Adult mouse hippocampal transcriptome changes associated with long-term behavioral and metabolic effects of gestational air pollution toxicity.

Gestational exposure to air pollution increases the risk of autism spectrum disorder and cognitive impairments with unresolved molecular mechanisms. This study exposed C57BL/6J mice throughout gestation to urban-derived nanosized particulate matter (nPM). Young adult male and female offspring were studied for behavioral and metabolic changes using forced swim test, fat gain, glucose tolerance, and hippocampal transcriptome. Gestational nPM exposure caused increased depressive behaviors, decreased neurogenesis in the dentate gyrus, and increased glucose tolerance in adult male offspring. Both sexes gained fat and body weight. Gestational nPM exposure induced 29 differentially expressed genes (DEGs) in adult hippocampus related to cytokine production, IL17a signaling, and dopamine degradation in both sexes. Stratification by sex showed twofold more DEGs in males than females (69 vs 37), as well as male-specific enrichment of DEGs mediating serotonin signaling, endocytosis, Gαi, and cAMP signaling. Gene co-expression analysis (WCGNA) identified a module of 43 genes with divergent responses to nPM between the sexes. Chronic changes in 14 DEGs (e.g., microRNA9-1) were associated with depressive behaviors, adiposity and glucose intolerance. These genes enriched neuroimmune pathways such as HMGB1 and TLR4. Based on cerebral cortex transcriptome data of neonates, we traced the initial nPM responses of HMGB1 pathway. In vitro, mixed glia responded to 24 h nPM with lower HMGB1 protein and increased proinflammatory cytokines. This response was ameliorated by TLR4 knockdown. In sum, we identified transcriptional changes that could be associated with air pollution-mediated behavioral and phenotypic changes. These identified genes merit further mechanistic studies for therapeutic intervention development.

Mouse brain transcriptome responses to inhaled nanoparticulate matter differed by sex and APOE in Nrf2-Nfkb interactions.

The neurotoxicity of air pollution is undefined for sex and APOE alleles. These major risk factors of Alzheimer's disease (AD) were examined in mice given chronic exposure to nPM, a nano-sized subfraction of urban air pollution. In the cerebral cortex, female mice had two-fold more genes responding to nPM than males. Transcriptomic responses to nPM had sex-APOE interactions in AD-relevant pathways. Only APOE3 mice responded to nPM in genes related to Abeta deposition and clearance (Vav2, Vav3, S1009a). Other responding genes included axonal guidance, inflammation (AMPK, NFKB, APK/JNK signaling), and antioxidant signaling (NRF2, HIF1A). Genes downstream of NFKB and NRF2 responded in opposite directions to nPM. Nrf2 knockdown in microglia augmented NFKB responses to nPM, suggesting a critical role of NRF2 in air pollution neurotoxicity. These findings give a rationale for epidemiologic studies of air pollution to consider sex interactions with APOE alleles and other AD-risk genes.

Toxicity of urban air pollution particulate matter in developing and adult mouse brain: Comparison of total and filter-eluted nanoparticles.

Air pollution (AirP) is associated with many neurodevelopmental and neurological disorders in human populations. Rodent models show similar neurotoxic effects of AirP particulate matter (PM) collected by different methods or from various sources. However, controversies continue on the identity of the specific neurotoxic components and mechanisms of neurotoxicity. We collected urban PM by two modes at the same site and time: direct collection as an aqueous slurry (sPM) versus a nano-sized sub-fraction of PM0.2 that was eluted from filters (nPM). The nPM lacks water-insoluble PAHs (polycyclic aromatic hydrocarbons) and is depleted by >50% in bioactive metals (e.g., copper, iron, nickel), inorganic ions, black carbon, and other organic compounds. Three biological models were used: in vivo exposure of adult male mice to re-aerosolized nPM and sPM for 3 weeks, gestational exposure, and glial cell cultures. In contrast to larger inflammatory responses of sPM in vitro, cerebral cortex responses of mice to sPM and nPM largely overlapped for adult and gestational exposures. Adult brain responses included induction of IFNγ and NF-κB. Gestational exposure to nPM and sPM caused equivalent depressive behaviors. Responses to nPM and sPM diverged for cerebral cortex glutamate receptor mRNA, systemic fat gain and insulin resistance. The shared toxic responses of sPM with nPM may arise from shared transition metals and organics. In contrast, gestational exposure to sPM but not nPM, decreased glutamatergic mRNAs, which may be attributed to PAHs. We discuss potential mechanisms in the overlap between nPM and sPM despite major differences in bulk chemical composition.

