permeability changes during spotted fever rickettsioses.

Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after Rickettsia rickettsii infection: regulation by nuclear transcription factor NF-kappaB.

Infection of endothelial cells (EC) with Rickettsia rickettsii results in Rocky Mountain spotted fever, an acute illness characterized by systemic inflammation. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for activating neutrophils and monocytes, respectively, and recruiting these circulating immune cells to the sites of inflammation. In this study, we have measured the expression and secretion of these chemokines during R. rickettsii infection of cultured human EC. In comparison to uninfected controls, increased mRNA expression of IL-8 and MCP-1 in R. rickettsii-infected EC was evident as early as 3 h and was sustained up to 21 h. Subsequent analysis of culture supernatants revealed significantly enhanced secretion of both chemokines at 3, 8, and 18 h post-infection (5-28-fold increase in IL-8 and 4-16-fold increase in MCP-1). The presence of peptide-aldehyde compound MG132 to inhibit proteasome-mediated degradation of the inhibitory protein IkappaBalpha and synthetic peptide SN-50 to inhibit the nuclear translocation of nuclear factor-kappa B (NF-kappaB) resulted in significant inhibition of the chemokine response. Also, T24 cells expressing a super-repressor mutant of IkappaBalpha (to render NF-kappaB inactivatable) secreted significantly lower quantities of IL-8 than mock-transfected cells. A neutralizing antibody against IL-1alpha or an IL-1 specific receptor antagonist had no effect on the early phase of R. rickettsii-induced NF-kappaB activation and IL-8/ MCP-1 secretion at 3 h. Both of these treatments, however, diminished late-phase NF-kappaB activation by about 33% and only partially suppressed the infection-induced chemokine release at 21 h. Thus, while chemokine response early during the infection likely depends on the direct activation of NF-kappaB, subtle autocrine effects of newly synthesized IL-1alpha may contribute, in part, to the control of NF-kappaB activation and chemokine production at later times. These findings implicate a prominent role for host EC in recruiting immune cells to the site of inflammation during Rickettsia infection and provide important insights to further our understanding of the pathogenesis of spotted fever group rickettsioses.

Activation of p38 stress-activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response.

Rickettsia rickettsii, a Gram-negative and obligate intracellular bacterium, preferentially infects the vascular endothelium during human infections leading to inflammation and dysfunction. The aim of this study was to determine whether R. rickettsii infection of endothelial cells (EC) activates p38 and/or c-jun N-terminal kinases (JNK) mitogen-activated protein (MAP) kinase, key regulatory proteins that control the response to inflammatory stimuli. We show that infection of cultured human EC results in the dose-dependent activation of p38, as assessed by increased phosphorylation and activity, without affecting the status of JNK. Rickettsia inactivation by heat or formaldehyde abolished the activation of p38 kinase and inhibition of cellular invasion by infection at low temperature, pre-treatment of host EC with cytochalasin D, or pre-incubation of rickettsiae with an irreversible phospholipase inhibitor led to a diminished p38 phosphorylation, suggesting requirement of invasion by viable rickettsiae for this host cell response. SB 203580, a p38-specific inhibitor, had no effect on infection-induced activation of the ubiquitous transcriptional regulator nuclear factor-kappa B, but effectively reduced the expression and secretion of important chemoattractant cytokines interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 by R. rickettsii-infected EC. Selective inhibition of p38 activity may be exploited as an anti-inflammatory target to prevent rickettsial vasculitis and to develop new and improved chemotherapeutic agents.