mRNA Expression Of Pro-inflammatory Cytokines Research Articles

Establishment of a chronic biliary disease mouse model with cholecystoduodenal anastomosis for intestinal microbiome preservation

BACKGROUND Chronic biliary disease, including cholangitis and cholecystitis, is attributed to ascending infection by intestinal bacteria. Development of a mouse model for bile duct inflammation is imperative for the advancement of novel therapeutic approaches. Current models fail to replicate the harmful bacterial influx to the biliary tract observed in humans and spread of inflammation to the liver. Therefore, we aimed to establish an animal model of biliary disease that faithfully replicates the mechanisms of human diseases. AIM To establish a cholecystoduodenal anastomosis model capable of mimicking the mechanisms of ascending infection and inflammation observed in human biliary diseases. METHODS We established a mouse biliary disease model by directly connecting the gallbladder and duodenum, enabling ascending infection into the biliary tract without traversing the sphincter of Oddi. RESULTS In the cholecystoduodenal anastomosis mouse model, we observed impaired epithelial structure, wall thickening, and macrophage recruitment in the gallbladder. Despite the absence of postoperative antibiotics, we detected no changes in serum proinflammatory cytokine levels, indicating no systemic inflammation. Moreover, patency between the gallbladder and duodenum was confirmed via common bile duct ligation. Injection of patient-derived pathogenic bacteria into bile duct-ligated mice led to ascending infection, which significantly increased proinflammatory cytokine mRNA expression in the liver, duodenum, and ileum. These results indicate that our mouse model exhibited a direct connection between the gallbladder and duodenum, leading to ascending infection and closely mimicking the clinical features of biliary diseases observed in humans. CONCLUSION The cholecystoduodenal anastomosis mouse model is an effective chronic biliary disease model with significant relevance in the development of microbiome-based therapies for the prevention and treatment of biliary disease.

CaMKII-dependent non-canonical RIG-I pathway promotes influenza virus propagation in the acute-phase of infection.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is one of hundreds of host-cell factors involved in the propagation of type A influenza virus (IAV), although its mechanism of action is unknown. Here, we identified CaMKII inhibitory peptide M3 by targeting its kinase domain using affinity-based screening of a tailored random peptide library. M3 inhibited IAV cytopathicity and propagation in cells by specifically inhibiting the acute-phase activation of retinoic acid-inducible gene I (RIG-I), which is uniquely regulated by CaMKII. Downstream of the RIG-I pathway activated TBK1 and then IRF3, which induced small but sufficient amounts of transcripts of the genes for IFN α/β to provide the capped 5'-ends that were used preferentially as primers to synthesize viral mRNAs by the cap-snatching mechanism. Importantly, knockout of RIG-I in cells almost completely inhibited the expression of IFN mRNAs and subsequent viral NP mRNA early in infection (up to 6 h after infection), which then protected cells from cytopathicity 24 h after infection. Thus, CaMKII-dependent acute-phase activation of RIG-I promoted IAV propagation, whereas the canonical RIG-I pathway stimulated antiviral activity by inducing large amounts of mRNA for IFNs and then for antiviral proteins later in infection. Co-administration of M3 with IAV infection rescued mice from the lethality and greatly reduced proinflammatory cytokine mRNA expression in the lung, indicating that M3 is highly effective against IAV in vivo. Thus, regulation of the CaMKII-dependent non-canonical RIG-I pathway may provide a novel host-factor-directed antiviral therapy.IMPORTANCEThe recent emergence of IAV strains resistant to commonly used therapeutic agents that target viral proteins has exacerbated the need for innovative strategies. Here, we originally identified CaMKII-inhibitory peptide M3, which efficiently inhibits IAV-lethality in vitro and in vivo. M3 specifically inhibited the acute-phase activation of RIG-I, which is a novel pathway to promote IAV propagation. Thus, this pathway acts in an opposite manner compared with the canonical RIG-I pathway, which plays essential roles in antiviral innate immune response later in infection. The CaMKII-dependent non-canonical RIG-I pathway can be a promising and novel drug target for the treatment of infections.

Anti-Inflammatory Effects of Apostichopus japonicus Extract in Porphyromonas gingivalis-Stimulated RAW 264.7 Cells

Apostichopus japonicus has been used both as a food and in traditional medicine. However, its anti-inflammatory effects in periodontal diseases have not been studied. We examined the anti-inflammatory properties of Apostichopus japonicus extract in RAW 264.7 cells stimulated by Porphyromonas gingivalis. The cytotoxicity of Apostichopus japonicus extract was evaluated using the MTS assay. Its effect on NO production was then measured using the NO assay. The mRNA expression of inducible nitric oxide synthase (iNOS) and the pro-inflammatory cytokines IL-1β and IL-6 were assessed using quantitative real-time PCR (qRT-PCR). Western blotting was performed to investigate the expression of regulatory proteins involved in the NF-κB and MAPK signaling pathways. Apostichopus japonicus extract significantly inhibited NO production without cytotoxicity in RAW 264.7 cells. Following Porphyromonas gingivalis stimulation, treatment with the extract decreased iNOS mRNA expression and protein levels, which are responsible for NO production. The extract also suppressed the mRNA expression of pro-inflammatory cytokines. Additionally, Apostichopus japonicus extract inhibited NF-κB activation by regulating signaling molecules such as IKK and IκBα, while also preventing the phosphorylation of MAPK, including ERK, p38, and JNK, showing anti-inflammatory potential. Therefore, it may be a promising natural candidate for the development of new preventive and therapeutic strategies for periodontitis.

Paraprobiotic Levilactobacillus brevis KU15151 exhibits antioxidative and anti-inflammatory activities in LPS-induced A549 cells

Levilactobacillus brevis KU15151, isolated from kimchi, has been reported in previous studies to possess probiotic properties. Here, we sought to explore the potential of heat-killed L. brevis KU15151 in improving respiratory health by identifying its antioxidant and anti-inflammatory effects in LPS-induced A549 cells. Inactivated L. brevis KU15151 exhibited strong DPPH and ABTS radical-scavenging abilities (48.78 ± 3.95 % and 69.08 ± 1.09 %) and effectively reduced the production of reactive oxygen species (25.32 %). In addition, it was found to have anti-inflammatory effects by inhibiting phosphorylation of ERK 1/2 (0.556), JNK (0.476), p38 MAPK (0.580), p65 (0.579), and IκB-α (1.170), which are involved in MAPK and NF-κB signaling. It also suppressed the mRNA expression of pro-inflammatory cytokines (0.173–0.617), which are important factors in respiratory diseases. IL-6 (19.47 %) and eotaxin (50.19 %) levels were reduced as measured by ELISA. Therefore, heat-killed L. brevis KU15151 is expected to improve respiratory health.

Activation of glial cells induces proinflammatory properties in brain capillary endothelial cells in vitro

Neurodegenerative diseases are often accompanied by neuroinflammation and impairment of the blood-brain barrier (BBB) mediated by activated glial cells through their release of proinflammatory molecules. To study the effects of glial cells on mouse brain endothelial cells (mBECs), we developed an in vitro BBB model with inflammation by preactivating mixed glial cells (MGCs) with lipopolysaccharide (LPS) before co-culturing with mBECs to study the influence of molecules released by activated MGCs. The response of the mBECs to activated MGCs was compared to direct stimulation with LPS. The cytokine profile of activated MGCs was analyzed together with their effects on the mBEC’s integrity, expression of tight junction proteins, adhesion molecules, and BBB-specific transport proteins. Stimulation of MGCs significantly upregulated mRNA expression and secretion of several pro-inflammatory cytokines. Co-culturing mBECs with pre-stimulated MGCs significantly affected the barrier integrity of mBECs similar to direct stimulation with LPS. The gene expression levels of tight junction proteins were unaltered, but tight junction proteins revealed rearrangements with respect to subcellular distribution. Compared to direct stimulation with LPS, the expression of cell-adhesion molecules was significantly increased when mBECs were co-cultured with prestimulated MGCs and thus pre-activating MGCs transforms mBECs into a proinflammatory phenotype.

Empagliflozin Attenuates High-Glucose-Induced Astrocyte Activation and Inflammation via NF-κB Pathway.

Sodium-glucose cotransporter-2 (SGLT2) inhibitors regulate blood glucose levels in patients with type 2 diabetes mellitus and may also exert anti-inflammatory and anti-atherosclerotic effects by promoting M2 macrophage polarization. Although SGLT2 is expressed in brain regions that influence glucose balance and cognitive function, its roles in the central nervous system are unclear. This study investigated the effects of empagliflozin (EMPA), an SGLT2 inhibitor, on hypothalamic inflammation associated with metabolic diseases. Mice were subjected to a high-fat diet (HFD) for varying durations (3 d, 3 weeks, and 16 weeks) and treated with EMPA for 3 weeks (NFD, NFD + EMPA, HFD, HFD + EMPA; n = 5/group). EMPA regulated the expression of astrocyte markers and pro-inflammatory cytokine mRNA in the hypothalamus of HFD-induced mice, which was linked to regulation of the NF-κB pathway. Under hyperglycemic conditions, EMPA may mitigate hypothalamic inflammation by modulating astrocyte activation via the NF-κB pathway. Our findings demonstrated that EMPA possesses therapeutic potential beyond merely lowering blood glucose levels, opening new avenues for addressing inflammation and providing neuroprotection in metabolic disease management.

Kjellmaniella crassifolia Reduces Lipopolysaccharide-Induced Inflammation in Caco-2 Cells and Ameliorates Loperamide-Induced Constipation in Mice.

Gastrointestinal disorders are widespread globally, with inflammatory diseases being particularly prominent. This study aimed to investigate the effect of Kjellmaniella crassifolia hot water extract (KCH) on lipopolysaccharide (LPS)-induced inflammation in human intestinal epithelial (Caco-2) cells and loperamide-induced constipation in BALB/c mice. The study's findings revealed that KCH dose-dependently increased the cell viability and reduced the NO production by decreasing the iNOS and COX-2 expression in LPS-stimulated Caco-2 cells. Also, KCH downregulated the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) by regulating the activation of MAPK and NF-κB signaling pathways in LPS-stimulated Caco-2 cells. In addition, KCH increased the expression levels of tight junction proteins, occludin, ZO-1, and claudin-1 in a dose-dependent manner. Furthermore, in vivo study outcomes demonstrated that KCH improved intestinal transit, increased fecal moisture content, and reduced fecal impaction in constipated mice. KCH decreased the mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α), thereby increasing the expression levels of intestinal tight junction proteins (occludin, ZO-1, and claudin-1) in the small intestine tissues of the experimental mice. These proteins may help regulate intestinal motility and improve stool passage, thus reducing constipation. These findings suggest that KCH could be a promising functional food ingredient for managing intestinal inflammation, inflammation-related disorders, constipation, and the pathophysiology of constipation.

Immune mRNA Expression and Fecal Microbiome Composition Change Induced by Djulis (Chenopodium formosanum Koidz.) Supplementation in Aged Mice: A Pilot Study.

Background and Objectives: The aging process has always been associated with a higher susceptibility to chronic inflammatory lung diseases. Several studies have demonstrated the gut microbiome's influence on the lungs through cross-talk or the gut-lungs axis maintaining nutrient-rich microenvironments. Taiwan djulis (Chenopodium formosanum Koidz.) provides antioxidant and anti-inflammatory characteristics that could modulate the gut microbiome. This could induce the gut-lung axis through microbial cross-talk, thus favoring the modulation of lung inflammation. Materials and Methods: Here, we investigate the immune mRNA expression in the spleen, fecal microbiome composition, and hyperplasia of the bronchial epithelium in aged 2-year-old BALB/c mice after 60 days of supplementation of djulis. Results: The pro-inflammatory cytokines IFN-γ, TNF-α, and IL-1β, T; cells CD4 and CD8; and TLRs TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 were reduced in their mRNA expression levels, while the anti-inflammatory cytokines IL-2, IL-4, and IL-10 were highly expressed in the C. formosanum-treated group. Interestingly, the fecal microbiome composition analysis indicated higher diversity in the C. formosanum-treated group and the presence of butyrate-producing bacteria that are beneficial in the gut microbiome. The histopathology showed reduced hyperplasia of the bronchial epithelium based on the degree of lesions. Conclusions: Our findings suggest that Taiwan djulis can modulate the gut microbiome, leading to microbial cross-talk; reducing the mRNA expression of pro-inflammatory cytokines, T cells, and TLRs; and increasing anti-inflammatory cytokines in the spleen, as cytokines migrate in the lungs, preventing lung inflammation damage in aged mice or the gut-lung axis. Thus, Taiwan djulis could be considered a beneficial dietary component for the older adult population. The major limitation includes a lack of protein validation of cytokines and TLRs and quantification of the T cell population in the spleen as a marker of the gut-lung axis.

Anti-inflammation Mechanisms of Flavones Are Highly Sensitive to the Position Isomers of Flavonoids: Acacetin vs Biochanin A.

