Comparative effects of the herbal constituent parthenolide (Feverfew) on lipopolysaccharide-induced inflammatory gene expression in murine spleen and liver

BACKGROUND: Parthenolide, a major sesquiterpene lactone present in extracts of the herb Feverfew, has been investigated for its inhibitory effects on mediators of inflammation, including the proinflammatory cytokines. Although parthenolide's anti-inflammatory effects have been investigated in vitro, little in vivo data are available. Moreover, the molecular mechanisms for these inhibitory effects are not fully understood. The objective of this study was to test the hypothesis that parthenolide suppresses lipopolysaccharide (LPS)-induced serum (interleukin) IL-6, tumor necrosis factor (TNF)-α, IL-1β and cyclooxygenase (COX)-2 expression in mice as indicated by reduced splenic and liver mRNA levels. METHODS: Mice were co-treated i.p. with LPS (1 mg/kg bw) and parthenolide (5 mg/kg bw) and blood, spleen and liver collected. Serum was analyzed for IL-6, TNF-α and IL-1β by ELISA. Total RNA was extracted from spleen and liver, and real-time RT-PCR was used to determine relative mRNA expression of IL-1β, IL-6, TNF-α and COX-2. RESULTS: LPS induced increases in serum IL-6 and TNF-α concentrations with only IL-6 being suppressed in parthenolide-treated mice. Induction of IL-6 mRNA was reduced, TNF-α and COX-2 mRNAs unchanged, and IL-1β mRNA increased in spleens of parthenolide plus LPS co-treated animals compared to LPS-only. No significant differences were observed in inflammatory gene expression between these two groups in liver samples. Overall, mRNA expression of each proinflammatory gene was much higher in spleen when compared to liver. CONCLUSION: In summary, only one gene, IL-6, was modestly suppressed by parthenolide co-exposure which contrasts with many in vitro studies suggesting anti-inflammatory effects of this compound. Also, LPS evoked greater effects in spleen than liver on expression of proinflammatory genes. Further study of the effects of parthenolide and other herbal constituents on inflammatory gene expression using model animal systems as described here are critical to evaluating efficacy of such supplements as well as elucidating their mechanisms of action

Impact of environmental factors on growth and satratoxin G production by strains of Stachybotrys chartarum

The black mould Stachybotrys chartarum and its mycotoxins have been linked to damp building-associated illnesses. The objective of this study was to determine the effects of water availability (water activity, aw) and temperature on growth and production of satratoxin G (SG) by a macrocyclic trichothecene-producing strain (IBT 7711) and non-producing strain (IBT 1495) of S. chartarum. Growth studies were carried out on potato dextrose agar modified with glycerol to 0.995-0.92 aw at 10-37 °C. Growth extension was measured and the cultures were extracted after 10 days and a competitive enzyme-linked immunosorbent assay (ELISA) method used to quantify the SG content. Growth was optimal at 25 to 30 °C at 0.995 aw, but this was modified to 0.98 aw at 30 °C for both strains (1.4- 1.6 mm/day, respectively). The ELISA method revealed that, in contrast to growth, SG production was maximal at 20 °C with highest production at 0.98 aw (approximately 250 μg/g mycelia). When water was freely available (0.995 aw), SG was maximally produced at 15 °C and decreased as temperature was increased. Interestingly, the strain classified as a non-toxigenic produced very low amounts of SG (<1.6 μg/g mycelia) that were maximal at 25 °C and 0.98 aw. Contour maps for growth and SG production were developed from these data sets. These data have shown, for the first time, that growth and SG production profiles are very different in relation to key environmental conditions in the indoor environment. This will be very useful in practically determining the risk from exposure to S. chartarum and its toxins in the built env

Satratoxin G from the Black Mold Stachybotrys chartarum Evokes Olfactory Sensory Neuron Loss and Inflammation in the Murine Nose and Brain

