In Utero Exposure to Environmental Tobacco Smoke Increases Neuroinflammation in Offspring

The embryonic stage is the most vulnerable period for congenital abnormalities. Due to its prolonged developmental course, the central nervous system (CNS) is susceptible to numerous genetic, epigenetic, and environmental influences. During embryo implantation, the CNS is more vulnerable to external influences such as environmental tobacco smoke (ETS), increasing the risk for delayed fetal growth, sudden infant death syndrome, and immune system abnormalities. This study aimed to evaluate the effects of in utero exposure to ETS on neuroinflammation in the offspring of pregnant mice challenged or not with lipopolysaccharide (LPS). After the confirmation of mating by the presence of the vaginal plug until offspring birth, pregnant C57BL/6 mice were exposed to either 3R4F cigarettes smoke (Kentucky University) or compressed air, twice a day (1h each), for 21 days. Enhanced glial cell and mixed cell cultures were prepared from 3-day-old mouse pups. After cell maturation, both cells were stimulated with LPS or saline. To inhibit microglia activation, minocycline was added to the mixed cell culture media 24 h before LPS challenge. To verify the influence of in utero exposure to ETS on the development of neuroinflammatory events in adulthood, a different set of 8-week-old animals was submitted to the Autoimmune Experimental Encephalomyelitis (EAE) model. The results indicate that cells from LPS-challenged pups exposed to ETS in utero presented high levels of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNFα) and decreased cell viability. Such a proinflammatory environment could modulate fetal programming by an increase in microglia and astrocytes miRNA155. This scenario may lead to the more severe EAE observed in pups exposed to ETS in utero.

The Amino Acids Sensing and Utilization in Response to Dietary Aromatic Amino Acid Supplementation in LPS-Induced Inflammation Piglet Model

Dietary supplementation with aromatic amino acids (AAAs) has been demonstrated to alleviate intestinal inflammation induced by lipopolysaccharide (LPS) in the piglets. But the mechanism of AAA sensing and utilization under inflammatory conditions is not well-understood. The study was conducted with 32 weanling piglets using a 2 × 2 factorial arrangement (diet and LPS challenge) in a randomized complete block design. Piglets were fed as basal diet or the basal diet supplemented with 0.16% tryptophan (Trp), 0.41% phenylalanine (Phe), and 0.22% tyrosine (Tyr) for 21 days. The results showed that LPS treatment significantly reduced the concentrations of cholecystokinin (CCK) and total protein but increased leptin concentration, the activities of alanine transaminase, and aspartate aminotransferase in serum. Dietary supplementation with AAAs significantly increased the serum concentrations of CCK, peptide YY (PYY), and total protein but decreased the blood urea nitrogen. LPS challenge reduced the ileal threonine (Thr) digestibility, as well as serum isoleucine (Ile) and Trp concentrations, but increased the serum concentrations of Phe, Thr, histidine (His), alanine (Ala), cysteine (Cys), and serine (Ser) (P < 0.05). The serum-free amino acid concentrations of His, lysine (Lys), arginine (Arg), Trp, Tyr, Cys, and the digestibilities of His, Lys, Arg, and Cys were significantly increased by feeding AAA diets (P < 0.05). Dietary AAA supplementation significantly increased the serum concentrations of Trp in LPS-challenged piglets (P < 0.05). In the jejunal mucosa, LPS increased the contents of Ala and Cys, and the mRNA expressions of solute carrier (SLC) transporters (i.e., SLC7A11, SLC16A10, SLC38A2, and SLC3A2), but decreased Lys and glutamine (Gln) contents, and SLC1A1 mRNA expression (P < 0.05). In the ileal mucosa, LPS challenge induced increasing in SLC7A11 and SLC38A2 and decreasing in SLC38A9 and SLC36A1 mRNA expressions, AAAs supplementation significantly decreased mucosal amino acid (AA) concentrations of methionine (Met), Arg, Ala, and Tyr, etc. (P < 0.05). And the interaction between AAAs supplementation and LPS challenge significantly altered the expressions of SLC36A1 and SLC38A9 mRNA (P < 0.05). Together, these findings indicated that AAAs supplementation promoted the AAs absorption and utilization in the small intestine of piglets and increased the mRNA expressions of SLC transports to meet the high demands for specific AAs in response to inflammation and immune response.