Acacetin (ACA) and biochanin A (BCA) are isomeric monomethoxyflavones with different structural positions of the 4'-methoxy-phenyl group. Both of them are present in many commonly consumed foods, such as citrus fruits and vegetables, and have been discovered with anti-inflammatory activities, but their mechanisms of action are not clearly elucidated at the molecular level. Herein, we reported the structure-activity relationship of ACA and BCA regarding their potency in inhibiting nitric oxide (NO) production, proinflammatory enzyme expression, and mRNA expression of proinflammatory cytokines in the lipopolysaccharide (LPS)-induced RAW 264.7 cells. Furthermore, transcriptome analysis was conducted to map out the overall pathways impacted by these two isomers. Our results showed that ACA possessed higher suppressive activity in nitric oxide (NO) production (IC50 = 23.93 ± 1.74 μM for ACA and IC50 = 71.41 ± 8.07 μM for BCA), inducible nitric oxide synthase (iNOS) expression, and proinflammatory interleukin (IL)-1β mRNA expression, but lower inhibitory activity in IL-6 mRNA expression as compared with BCA. Although two compounds were revealed to bind to c-Src protein according to drug affinity responsive target stability (DARTS) and western blotting results, molecular docking and molecular dynamics (MD) simulation showed that they had different binding sites, which subsequently resulted in different signaling transduction. ACA primarily exerted anti-inflammatory activity through the mitogen-activated protein kinase (MAPK) signaling pathway, the tumor necrosis factor (TNF) signaling pathway, and the hypoxia-inducible factor (HIF)-1 signaling pathway, while BCA was particularly involved in the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway, the nuclear factor kappa B (NF-κB) signaling pathway, the TNF signaling pathway, and the toll-like receptor (TLR) signaling pathway. Moreover, two isomers remained stable in the cell culture medium and did not encounter the biotransformation process in RAW 264.7 cells. However, BCA exhibited insufficient cellular uptake ability and transport efficiency in macrophages compared to ACA. Taken together, ACA is more promising for controlling inflammation and worthy of further study for human use.

Eutypetides A—E, Structurally Intriguing Polyketides Formed by Intramolecular [4+2] Cycloaddition from Marine‐Derived Fungus Eutypella sp. F0219

Comprehensive SummaryFour polycyclic ten‐membered lactones possessing unprecedented 10/6/5 tricyclic ring skeleton, named eutypetides A—D (1—4), and an intriguing polyketide containing a hexahydroisobenzofuran‐1(3H)‐one motif, named eutypetide E (5) were isolated from the marine‐derived fungus Eutypella sp. F0219, together with three new related biosynthetic polyketides eutypetides F—H (6—8). The absolute configurations of 1—5 were unequivocally determined by single‐crystal X‐ray diffraction analyses (Cu Kα), modified Mosher's method and electronic circular dichroism (ECD) calculations. Eutypetides G (7) showed remarkable anti‐inflammatory activity and could reduce the mRNA expression of proinflammatory cytokines IL‐1β, IL‐6, TNF‐α, and iNOS induced by LPS. Most notably, compounds 1—4 were formed biogenetically from 6—7 via the key intramolecular [4+2] cycloaddition, while compound 5 could be constructed biogenetically from 8 through the intramolecular [4+2] cycloaddition. All the above eight polyketides are proposed to originate from a C10 and a C6 fatty acid.

Immunotoxic response of bio-based plastic on early life stage zebrafish (Danio rerio): A safe alternative to petroleum-based plastics?

Bio-based plastics are marketed as environmentally friendly alternatives to petroleum-based plastics, although they require specific composting conditions for degradation, which leads to their accumulation in the environment and potential risks to aquatic organisms. We hypothesized that the accumulation of bio-based plastics may induce immunotoxic responses in fish. Our research focused on the accumulation and immunotoxicity of 80 nm polylactic acid (PLA) and polystyrene (PS) (0.1–10 mg/L) on early life stage zebrafish (Danio rerio) exposed for 7 days. Compared to PS, there was a higher accumulation of PLA in larvae. Exposure to PLA resulted in a significant increase in neutrophils and macrophages, while immune protein levels such as Complement 3 (C3), Immunoglobulin M (IgM), and C-reactive protein (CRP) were significantly reduced. Furthermore, the mRNA expression of pro-inflammatory cytokines, including tnf-α and il-6, were significantly elevated in PLA treatments. Additionally, PLA-exposed zebrafish were more susceptible to infection by Vibrio parahaemolyticus. Interestingly, at the same concentration, exposures to PS did not induce significant changes in macrophages or immune protein levels, C3 and IgM. This suggests that PLA has a greater immunotoxic response relative to PS. Our research findings contradict the popular belief that bio-based plastics are non-toxic and harmless, which may have potential risk to aquatic organisms.

Modulation of Inflammation in McCoy Cells by Zinc Nanoparticles Conjugated With β-Chitosan.

This study investigated biosynthetically derived β-chitosan-derived zinc nanoparticles (β-Ch-Zn NPs) for their potential anti-inflammatory properties on McCoy cells.β-Ch-Zn NPs were synthesized usinga green chemistry approach, and their characterization confirmed successful synthesis, appropriate size, and morphology. The study aimed to evaluate the cytotoxicity ofβ-Ch-Zn NPs and their effects on inflammatory responses in McCoy cells stimulated with lipopolysaccharide (LPS). β-Ch-Zn NPs were synthesizedand characterized using Fourier-transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-Vis) spectroscopy, and X-ray diffraction (XRD) to confirm their structural and morphological properties. The cytotoxicity ofβ-Ch-Zn NPs was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay at various concentrations to determine safe doses for subsequent experiments. To induce inflammation, McCoy cells were pretreated with β-Ch-Zn NPs at different concentrations before LPS stimulations. Gene expression analysis using quantitative real-time polymerase chain reaction was performed to measure the messenger RNA (mRNA) levels of proinflammatory cytokine. FTIR, UV-Vis spectroscopy, and XRD confirmed the successful synthesis ofβ-Ch-Zn NPs with the desired size and morphology. The MTT assay demonstrated concentration-dependent cytotoxicity of β-Ch-Zn NPs, indicating safety for cellular studies. Pretreatment withβ-Ch-Zn NPs significantly downregulated the mRNA expression of proinflammatory cytokines. The nanoparticles effectively downregulate proinflammatory cytokines and promote anti-inflammatory pathways, as evidenced by the significant reduction in interleukin (IL)-2, IL-6, hypoxia-inducible factor, and nuclear factor kappa B expression in a dose-dependent manner. This study demonstrated that biosynthetically derivedβ-Ch-Zn NPs exhibit potent anti-inflammatory effects in McCoy cells. These findings underscore the therapeutic potential ofβ-Ch-Zn NPs for treating inflammatory conditions and support further investigation into their in vivo efficacy and safety.

Abstract 59: Beclin-1-Dependent Autophagy Prevents Hyperaldosternism-Induced Endothelial Dysfunction And Hypertension

Hyperaldosteronism (HA), characterized by the overproduction of aldosterone (Aldo), leads to cardiovascular damage, including endothelial dysfunction. Beclin-1 (Becn1) is a key protein regulating autophagy, a recycling process for unwanted components. Whether HA disturbs autophagic flux in endothelial cells via Becn1 is unknown. We hypothesize that Becn1-dependent autophagic flux will improve endothelial function and inhibit the hypertension (HTN) in HA. 10-to-12-week-old male C57BL/6J (WT) or Becn1 knock-in mice (Becn1 Tg ) were infused with aldosterone for 14 days (600 µg/kg/day). Vascular function was studied in mesenteric arteries (MA) and blood pressure was analyzed by radiotelemetry. HA disrupted MA autophagic flux characterized by accumulation of LC3A/B and p62 and reduction in Becn1 expression and activity followed by suppressed acetylcholine (ACh)-induced vasodilation (% relaxation) in WT [WT: 91.5±1.1 vs HA: 56.7±2.7], whereas Becn1 Tg mice were protected from HA-induced HTN [mean blood pressure (mmHg) – WT_Aldo: 149.1±1.2 vs BCN1_Aldo: 118.1±0.8] and endothelial dysfunction [HA_Becn1 Tg : 92.8±2.9]. Given the link between the immune system and cardiovascular diseases, we investigated whether HA reduces autophagic flux and BCN1 in immune cells. HA led to the accumulation of LC3A/B and p62, and decreased BCN1 activity in peritoneal cavity cells of WT mice, however bone marrow transplant from Becn1 Tg mice into WT mice did not confer protection against HA-induced changes in vascular reactivity [WT to WT_HA: 51.2±1.3 vs Becn1 Tg to WT_HA: 53.8±1.9]. As a pharmacological approach, we accelerated autophagic flux by treating mice with spermidine (3 mM for 7 days), which subsequently improved endothelial function in HA [HA: 52.7±3.9 vs HA+spermidine: 88.9±3.9]. To assess the direct impact of Aldo on disrupting autophagy flux in endothelial cells, we treated mesenteric endothelial cells (MEC) with Aldo (0.1 µM for 24 hours). This treatment resulted in the accumulation of LC3A/B and p62 proteins and a reduction in BCN1 activity, accompanied by an upregulation in the mRNA expression of pro-inflammatory cytokines. All these effects were prevented by spermidine (100 uM for 3h). These findings reveal a novel pathway in HA-associated endothelial dysfunction and HTN, where Aldo disrupts autophagic flux and inflammation by suppressing BCN1 activity, positioning BCN1 as a pharmacological target for cardiovascular outcomes in HA.

Developing a mouse model of human coronavirus NL63 infection: comparison with rhinovirus-A1B and effects of prior rhinovirus infection.

Human coronavirus (HCoV)-NL63 causes respiratory tract infections in humans and uses angiotensin-converting enzyme 2 (ACE2) as a receptor. We sought to establish a mouse model of HCoV-NL63 and determine whether prior rhinovirus (RV)-A1B infection affected HCoV-NL63 replication. HCoV-NL63 was propagated in LLC-MK2 cells expressing human ACE2. RV-A1B was grown in HeLa-H1 cells. C57BL6/J or transgenic mice expressing human ACE2 were infected intranasally with sham LLC-MK2 cell supernatant or 1 × 105 tissue culture infectious dose (TCID50) units HCoV-NL63. Wild-type mice were infected with 1 × 106 plaque-forming units (PFU) RV-A1B. Lungs were assessed for vRNA, bronchoalveolar lavage (BAL) cells, histology, HCoV-NL63 nonstructural protein 3 (nsp3), and host gene expression by next-generation sequencing and qPCR. To evaluate sequential infections, mice were infected with RV-A1B followed by HCoV-NL63 infection 4 days later. We report that hACE2 mice infected with HCoV-NL63 showed evidence of replicative infection with increased levels of vRNA, BAL neutrophils and lymphocytes, peribronchial and perivascular infiltrates, and expression of nsp3. Viral replication peaked 3 days after infection and inflammation persisted 6 days after infection. HCoV-NL63-infected hACE2 mice showed increased mRNA expression of IFNs, IFN-stimulated proteins, and proinflammatory cytokines. Infection with RV-A1B 4 days before HCoV-NL63 significantly decreased both HCoV-NL63 vRNA levels and airway inflammation. Mice infected with RV-A1B prior to HCoV-NL63 showed increased expression of antiviral proteins compared with sham-treated mice. In conclusion, we established a mouse model of HCoV-NL63 replicative infection characterized by relatively persistent viral replication and inflammation. Prior infection with RV-A1B reduced HCoV-NL63 replication and airway inflammation, indicative of viral interference.NEW & NOTEWORTHY We describe a mouse model of human coronavirus (HCoV) infection. Infection of transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) with HCoV-NL63 produced a replicative infection with peribronchial inflammation and nonstructural protein 3 expression. Mice infected with RV-A1B 4 days before HCoV-NL63 showed decreased HCoV-NL63 replication and airway inflammation and increased expression of antiviral proteins compared with sham-treated mice. This research may shed light on human coronavirus infections, viral interference, and viral-induced asthma exacerbations.

Antioxidative and immunity-enhancing effects of heat-killed probiotic Enterococcus faecium KU22001 without toxin or antibiotic resistance

This study evaluated the probiotic properties, safety profile, and antioxidative and immune system-enhancing effects of Enterococcus faecium strains isolated from human infant feces. E. faecium KU22001, E. faecium KU22002, and E. faecium KU22005 exhibited potential probiotic properties; however, to eliminate concerns about toxin production and antibiotic resistance, the E. faecium strains were heat-treated prior to experimental usage. E. faecium KU22001 showed the highest antioxidant activity and lowest reactive oxygen species production among the three strains. The immune system-enhancing effects of heat-killed E. faecium strains were evaluated using a nitric oxide assay. E. faecium KU22001 induced an increase in the mRNA expression of inducible nitric oxide synthase, cyclooxygenase-2, and proinflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in RAW 264.7 cells. Furthermore, E. faecium KU22001 activated the mitogen-activated protein kinase pathway, which was a key regulator of the immune system. These results demonstrate the potential use of E. faecium KU22001 as a multifunctional food material.

In mouse model of mixed granulocytic asthma with corticosteroid refractoriness, Bronchom mitigates airway hyperresponsiveness, inflammation and airway remodeling

BackgroundAsthma is a heterogeneous, inflammatory disease with several phenotypes and endotypes. Severe asthmatics often exhibit mixed granulocytosis with reduced corticosteroid sensitivity. Bronchom is a newly developed Ayurvedic prescription medicine, indicated for the treatment of obstructive airway disorders. The purpose of the present study was to evaluate the in-vivo efficacy of Bronchom in mouse model of mixed granulocytic asthma with steroidal recalcitrance.MethodsHigh-performance thin layer chromatography (HPTLC) and Ultra-high performance liquid chromatography (UHPLC) were employed to identify and quantitate the phytometabolites present in Bronchom. The preclinical effectiveness of Bronchom was assessed in house dust mite (HDM) and Complete Freund’s adjuvant (CFA)-induced mixed granulocytic asthma model in mice. High dose of dexamethasone was tested parallelly. Specific-pathogen-free C57BL/6 mice were immunized with HDM and CFA and nineteen days later, they were intranasally challenged with HDM for four consecutive days. Then the mice were challenged with nebulized methacholine to evaluate airway hyperresponsiveness (AHR). Inflammatory cell influx was enumerated in the bronchoalveolar lavage fluid (BALF) followed by lung histology. Additionally, the concentrations of Th2 and pro-inflammatory cytokines was assessed in the BALF by multiplexed immune assay. The mRNA expression of pro-inflammatory cytokines and Mucin 5AC (MUC5AC) was also evaluated in the lung.ResultsHPTLC fingerprinting and UHPLC quantification of Bronchom revealed the presence of bioactive phytometabolites, namely, rosmarinic acid, gallic acid, methyl gallate, piperine, eugenol and glycyrrhizin. Bronchom effectively reduced AHR driven by HDM-CFA and the influx of total leukocytes, eosinophils and neutrophils in the BALF. In addition, Bronchom inhibited the infiltration of inflammatory cells in the lung as well as goblet cell metaplasia. Further, it also suppressed the elevated levels of Th2 cytokines and pro-inflammatory cytokines in the BALF. Similarly, Bronchom also regulated the mRNA expression of pro-inflammatory cytokines as well as MUC5AC in mice lungs. Reduced effectiveness of a high dose of the steroid, dexamethasone was observed in the model.ConclusionsWe have demonstrated for the first time the robust pharmacological effects of an herbo-mineral medicine in an animal model of mixed granulocytic asthma induced by HDM and CFA. The outcomes suggest the potential utility of Bronchom in severe asthmatics with a mixed granulocytic phenotype.

Abstract Mo137: Modulation of the Inflammatory and Fibrotic Effects of Angiotensin on Cardiomyocytes through YAP-mediated Transcription.

Introduction: Signaling through the transcriptional co-activator Yes-associated protein (YAP) has been shown to protect the myocardium against ischemic or mechanical stress. Research Question: In light of the critical role of inflammation on adverse cardiac responses we examined the possibility that YAP regulates inflammatory responses to Angiotensin II (AngII)-mediated stress. Approach: Cardiomyocyte specific YAP-KO mice were generated, implanted with AngII minipumps, infused with AngII (1.5 mg/kg/min) or vehicle for periods of 3 hrs to 3 days and hearts collected for analysis. Results: YAP deletion markedly enhanced AngII-induced mRNA expression of pro-inflammatory cytokines and chemokines (e.g. CCL2, CXCL2, IL-6) at 3 or 24 hrs in the cardiomyocyte, but not non-myocyte cell fraction. Expression of mRNA for the hypertrophic genes ANP and MHC-β, in contrast, was decreased in YAP KO mice. After 3 days of AngII infusion there was enhanced mRNA for pro-fibrotic genes (Col1a1, Col3a1 and Periostin) and increased staining for COL1A1 and F4/80, indicative of increased fibrosis and inflammatory cell recruitment in the absence of YAP signaling. Single cell RNA sequencing revealed specific subsets of genes that were upregulated in the cardiomyocyte subcluster from YAP KO mice. These included pro-inflammatory cytokines, cJun family genes, TLR4 and, surprisingly, CaMKIId. Elevated activation of CaMKIIδ in response to AngII in YAP KO hearts was confirmed by increased phosphorylation of phospholamban. Cardiomyocytes expressing siYAP also showed enhanced expression of TLR4 and CaMKIIδ. Conclusion: Upregulated expression of cJun, TLR4 and CaMKII occurs in the absence of YAP and this conspires to create a greatly enhanced inflammatory milieu which fuels adverse responses to external insults. YAP activation serves to suppress these responses and thus temper development of maladaptive inflammation.

Effects of parity and early pregnancy on peripheral blood leukocytes in dairy cattle

Subfertility remains a major problem in the dairy industry. Only 35-40% of high-yielding dairy cows and 55-65% of nonlactating heifers become pregnant after their first service. The immune system plays a critical role in the establishment of pregnancy. However, it can also create challenges for embryo survival and contribute to reduced fertility. We conducted 2 separate experiments to characterize changes in subsets of peripheral blood leukocytes (PBL) and their phenotype over the estrous cycle and early pregnancy in heifers and cows. We used flow cytometry and RT-qPCR to assess protein and mRNA expression of molecules important for immune function. We observed that CD14+ monocytes and CD3+ T cells tended to be affected by pregnancy status in heifers, whereas CD8B+ lymphocytes and NCR1+ natural killer (NK) cells were affected during early pregnancy in cows. Changes in expression of immune function proteins appeared to be greater in heifers than cows. To compare the most striking differences between heifers and cows observed in the initial experiments, we conducted a third experiment where PBL sampled from heifers and cows were simultaneously collected and analyzed under the same experimental conditions. Our results indicate that, compared with heifers, cows had greater mRNA expression of proinflammatory cytokines (IFNG and IL6) and AHR protein along with greater percentage of MM20A+ neutrophils and myeloid cells expressing SIRPA, ITGAM and ITGAX. Moreover, animals that failed to become pregnant showed altered expression of anti-inflammatory molecules compared with cyclic and pregnant animals. Overall, these findings support the hypothesis that early pregnancy signaling alters the phenotype of immune cells in the peripheral blood and that there are differences in the peripheral immune response to pregnancy between cows and heifers. Because cows have lower conception rates than heifers, it is possible that a more proinflammatory immune status in peripheral blood may play a role in embryo loss.

SGLT2 Inhibitors Empagliflozin and Canagliflozin Ameliorate Allergic Asthma Responses in Mice.

Inhibitors of sodium/glucose cotransporter 2 (SGLT2), such as empagliflozin and canagliflozin, have been widely used to block glucose reabsorption in the proximal tubules of kidneys in patients with diabetes. A meta-analysis suggested that SGLT2 inhibitors are associated with a decreased risk of asthma development. Therefore, we investigated whether SGLT2 inhibitors could suppress allergic asthma. Empagliflozin and canagliflozin suppressed the in vitro degranulation reaction induced by antigens in a concentration-dependent manner in RBL-2H3 mast cells. Empagliflozin and canagliflozin were administered to BALB/c mice sensitized to ovalbumin (OVA). The administration of empagliflozin or canagliflozin significantly suppressed OVA-induced airway hyper-responsiveness and increased the number of immune cells and pro-inflammatory cytokine mRNA expression levels in bronchoalveolar lavage fluid. The administration of empagliflozin and canagliflozin also suppressed OVA-induced histopathological changes in the lungs. Empagliflozin and canagliflozin also suppressed serum IgE levels. These results suggested that empagliflozin and canagliflozin may be applicable for the treatment of allergic asthma by suppressing immune responses.

Antiretroviral drug dolutegravir induces inflammation at the mouse brain barriers.

Integrase strand transfer inhibitors (INSTIs) based antiretroviral therapy (ART) is currently used as first-line regimen to treat HIV infection. Despite its high efficacy and barrier to resistance, ART-associated neuropsychiatric adverse effects remain a major concern. Recent studies have identified a potential interaction between the INSTI, dolutegravir (DTG), and folate transport pathways at the placental barrier. We hypothesized that such interactions could also occur at the two major blood-brain interfaces: blood-cerebrospinal fluid barrier (BCSFB) and blood-brain barrier (BBB). To address this question, we evaluated the effect of two INSTIs, DTG and bictegravir (BTG), on folate transporters and receptor expression at the mouse BCSFB and the BBB invitro, exvivo and invivo. We demonstrated that DTG but not BTG significantly downregulated the mRNA and/or protein expression of folate transporters (RFC/SLC19A1, PCFT/SLC46A1) in human and mouse BBB models invitro, and mouse brain capillaries exvivo. Our invivo study further revealed a significant downregulation in Slc19a1 and Slc46a1 mRNA expression at the BCSFB and the BBB following a 14-day DTG oral treatment in C57BL/6 mice. However, despite the observed downregulatory effect of DTG in folate transporters/receptor at both brain barriers, a 14-day oral treatment of DTG-based ART did not significantly alter the brain folate level in animals. Interestingly, DTG treatment robustly elevated the mRNA and/or protein expression of pro-inflammatory cytokines and chemokines (Cxcl1, Cxcl2, Cxcl3, Il6, Il23, Il12) in primary cultures of mouse brain microvascular endothelial cells (BBB). DTG oral treatment also significantly upregulated proinflammatory cytokines and chemokine (Il6, Il1β, Tnfα, Ccl2) at the BCSFB in mice. We additionally observed a downregulated mRNA expression of drug efflux transporters (Abcc1, Abcc4, and Abcb1a) and tight junction protein (Cldn3) at the CP isolated from mice treated with DTG. Despite the structural similarities, BTG only elicited minor effects on the markers of interest at both the BBB and BCSFB. In summary, our current data demonstrates that DTG but not BTG strongly induced inflammatory responses in a rodent BBB and BCSFB model. Together, these data provide valuable insights into the mechanism of DTG-induced brain toxicity, which may contribute to the pathogenesis of DTG-associated neuropsychiatric adverse effect.