Satratoxin G (SG) is a macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum, the “black mold” suggested to contribute etiologically to illnesses associated with water-damaged buildings. Using an intranasal instillation model in mice, we found that acute SG exposure specifically induced apoptosis of olfactory sensory neurons (OSNs) in the olfactory epithelium. Dose–response analysis revealed that the no-effect and lowest-effect levels at 24 hr postinstillation (PI) were 5 and 25 μg/kg body weight (bw) SG, respectively, with severity increasing with dose. Apoptosis of OSNs was identified using immunohistochemistry for caspase-3 expression, electron microscopy for ultrastructural cellular morphology, and real-time polymerase chain reaction for elevated expression of the proapoptotic genes Fas, FasL, p75NGFR, p53, Bax, caspase-3, and CAD. Time-course studies with a single instillation of SG (500 μg/kg bw) indicated that maximum atrophy of the olfactory epithelium occurred at 3 days PI. Exposure to lower doses (100 μg/kg bw) for 5 consecutive days resulted in similar atrophy and apoptosis, suggesting that in the short term, these effects are cumulative. SG also induced an acute, neutrophilic rhinitis as early as 24 hr PI. Elevated mRNA expression for the proinflammatory cytokines tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1 and the chemokine macrophage-inflammatory protein-2 (MIP-2) were detected at 24 hr PI in both the ethmoid turbinates of the nasal airways and the adjacent olfactory bulb of the brain. Marked atrophy of the olfactory nerve and glomerular layers of the olfactory bulb was also detectable by 7 days PI along with mild neutrophilic encephalitis. These findings suggest that neurotoxicity and inflammation within the nose and brain are potential adverse health effects of exposure to satratoxins and Stachybotrys in the indoor air of water-damaged buildings

Mapping of Dynamic Transcriptome Changes Associated With Silica-Triggered Autoimmune Pathogenesis in the Lupus-Prone NZBWF1 Mouse

Crystalline silica (cSiO2) is a widely recognized environmental trigger of autoimmune disease. In the lupus-prone female NZBWF1 mouse, airway exposure to cSiO2 triggers pulmonary ectopic lymphoid neogenesis, systemic autoantibody elevation, and glomerulonephritis. Here we tested the hypothesis that upregulation of adaptive immune function genes in the lung precedes cSiO2-triggering of autoimmune disease in this model. The study include three groups of mice, as follows: (1) necropsied 1 d after a single intranasal instillation of 1 mg cSiO2 or vehicle, (2) necropsied 1 d after four weekly single instillations of 1 mg cSiO2 or vehicle, or (3) necropsied 1, 5, 9, or 13 weeks after four weekly single instillations of 1 mg cSiO2 or vehicle. NanoString nCounter analysis revealed modest transcriptional changes associated with innate and adaptive immune response as early as 1 d after a single cSiO2 instillation. These responses were greatly expanded after four weekly cSiO2 instillations. Concurrent with ectopic lymphoid neogenesis, dramatic increases in mRNAs associated with chemokine release, cytokine production, sustained interferon activity, complement activation, and adhesion molecules were observed. As disease progressed, expression of these genes persisted and was further amplified. Consistent with autoimmune pathogenesis, the time between 5 and 9 weeks post-instillation reflected an important transition period where considerable immune gene upregulation in the lung was observed. Upon termination of the chronic study (13 weeks), cSiO2-induced changes in transcriptome signatures were similarly robust in kidney as compared to the lung, but more modest in spleen. Transcriptomic signatures in lung and kidney were indicative of infiltration and/or expansion of neutrophils, macrophages, dendritic cells, B cells, and T cells that corresponded with accelerated autoimmune pathogenesis. Taken together, airway exposure to cSiO2 elicited aberrant mRNA signatures for both innate and adaptive immunity that were consistent with establishment of the lung as the central autoimmune nexus for launching systemic autoimmunity and ultimately, kidney injury

Health Related Quality of Life and Return to Work After Minor Extremity Injuries: a Longitudinal Study Comparing Upper Versus Lower Extremity Injuries