Expression of genes associated with fertility in the uterus and oviduct of heifers challenged with lipopolysaccharide

Summary Lipopolysaccharide (LPS) endotoxemia has been negatively associated with fertility. This study aimed to investigate the effect of LPS-induced inflammation on gene expression associated with bovine fertility in the uterus and oviduct. Sixteen healthy heifers were divided into two groups. The LPS group (n = 8) received two intravenous (i.v.) injections of 0.5 µg/kg of body weight of LPS with a 24-h interval, and the control group (n = 8) received two i.v. injections of saline solution with the same interval of time. All the animals had the follicular wave synchronized. Three days after the second injection of LPS, all animals were slaughtered and uterine and oviduct samples were collected. Gene expression associated with inflammatory response, thermal and oxidative stresses, oviduct environment quality, and uterine environment quality was evaluated. Body temperature and leucogram demonstrated that LPS induced an acute systemic inflammatory response. In the uterus, the expression of PTGS2 and NANOG genes was downregulated by the LPS challenge. However, no change in expression was observed in the other evaluated genes in the uterus, nor those evaluated in the oviduct. In conclusion, the inflammatory process triggered by LPS did not persist in the uterus and oviduct 3 days after challenge with LPS. Nonetheless, reduction in PTGS2 and NANOG expression in the uterus suggested that, indirectly, LPS may have a prolonged effect, which may affect corpus luteum and endometrial functions.

Differential Macrophage Phenotype Rewired by Hantaan Virus Constrains the Magnitude of Inflammatory Responses in Murine versus Humans

Hantaan virus (HTNV) is principally maintained and transmitted by rodents in nature, the infection of which is non-pathogenic in the field or laboratory mouse, but can cause hemorrhagic fever with renal syndrome (HFRS) in human beings, a severe systemic inflammatory disease with high mortality. It remains obscure how HTNV infection leads to disparate outcomes in distinct species. Here, we revealed a differential immune status in murine versus humans post HTNV infection, which was orchestrated by the macrophage reprogramming process and characterized by late-phase inactivation of NF-κB signaling. In HFRS patients, the immoderate and continuous activation of inflammatory monocyte/macrophage (M1) launched TNFα-centered cytokine storm and aggravated host immunopathologic injury, which can be life-threatening; however, in field or laboratory mice, the M1 activation and TNFα release were significantly suppressed at the late infection stage of HTNV, restricting excessive inflammation and blocking viral disease process, which also protected mice from secondary LPS challenge or polymicrobial sepsis. Mechanistically, we found that murine macrophage phenotype was dynamically manipulated by HTNV via the Notch-lncRNA-p65 axis. At the early stage of HTNV infection, the intracellular domain of Notch receptor (NICD) was activated by viral nucleocapsid (NP) stimulation and potentiated the NF-κB pathway by associating with and facilitating the interaction between IKKβ and p65. At the late stage, Notch signaling launched the expression of diverse murine-specific long non-coding RNAs (lncRNAs) and attenuated M1 polarization. Among them, lncRNA 30740.1 (termed as lnc-ip65, an inhibitor of p65) bound to p65 and hindered its phosphorylation, exerting negative feedback on the NF-κB pathway. Genetic ablation of lnc-ip65 shifted the balance of macrophage polarization from a pro-resolution to an inflammatory phenotype, leading to superabundant production of pro-inflammatory cytokines and increasing mice susceptibility to HTNV infection or bacterial sepsis. Collectively, our findings identify an immune braking function and mechanism for murine lncRNAs in inhibiting p65-mediated M1 activation, opening a novel therapeutic avenue of controlling the magnitude of immune responses for HFRS and other inflammatory diseases.