Cell-based assays that predict in vivo neurotoxicity of urban ambient nano-sized particulate matter.

Exposure to urban ambient particulate matter (PM) is associated with risk of Alzheimer's disease and accelerated cognitive decline in normal aging. Assessment of the neurotoxic effects caused by urban PM is complicated by variations of composition from source, location, and season. We compared several in vitro cell-based assays in relation to their in vivo neurotoxicity for NF-κB transcriptional activation, nitric oxide induction, and lipid peroxidation. These studies compared batches of nPM, a nanosized subfraction of PM2.5, extracted as an aqueous suspension, used in prior studies. In vitro activities were compared with in vivo responses of mice chronically exposed to the same batch of nPM. The potency of nPM varied widely between batches for NF-κB activation, analyzed with an NF-κB reporter in human monocytes. Three independently collected batches of nPM had corresponding differences to responses of mouse cerebral cortex to chronic nPM inhalation, for levels of induction of pro-inflammatory cytokines, microglial activation (Iba1), and soluble Aß40 & -42 peptides. The in vitro responses of BV2 microglia for NO-production and lipid peroxidation also differed by nPM batch, but did not correlate with in vivo responses. These data confirm that batches of nPM can differ widely in toxicity. The in vitro NF-κB reporter assay offers a simple, high throughput screening method to predict the in vivo neurotoxic effects of nPM exposure.

Air Pollution Alters Caenorhabditis elegans Development and Lifespan: Responses to Traffic-Related Nanoparticulate Matter.

Air pollution is a heterogeneous environmental toxicant that impacts humans throughout their life. We introduce Caenorhabditis elegans as a valuable air pollution model with its short lifespan, medium-throughput capabilities, and highly conserved biological pathways that impact healthspan. We exposed developmental and adult life stages of C. elegans to airborne nano-sized particulate matter (nPM) produced by traffic emissions and measured biological and molecular endpoints that changed in response. Acute nPM did not cause lethality in C. elegans, but short-term exposure during larval stage 1 caused delayed development. Gene expression responses to nPM exposure overlapped with responses of mouse and cell culture models of nPM exposure in previous studies. We showed further that the skn-1/Nrf2 antioxidant response has a role in the development and hormetic effects of nPM. This study introduces the worm as a new resource and complementary model for mouse and cultured cell systems to study air pollution toxicity across the lifespan.

Induction of a robust immune response against avian influenza virus following transdermal inoculation with H5-DNA vaccine formulated in modified dendrimer-based delivery system in mouse model.

This study was aimed to evaluate the immunogenicity of recombinant plasmid deoxyribonucleic acid (DNA), pBud-H5-green fluorescent protein (GFP)-interferon-regulatory factor (IRF)3 following delivery using polyamidoamine (PAMAM) dendrimer and transactivator of transcription (TAT)-conjugated PAMAM dendrimer as well as the effect of IRF3 as the genetic adjuvant. BALB/c mice were vaccinated transdermally with pBud-H5-GFP, PAMAM/pBud-H5-GFP, TAT-PAMAM/pBud-H5-GFP, and TAT-PAMAM/pBud-H5-GFP-IRF3. The expression analysis of H5 gene from the blood by using quantitative real-time reverse transcriptase polymerase chain reaction confirmed the ability of PAMAM dendrimer as a carrier for gene delivery, as well as the ability of TAT peptide to enhance the delivery efficiency of PAMAM dendrimer. Mice immunized with modified PAMAM by TAT peptide showed higher hemagglutination inhibition titer, and larger CD3+/CD4+ T cells and CD3+/CD8+ T cells population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2, IL-15, IL-12, IL-6, and tumor necrosis factor-α compared with those immunized with native PAMAM. These results suggest that the function of TAT peptide as a cell-penetrating peptide is able to enhance the gene delivery, which results in rapid distribution of H5 in the tissues of the immunized mice. Furthermore, pBud-H5-GFP co-expressing IRF3 as a genetic adjuvant demonstrated the highest hemagglutination inhibition titer besides larger CD3+/CD4+ and CD3+/CD8+ T cells population, and strong Th1-like cytokine responses among all the systems tested. In conclusion, TAT-PAMAM dendrimer-based delivery system with IRF3 as a genetic adjuvant is an attractive transdermal DNA vaccine delivery system utilized to evaluate the efficacy of the developed DNA vaccine in inducing protection during challenge with virulent H5N1 virus.

Expression profiles of immune mediators in feline Coronavirus-infected cells and clinical samples of feline Coronavirus-positive cats.

BACKGROUND: There are two biotypes of feline coronavirus (FCoV): the self-limiting feline enteric coronavirus (FECV) and the feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP), a fatal disease associated with cats living in multi-cat environments. This study provides an insight on the various immune mediators detected in FCoV-positive cats which may be responsible for the development of FIP. RESULTS: In this study, using real-time PCR and multiplex bead-based immunoassay, the expression profiles of several immune mediators were examined in Crandell-Reese feline kidney (CRFK) cells infected with the feline coronavirus (FCoV) strain FIPV 79-1146 and in samples obtained from FCoV-positive cats. CRFK cells infected with FIPV 79-1146 showed an increase in the expression of interferon-related genes and pro-inflammatory cytokines such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, and IL8. In addition, an increase in the expression of the above cytokines as well as GM-CSF and IFNγ was also detected in the PBMC, serum, and peritoneal effusions of FCoV-positive cats. Although the expression of MX1 and viperin genes was variable between cats, the expression of these two genes was relatively higher in cats having peritoneal effusion compared to cats without clinically obvious effusion. Higher viral load was also detected in the supernatant of peritoneal effusions compared to in the plasma of FCoV-positive cats. As expected, the secretion of IL1ß, IL6 and TNFα was readily detected in the supernatant of peritoneal effusions of the FCoV-positive cats. CONCLUSIONS: This study has identified various pro-inflammatory cytokines and interferon-related genes such as MX1, viperin, CXCL10, CCL8, RANTES, KC, MCP1, IL8, GM-CSF and IFNγ in FCoV-positive cats. With the exception of MX1 and viperin, no distinct pattern of immune mediators was observed that distinguished between FCoV-positive cats with and without peritoneal effusion. Further studies based on definitive diagnosis of FIP need to be performed to confirm the clinical importance of this study.

Edible bird's nest modulate intracellular molecular pathways of influenza A virus infected cells.

BACKGROUND: Edible Bird's Nest (EBN) as a popular traditional Chinese medicine is believed to have health enhancing and antiviral activities against influenza A virus (IAV); however, the molecular mechanism behind therapeutic effects of EBN is not well characterized. METHODS: In this study, EBNs that underwent different enzymatic preparation were tested against IAV infected cells. 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50) of the EBNs against IAV strain A/Puerto Rico/8/1934(H1N1) were determined by HA and MTT assays. Subsequently, the sialic acid content of the used EBNs were analyzed by fluorometric HPLC. Western Blotting and immunofluorescent staining were used to investigate the effects of EBNs on early endosomal trafficking and autophagy process of influenza virus. RESULTS: This study showed that post inoculations of EBNs after enzymatic preparations have the highest efficacy to inhibit IAV. While CC50 of the tested EBNs ranged from 27.5-32 mg/ml, the IC50 of these compounds ranged between 2.5-4.9 mg/ml. EBNs could inhibit IAV as efficient as commercial antiviral agents, such as amantadine and oseltamivir with different mechanisms of action against IAV. The antiviral activity of these EBNs correlated with the content of N-acetyl neuraminic acid. EBNs could affect early endosomal trafficking of the virus by reducing Rab5 and RhoA GTPase proteins and also reoriented actin cytoskeleton of IAV infected cells. In addition, for the first time this study showed that EBNs can inhibit intracellular autophagy process of IAV life cycle as evidenced by reduction of LC3-II and increasing of lysosomal degradation. CONCLUSIONS: The results procured in this study support the potential of EBNs as supplementary medication or alternative to antiviral agents to inhibit influenza infections. Evidently, EBNs can be a promising antiviral agent; however, these natural compounds should be screened for their metabolites prior to usage as therapeutic approach.

In vitro and in vivo mechanism of immunomodulatory and antiviral activity of Edible Bird's Nest (EBN) against influenza A virus (IAV) infection.

ETHNOPHARMACOLOGICAL RELEVANCE: For centuries, Edible Bird Nest (EBN) has been used in treatment of variety of respiratory diseases such as flu and cough as a Chinese natural medicine. AIM OF THE STUDY: This natural remedy showed the potential to inhibit influenza A virus (IAV). However, little is known about the mechanism of this process and also the evaluation of this product in an animal model. Hence, the current study was designed to elucidate the antiviral and immunomodulatory effects of EBN against IAV strain A/Puerto Rico/8/1934 (H1N1). MATERIALS AND METHODS: First, influenza infected MDCK cells treated with EBNs from two locations of Malaysia (Teluk Intan and Gua Madai) that prepared with different enzymatic preparations were analyzed by RT-qPCR and ELISA for detection of viral and cytokines genes. The sialic acid composition of these EBNs was evaluated by H-NMR. Subsequently, after toxicity evaluation of EBN from Teluk Intan, antiviral and immunomodulatory effects of this natural product was evaluated in BALB/c mice by analysis of the viral NA gene and cytokine expressions in the first week of the infection. RESULTS: EBN showed high neuraminidase inhibitory properties in both in vitro and in vivo, which was as effective as Oseltamivir phosphate. In addition, EBN decreased NS1 copy number (p<0.05) of the virus along with high immunomodulatory effects against IAV. Some of the immune changes during treatment of IAV with EBN included significant increase in IFNγ, TNFα, NFκB, IL2, some proinflammatory cytokines like IL1ß, IL6, and cytokines with regulatory properties like IL10, IL27, IL12, CCL2 and IL4 depends on the stage of the infection. EBNs from two locations contained different composition of sialic acid and thymol derivatives, which gave them different antiviral properties. EBN from Gua Madai that contained more acetylated sialic acid (Neu2,4,7,8,9 Ac6) showed higher antiviral activity. CONCLUSION: The findings of this study support the antiviral activity of EBN against influenza virus and validate the traditional usage of this natural remedy by elucidation of toxicity and the molecular mechanism of action.

Apoptosis transcriptional mechanism of feline infectious peritonitis virus infected cells.

Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection (hpi). FIPV infected cells underwent early apoptosis at 9 hpi (p < 0.05) followed by late apoptosis at 12 hpi (p < 0.05) and necrosis from 24 hpi (p < 0.05). Then, next generation sequencing was performed on 9 hpi and control uninfected cells by Illumina analyzer. An aggregate of 4546 genes (2229 down-regulated and 2317 up-regulated) from 17 cellular process, 11 molecular functions and 130 possible biological pathways were affected by FIPV. 131 genes from apoptosis cluster (80 down-regulated and 51 up-regulated) along with increase of apoptosis, p53, p38 MAPK, VEGF and chemokines/cytokines signaling pathways were probably involved in apoptosis process. Six of the de-regulated genes expression (RASSF1, BATF2, MAGEB16, PDCD5, TNFα and TRAF2) and TNFα protein concentration were analyzed by RT-qPCR and ELISA, respectively, at different time-points. Up-regulations of both pro-apoptotic (i.e. PDCD5) and anti-apoptotic (i.e. TRAF2) were detected from first hpi and continuing to deregulate during apoptosis process in the infected cells.