Purpose: To investigate the impact on health related quality of life (HRQL) during the first year after minor extremity injury and to determine whether there is a difference in recovery patterns and return to work between upper extremity injuries (UEI) and lower extremity injuries (LEI). Method: A total of 181 adults’ age 18 years or older randomly selected from patients admitted to an emergency department with minor injuries were studied. HRQL was measured using the Functional Status Questionnaire (FSQ) at 1–2 weeks, 3, 6, and 12-months post-injury. Pre-injury FSQ scores were measured retrospectively at admission. A quasi-least square (QLS) model was constructed to examine differences of FSQ scores at each measuring point for UEI and LEI. Results: Fractures of the knee/lower leg (25%) were the most frequently injured body area. Slips or falls (57%) and traffic-related events (22%) were the most common injury causes. The mean ISS was 4.2 (SD 0.86). Both groups had significant declines in the FSQ scores physical and social functioning at 1–2 weeks after injury. Patients with UEI made larger improvements in the first 3 months post-injury versus patients with LEI whose improvements extended over the first 6 months. None of the groups reached the pre-injury FSQ scores during the first post-injury year except in the subscale work performance where UEI exceeded the pre-injury scores. At 12 months post-injury, significant lower FSQ scores remained in the LEI group compared to the UEI group in intermediate activities of daily living (p = 0.036, d 0.4) and work performance (p = 0.004, d 0.7). The return to work at 3 months and 12 months were 76% and 88% for UEI and 58% and 77% for LEI. No significant differences were found between groups in the FSQ scale mental health and social interaction. Conclusions: LEI had the highest impact on HRQL and return to work during the first year which exceeded the consequences of UEI. These findings contribute to the information about the consequences of injury in order to give sufficient prognostic information to patients and different stakeholders. Future investigations should aim to investigate specific minor extremity injuries and identify factors that facilitate recovery and return to work

Deoxynivalenol-Induced Proinflammatory Gene Expression: Mechanisms and Pathological Sequelae

The trichothecene mycotoxin deoxynivalenol (DON) is commonly encountered in human cereal foods throughout the world as a result of infestation of grains in the field and in storage by the fungus Fusarium. Significant questions remain regarding the risks posed to humans from acute and chronic DON ingestion, and how to manage these risks without imperiling access to nutritionally important food commodities. Modulation of the innate immune system appears particularly critical to DON’s toxic effects. Specifically, DON induces activation of mitogen-activated protein kinases (MAPKs) in macrophages and monocytes, which mediate robust induction of proinflammatory gene expression—effects that can be recapitulated in intact animals. The initiating mechanisms for DON-induced ribotoxic stress response appear to involve the (1) activation of constitutive protein kinases on the damaged ribosome and (2) autophagy of the chaperone GRP78 with consequent activation of the ER stress response. Pathological sequelae resulting from chronic low dose exposure include anorexia, impaired weight gain, growth hormone dysregulation and aberrant IgA production whereas acute high dose exposure evokes gastroenteritis, emesis and a shock-like syndrome. Taken together, the capacity of DON to evoke ribotoxic stress in mononuclear phagocytes contributes significantly to its acute and chronic toxic effects in vivo. It is anticipated that these investigations will enable the identification of robust biomarkers of effect that will be applicable to epidemiological studies of the human health effects of this common mycotoxin

Docosahexaenoic Acid Consumption Impedes Early Interferon- and Chemokine-Related Gene Expression While Suppressing Silica-Triggered Flaring of Murine Lupus

Exposure of lupus-prone female NZBWF1 mice to respirable crystalline silica (cSiO2), a known human autoimmune trigger, initiates loss of tolerance, rapid progression of autoimmunity, and early onset of glomerulonephritis. We have previously demonstrated that dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) suppresses autoimmune pathogenesis and nephritis in this unique model of lupus flaring. In this report, we utilized tissues from prior studies to test the hypothesis that DHA consumption interferes with upregulation of critical genes associated with cSiO2-triggered murine lupus. A NanoString nCounter platform targeting 770 immune-related genes was used to assess the effects cSiO2 on mRNA signatures over time in female NZBWF1 mice consuming control (CON) diets compared to mice fed diets containing DHA at an amount calorically equivalent to human consumption of 2 g per day (DHA low) or 5 g per day (DHA high). Experimental groups of mice were sacrificed: (1) 1 d after a single intranasal instillation of 1 mg cSiO2 or vehicle, (2) 1 d after four weekly single instillations of vehicle or 1 mg cSiO2, and (3) 1, 5, 9, and 13 weeks after four weekly single instillations of vehicle or 1 mg cSiO2. Genes associated with inflammation as well as innate and adaptive immunity were markedly upregulated in lungs of CON-fed mice 1 d after four weekly cSiO2 doses but were significantly suppressed in mice fed DHA high diets. Importantly, mRNA signatures in lungs of cSiO2-treated CON-fed mice over 13 weeks reflected progressive amplification of interferon (IFN)- and chemokine-related gene pathways. While these responses in the DHA low group were suppressed primarily at week 5, significant downregulation was observed at weeks 1, 5, 9, and 13 in mice fed the DHA high diet. At week 13, cSiO2 treatment of CON-fed mice affected 214 genes in kidney tissue associated with inflammation, innate/adaptive immunity, IFN, chemokines, and antigen processing, mostly by upregulation; however, feeding DHA dose-dependently suppressed these responses. Taken together, dietary DHA intake in lupus-prone mice impeded cSiO2-triggered mRNA signatures known to be involved in ectopic lymphoid tissue neogenesis, systemic autoimmunity, and glomerulonephritis