Cis-9, Trans-11 CLA Alleviates Lipopolysaccharide-Induced Depression of Fatty Acid Synthesis by Inhibiting Oxidative Stress and Autophagy in Bovine Mammary Epithelial Cells

Lipopolysaccharide (LPS) is the dominating endotoxin of Gram-negative bacteria, which can cause mastitis. Bovine mammary epithelial cells (BMECs), as major components of the mammary gland, usually suffer LPS challenge. Cis-9, trans-11 conjugated linoleic acid (CLA) has been reported to have anti-inflammatory characteristics, while its anti-oxidative ability to maintain cellular homeostasis in BMECs under LPS challenge is limited. Therefore, we studied whether cis-9, trans-11 CLA can restore the disturbance of cellular homeostasis indicated by the redox status and autophagy level caused by LPS and have an effect on cellular function- milk fat metabolism. For oxidative stress, LPS challenge promoted the formation of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and decreased the concentration of glutathione. Anti-oxidative signaling regulated by transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) was also depressed by LPS at the mRNA and protein level. However, cis-9, trans-11 CLA pretreatment downregulated the formation of ROS and TBARS and upregulated the expression of antioxidative enzymes. As a part of innate immunity, autophagy was also motivated by LPS challenge, while CLA decreased the autophagy level. LPS and H2O2 inhibited milk fat synthesis-related transcription factor sterol regulatory element binding protein (SREBP1), peroxisome proliferator activated receptor gamma (PPARG) and their downstream enzymes. Furthermore, 50 uM cis-9, trans-11 CLA promoted the mRNA and protein abundance of milk fat synthesis-related genes and lipid droplet formation in BMECs. In conclusion, LPS challenge disturbed the cellular homeostasis and depressed milk fat synthesis in BMECs; while cis-9, trans-11 CLA alleviated oxidative stress and decreased autophagy level, thus promoting milk fat synthesis, which offers a natural therapeutic strategy for mastitis.

Vitamin D Treatment Sequence Is Critical for Transcriptome Modulation of Immune Challenged Primary Human Cells

Microbe-associated molecular patterns, such as lipopolysaccharide (LPS) and β-glucan (BG), are surrogates of immune challenges like bacterial and fungal infections, respectively. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), supports the immune system in its fight against infections. This study investigated significant and prominent changes of the transcriptome of human peripheral blood mononuclear cells that immediately after isolation are exposed to 1,25(OH)2D3-modulated immune challenges over a time frame of 24-48 h. In this in vitro study design, most LPS and BG responsive genes are downregulated and their counts are drastically reduced when cells are treated 24 h after, 24 h before or in parallel with 1,25(OH)2D3. Interestingly, only a 1,25(OH)2D3 pre-treatment of the LPS challenge results in a majority of upregulated genes. Based on transcriptome-wide data both immune challenges display characteristic differences in responsive genes and their associated pathways, to which the actions of 1,25(OH)2D3 often oppose. The joined BG/1,25(OH)2D3 response is less sensitive to treatment sequence than that of LPS/1,25(OH)2D3. In conclusion, the functional consequences of immune challenges are significantly modulated by 1,25(OH)2D3 but largely depend on treatment sequence. This may suggest that a sufficient vitamin D status before an infection is more important than vitamin D supplementation afterwards.

Effects of mannan oligosaccharides and Lactobacillus mucosae on growth performance, immune response, and gut health of weanling pigs challenged with Escherichia coli lipopolysaccharides

Addition of pre- and probiotics may confer growth and health benefits when added to the diet of pigs. To determine the effects of feeding mannan oligosaccharide (MOS) and Lactobacillus mucosae (LM) as prebiotic and probiotic sources in weanling pigs under immune challenge, 96 weaned pigs were randomly allotted to 16 experimental pens within a 2 × 2 factorial arrangement of treatments. Control diets with or without 0.1% yeast-derived MOS were randomly assigned to pens and 109 cfu/pig LM broth or a control broth were top-dressed daily. Pigs were fed one of four dietary treatments (control, MOS, LM, and MOS+LM) in Phases I and II (days 0 to 7 and days 7 to 21 postweaning, respectively) and a common diet during Phase III (days 21 to 35 postweaning). On day 14, all pigs were challenged with 100 µg/kg body weight (BW) Escherichia coli lipopolysaccharide (LPS) via intraperitonial injection. Feed disappearance and pig BW were measured weekly. Blood and fecal samples were collected weekly, and additional blood samples were collected on days 1 and 3 post-LPS challenge. On days 15 and 21, one pig per pen was euthanized for collection of ileal mucosa and duodenal and ileal tissue samples. From days 0 to 14, feeding LM decreased gain-to-feed ratio (G:F; P < 0.05). An interaction between LM and MOS was observed for G:F on days 14 to 21 (P < 0.05); G:F in LM (715 g/kg) was greater compared with MOS+LM (P < 0.05; 600 g/kg) and control (P < 0.10; 615 g/kg), but was not different (P > 0.10) from MOS (674 g/kg). After pigs were fed a common diet (days 21 to 35), G:F was decreased (P < 0.05) in the LM treatment groups. Pigs fed diets that included MOS had increased serum immunoglobulin (Ig) G on days 1 and 3 post-LPS challenge and 2 wk after removal of treatments (P < 0.05) and on days 14 and 21 postweaning (P < 0.10) compared with pigs fed diets without MOS. On day 15, mucosal immunoglobulin G was increased (P < 0.05) in control vs. MOS and LM groups. Circulating IL-1β in control and MOS+LM pigs increased (P < 0.05) on day 1 post-LPS challenge but did not change (P > 0.10) in MOS and LM groups. On day 15, pigs fed LM had decreased (P < 0.05) ileal crypt depth compared with pigs fed the control diet. On day 21, fecal propionate and butyrate tended to be lower (P < 0.10) in pigs fed MOS vs. control and MOS+LM diet. These preliminary findings suggest that feeding LM alone improved feed efficiency and ileal morphological structure during the first week of LPS challenge; additionally, feeding LM and MOS may have beneficial effects relative to immune biomarkers.

Core genes involved in the regulation of acute lung injury and their association with COVID-19 and tumor progression: A bioinformatics and experimental study

Acute lung injury (ALI) is a specific form of lung damage caused by different infectious and non-infectious agents, including SARS-CoV-2, leading to severe respiratory and systemic inflammation. To gain deeper insight into the molecular mechanisms behind ALI and to identify core elements of the regulatory network associated with this pathology, key genes involved in the regulation of the acute lung inflammatory response (Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Ptx3, Socs3) were revealed using comprehensive bioinformatics analysis of whole-genome microarray datasets, functional annotation of differentially expressed genes (DEGs), reconstruction of protein-protein interaction networks and text mining. The bioinformatics data were validated using a murine model of LPS-induced ALI; changes in the gene expression patterns were assessed during ALI progression and prevention by anti-inflammatory therapy with dexamethasone and the semisynthetic triterpenoid soloxolone methyl (SM), two agents with different mechanisms of action. Analysis showed that 7 of 8 revealed ALI-related genes were susceptible to LPS challenge (up-regulation: Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Socs3; down-regulation: Cat) and their expression was reversed by the pre-treatment of mice with both anti-inflammatory agents. Furthermore, ALI-associated nodal genes were analysed with respect to SARS-CoV-2 infection and lung cancers. The overlap with DEGs identified in postmortem lung tissues from COVID-19 patients revealed genes (Saa1, Rsad2, Ifi44, Rtp4, Mmp8) that (a) showed a high degree centrality in the COVID-19-related regulatory network, (b) were up-regulated in murine lungs after LPS administration, and (c) were susceptible to anti-inflammatory therapy. Analysis of ALI-associated key genes using The Cancer Genome Atlas showed their correlation with poor survival in patients with lung neoplasias (Ptx3, Timp1, Serpine1, Plaur). Taken together, a number of key genes playing a core function in the regulation of lung inflammation were found, which can serve both as promising therapeutic targets and molecular markers to control lung ailments, including COVID-19-associated ALI.

Lycium barbarum polysaccharides alleviate LPS-induced inflammatory responses through PPARγ/MAPK/NF-κB pathway in bovine mammary epithelial cells

As the main component of the gram-negative bacterial cell wall, lipopolysaccharide (LPS) is well-documented as an inducer of inflammation in bovine mammary cells. Lycium barbarum (goji) polysaccharides (LBP) have been used in non-ruminants as prebiotics to improve growth performance, immune ability and antioxidant capacity. We aimed to investigate the underlying effects of LBPs on pro-inflammatory responses in LPS-stimulated primary bovine mammary epithelial cells (bMECs). Cells were isolated from mammary tissue of 3 lactating Holstein cows without clinical disease (30.26 ± 3.1 kg/d of milk yield; 175 ± 6 DIM). For the pre-experimental treatment, bMECs were precultured with serum-free medium for 12 h. Treatments were as follows: pretreatment with culture medium devoid of LPS or LBP for 30 h (CON); CON for 24 h followed by challenge with 2 μg/mL LPS for 6 h (LPS); pretreatment with 100 μg/mL or 300 μg/mL LBP for 24 h followed by LPS challenge (2 μg/mL) for 6 h (LBP(100)+LPS; LBP(300)+LPS). To further determine if the effect of LBP on immune-regulation is PPARγ activation-dependent, an inhibitor of PPARγ, GW9662, at a concentration of 1 μM was used. Cells treated with LBP at 100, 300 and 500 μg/mL had upregulated protein abundance of PPARγ, while PGC1α had a higher expression only at 300 μg/mL of LBP treatment. Compared with CON, cells pretreated with LBP at 100 and 300 μg/mL had greater protein abundance of SCD1 and SREBP1. EdU staining and cell wound healing assays showed that the negative effect of LPS alone on cell proliferation was reversed by pretreatment with LBP at both 100 and 300 μg/mL. Upregulation of gene and protein abundance of proinflammatory factors and cytokines (COX-2, NLRP3, TNF-α, IL-1β and IL-6) induced by LPS stimulation were alleviated by LBP pretreatment at 300 μg/mL (more than 2-fold decrease). Compared with LPS challenge alone, phosphorylation of proteins involved in NF-κB (IκBα and p65) and MAPK (p38, JNK and ERK) pathways was downregulated following LBP treatment. Additionally, inhibition of PPARγ by GW9662 weakened the protective effect of LBP on LPS-induced protein abundance of phosphorylated p65, COX-2, IL-1β and TNF-α. These results indicated that the protective effect of LBP on LPS-induced bMECs inflammatory responses is PPARγ activation-dependent. As such, this knowledge might help design strategies for intervening against the detrimental effects of bovine mastitis.

Surfactant protein A enhances the degradation of LPS-induced TLR4 in primary alveolar macrophages involving Rab7, β-arrestin2, and mTORC1

Respiratory infections by Gram-negative bacteria are a major cause of global morbidity and mortality. Alveolar macrophages (AMs) play a central role in maintaining lung immune homeostasis and host defense by sensing pathogens via pattern recognition receptors (PRR). The PRR Toll-like receptor (TLR) 4 is a key sensor of lipopolysaccharide (LPS) from Gram-negative bacteria. Pulmonary surfactant is the natural microenvironment of AMs. Surfactant protein A (SP-A), a multifunctional host defense collectin, controls LPS-induced pro-inflammatory immune responses at the organismal and cellular level via distinct mechanisms. We found that SP-A post-transcriptionally restricts LPS-induced TLR4 protein expression in primary AMs from healthy humans, rats, wild-type and SP-A -/- mice by further decreasing cycloheximide-reduced TLR4 protein translation and enhances the co-localization of TLR4 with the late endosome/lysosome. Both effects as well as the SP-A-mediated inhibition of LPS-induced TNFα release are counteracted by pharmacological inhibition of the small GTPase Rab7. SP-A-enhanced Rab7 expression requires β-arrestin2 and, in β-arrestin2 -/- AMs and after intratracheal LPS challenge of β-arrestin2 -/- mice, SP-A fails to enhance TLR4/lysosome co-localization and degradation of LPS-induced TLR4. In SP-A -/- mice, TLR4 levels are increased after pulmonary LPS challenge. SP-A-induced activation of mechanistic target of rapamycin complex 1 (mTORC1) kinase requires β-arrestin2 and is critically involved in degradation of LPS-induced TLR4. The data suggest that SP-A post-translationally limits LPS-induced TLR4 expression in primary AMs by lysosomal degradation comprising Rab7, β-arrestin2, and mTORC1. This study may indicate a potential role of SP-A-based therapeutic interventions in unrestricted TLR4-driven immune responses to lower respiratory tract infections caused by Gram-negative bacteria.