Consumption of the Total Western Diet Promotes Colitis and Inflammation-Associated Colorectal Cancer in Mice

Consumption of a Western type diet is a known risk factor for colorectal cancer. Our group previously developed the total Western diet (TWD) for rodents with energy and nutrient profiles that emulate a typical Western diet. In this study, we tested the hypothesis that consumption of the TWD would enhance colitis, delay recovery from gut injury and promote colon tumorigenesis. In multiple experiments using the azoxymethane + dextran sodium sulfate or ApcMin/+ mouse models of colitis-associated colorectal carcinogenesis (CAC), we determined that mice fed TWD experienced more severe and more prolonged colitis compared to their counterparts fed the standard AIN93G diet, ultimately leading to markedly enhanced colon tumorigenesis. Additionally, this increased tumor response was attributed to the micronutrient fraction of the TWD, and restoration of calcium and vitamin D to standard amounts ameliorated the tumor-promoting effects of TWD. Finally, exposure to the TWD elicited large scale, dynamic changes in mRNA signatures of colon mucosa associated with interferon (IFN) response, inflammation, innate immunity, adaptive immunity, and antigen processing pathways, among others. Taken together, these observations indicate that consumption of the TWD markedly enhanced colitis, delayed recovery from gut injury, and enhanced colon tumorigenesis likely via extensive changes in expression of immune-related genes in the colon mucosa

Influence of Total Western Diet on Docosahexaenoic Acid Suppression of Silica-Triggered Lupus Flaring in NZBWF1 Mice

Lupus is a debilitating multi-organ autoimmune disease clinically typified by periods of flare and remission. Exposing lupus-prone female NZBWF1 mice to crystalline silica (cSiO2), a known human autoimmune trigger, mimics flaring by inducing interferon-related gene (IRG) expression, inflammation, ectopic lymphoid structure (ELS) development, and autoantibody production in the lung that collectively accelerate glomerulonephritis. cSiO2-triggered flaring in this model can be prevented by supplementing mouse diet with the ω-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA). A limitation of previous studies was the use of purified diet that, although optimized for rodent health, does not reflect the high American intake of saturated fatty acid (SFA), ω-6 PUFAs, and total fat. To address this, we employed here a modified Total Western Diet (mTWD) emulating the 50th percentile U.S. macronutrient distribution to discern how DHA supplementation and/or SFA and ω-6 reduction influences cSiO2-triggered lupus flaring in female NZBWF1 mice. Six-week-old mice were fed isocaloric experimental diets for 2 wks, intranasally instilled with cSiO2 or saline vehicle weekly for 4 wks, and tissues assessed for lupus endpoints 11 wks following cSiO2 instillation. In mice fed basal mTWD, cSiO2 induced robust IRG expression, proinflammatory cytokine and chemokine elevation, leukocyte infiltration, ELS neogenesis, and autoantibody production in the lung, as well as early kidney nephritis onset compared to vehicle-treated mice fed mTWD. Consumption of mTWD containing DHA at the caloric equivalent to a human dose of 5 g/day dramatically suppressed induction of all lupus-associated endpoints. While decreasing SFA and ω-6 in mTWD modestly inhibited some disease markers, DHA addition to this diet was required for maximal protection against lupus development. Taken together, DHA supplementation at a translationally relevant dose was highly effective in preventing cSiO2-triggered lupus flaring in NZBWF1 mice, even against the background of a typical Western diet

Single cell analysis of docosahexaenoic acid suppression of sequential LPS-induced proinflammatory and interferon-regulated gene expression in the macrophage

Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24 h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4 h, and then subjected to scRNAseq using the 10X Chromium System. At 0 h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1, Hmox1, Chchd10) and metal homeostasis (e.g. Mt1, Mt2, Ftl1, Fth1), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1 h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4 h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf) and chemokine (e.g. Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages