Proinflammatory Cytokine Gene Expression Research Articles

Imbalanced IL10/TGF-β production by regulatory T-lymphocytes in patients with HTLV-1-associated myelopathy/ tropical spastic paraparesis

BackgroundHuman T-cell lymphotropic virus type 1 (HTLV-1), also denominated Human T-cell leukemia virus-1, induces immune activation and secretion of proinflammatory cytokines, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Regulatory T lymphocytes (Tregs) may control of inflammation through the production of regulatory cytokines, including IL10 and TGF-β. In this study we determined the frequencies of CD4 + and CD8 + Tregs in a HAM/TSP population, compared to asymptomatic carriers and uninfected individuals, as well as investigated the profiles of regulatory and inflammatory cytokines.MethodsAsymptomatic HTLV-1 carriers and HAM/TSP patients were matched by sex and age. The frequencies of IL10- and/or TGF-β-producing Tregs were quantified by flow cytometry. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify HTLV-1 proviral load and the mRNA expression of cytokines and cellular receptors in peripheral blood mononuclear cells.ResultsTotal frequencies of CD4 + Tregs, as well as the IL10-producing CD4 + and CD8 + Treg subsets, were statistically higher in patients with HAM/TSP compared to asymptomatic HTLV-1-infected individuals. In addition, a positive correlation was found between the frequency of CD4 + IL10 + Tregs and proviral load in the HAM/TSP patients evaluated. A positive correlation was also observed between gene expression of proinflammatory versus regulatory cytokines only in HAM / TSP group.ConclusionsA higher frequencies of IL10-producing Tregs were identified in patients with HAM/TSP. Imbalanced production of IL10 in relation to TGF-β may contribute to the increased inflammatory response characteristically seen in HAM/TSP patients.

Aconitate decarboxylase 1 (Acod1) suppresses inflammatory stimuli-induced immune responses in Japanese flounder (Paralichthys olivaceus)

Aconitate decarboxylase 1 (ACOD1, formerly termed as immunoresponsive gene 1) is an enzyme responsible for generation of anti-inflammatory metabolite itaconate through decarboxylation of cis-aconitate and plays a key role in inflammation regulation and anti-microbial host defense in mammals. However, the immune functions of Acod1 in fish remain largely undetermined. Here, we characterize a Acod1 homolog that is highly induced by LPS, poly(I:C) and extracellular ATP (eATP) challenges in the Japanese flounder Paralichthys olivaceus head kidney macrophages (HKMs). We find that Acod1 syntenic genes in P. olivaceus are similar with those in mammalian species and the deduced Japanese flounder ACOD1 protein shares similar protein domains and 3-D structures with its counterparts in mammals. We next show that itaconate derivatives dimethyl itaconate (DMI) and citraconate (Cit) can markedly down-regulate inflammatory stimuli, including LPS-, poly(I:C)- and eATP-induced inflammatory mediator nitric oxide (NO) production and gene expression of proinflammatory cytokines (IL-1β and TNF-α), chemokines (CXCL-8, CXCL-9 and CXCL-13), HMGB1 and inflammasome components (NLRP3, NLRC3, caspase1, ASC and GSDME), but up-regulate anti-inflammatory gene (IL-10 and SOCS1/3) expression in P. olivaceus HKMs. In addition, DMI and Cit treatment significantly inhibits Edwardsiella piscicida growth in P. olivaceus HKMs. Furthermore, knockdown of the endogenous Acod1 expression by siRNA silencing not only significantly increases LPS-, poly(I:C)- and eATP-induced proinflammatory gene expression but also decreases anti-inflammatory gene expression in the HKMs. In contrast, the LPS-, poly(I:C)- and eATP-indued proinflammatory gene expression and NO production are greatly attenuated in ACOD1 overexpressed Japanese flounder FG-9307 cells. Collectively, our studies demonstrate that the inducible fish Acod1 gene is a novel immune regulator in dampening inflammatory stimuli-induced immune responses and ACOD1-produced itaconate may play an essential role in anti-bacterial immunity in fish macrophages.

PNSC928, a plant-derived compound, specifically disrupts CtBP2-p300 interaction and reduces inflammation in mice with acute respiratory distress syndrome

BackgroundPrior research has highlighted the involvement of a transcriptional complex comprising C-terminal binding protein 2 (CtBP2), histone acetyltransferase p300, and nuclear factor kappa B (NF-κB) in the transactivation of proinflammatory cytokine genes, contributing to inflammation in mice with acute respiratory distress syndrome (ARDS). Nonetheless, it remains uncertain whether the therapeutic targeting of the CtBP2-p300-NF-κB complex holds potential for ARDS suppression.MethodsAn ARDS mouse model was established using lipopolysaccharide (LPS) exposure. RNA-Sequencing (RNA-Seq) was performed on ARDS mice and LPS-treated cells with CtBP2, p300, and p65 knockdown. Small molecules inhibiting the CtBP2-p300 interaction were identified through AlphaScreen. Gene and protein expression levels were quantified using RT-qPCR and immunoblots. Tissue damage was assessed via histological staining.Key findingsWe elucidated the specific role of the CtBP2-p300-NF-κB complex in proinflammatory gene regulation. RNA-seq analysis in LPS-challenged ARDS mice and LPS-treated CtBP2-knockdown (CtBP2KD), p300KD, and p65KD cells revealed its significant impact on proinflammatory genes with minimal effects on other NF-κB targets. Commercial inhibitors for CtBP2, p300, or NF-κB exhibited moderate cytotoxicity in vitro and in vivo, affecting both proinflammatory genes and other targets. We identified a potent inhibitor, PNSC928, for the CtBP2-p300 interaction using AlphaScreen. PNSC928 treatment hindered the assembly of the CtBP2-p300-NF-κB complex, substantially downregulating proinflammatory cytokine gene expression without observable cytotoxicity in normal cells. In vivo administration of PNSC928 significantly reduced CtBP2-driven proinflammatory gene expression in ARDS mice, alleviating inflammation and lung injury, ultimately improving ARDS prognosis.ConclusionOur results position PNSC928 as a promising therapeutic candidate to specifically target the CtBP2-p300 interaction and mitigate inflammation in ARDS management.

The Nurr1 ligand indole acetic acid hydrazide loaded onto ZnFe2O4 nanoparticles suppresses proinflammatory gene expressions in SimA9 microglial cells

The nuclear receptor-related factor 1 (Nurr1), an orphan nuclear receptor in microglia, has been recognized as a major player in attenuating the transcription of the pro-inflammatory genes to maintain CNS homeostasis. In this study, we investigate Nurr1 trans-repression activity by targeting this receptor with one of the indole derivatives 3-Indole acetic acid hydrazide (IAAH) loaded onto zinc iron oxide (ZnFe2O4) NPs coated with PEG. XRD, SEM, FTIR, UV–Vis spectroscopy, and DLS were used to characterize the synthesized IAAH-NPs. The anti-inflammatory properties of IAAH-NPs on LPS-stimulated SimA9 microglia were assayed by measuring pro-inflammatory cytokine gene expressions and protein levels using RT-PCR and ELISA, respectively. As a result, IAAH-NPs showed an ability to suppress pro-inflammatory genes, including IL-6, IL-1β, and TNF-α in LPS-stimulated SimA9 via targeting Nurr1. The current study suggests that ZnFe2O4 NPs as a delivery system can increase the efficiency of cellular uptake and enhance the IAAH ability to inhibit the pro-inflammatory cytokines. Collectively, we demonstrate that IAAH-NPs is a potential modulator of Nurr1 that combines nanotechnology as a delivery system to suppress neuroinflammation in CNS which opens a window for possible ambitious neuroprotective therapeutic approaches to neuro disorders.

Human milk affects TLR4 activation and LPS-induced inflammatory cytokine expression in Caco-2 intestinal epithelial cells

Human milk (HM) components affect immune cell toll-like receptor 4 (TLR4) signaling. However, studies examining the immunomodulatory impacts of HM on TLR4 signaling in intestinal epithelial cells (IECs) are limited. This study utilized both a TLR4 reporter cell line and a Caco-2 IEC model to examine the effects of HM on lipopolysaccharide (LPS)-induced TLR4 activation and cytokine responses, respectively. Additionally, we performed fast protein liquid chromatography and mass spectrometry to identify a HM component that contributes to the effect of HM on LPS/TLR4 signaling. HM enhances LPS-induced TLR4 signaling as well as LPS-induced IEC gene expression of pro-inflammatory cytokines and negative regulators of NF-κB. Human serum albumin (HSA) present in HM contributes to these effects. HSA within HM synergizes with LPS to induce IEC gene expression of pro-inflammatory cytokines and negative regulators of NF-κB. Altogether, this study provides mechanistic evidence behind the immunomodulatory function of HM on IECs, which may contribute to an enhanced immune response in breast-fed neonates.

Nonstructural Protein A238L of the African Swine Fever Virus (ASFV) Enhances Antiviral Immune Responses by Activating the TBK1-IRF3 Pathway.

African swine fever virus (ASFV) is a double-stranded DNA virus with an envelope. ASFV has almost the largest genome among all DNA viruses, and its mechanisms of immune evasion are complex. Better understanding of the molecular mechanisms of ASFV genes will improve vaccine design. A238L, a nonstructural protein of ASFV, inhibits NF-κB activation by suppressing the HAT activity of p300. Whether A238L also affects the transcriptional activity of IRF3 remains unexplored. Here we first confirmed the ability of A238L to suppress NF-κB-activity in L929 cells. A238L inhibits the expression of proinflammatory cytokine genes. In contrast, A238L increased the phosphorylation levels of TBK1 and IRF3 in three different cell lines. A238L increases the IRF3-driven promoter activity and induces IRF3 nuclear translocation. Furthermore, A238L enhanced innate antiviral immunity in the absence or presence of poly d (A:T) or poly (I:C) stimulation, or herpes simplex virus type 1 (HSV-1) or Sendai virus (SeV) infection. This study reveals a previously unrecognized role of A238L in promoting antiviral immune responses by TBK1-IRF3 pathway activation.

Characterization of exosome-mediated propagation of systemic inflammatory responses into the Central Nervous System.

The mechanisms through which systemic inflammation exerts its effect on the CNS are still not completely understood. Exosomes are small (30 to 100 nanometers) membrane-bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell-to-cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs, and cytokines both locally and systemically to the neighboring as well as distant cells to further modulate their transcriptional and/or translational states and affect the functional phenotype of those cells that have taken up these exosomes. The role of circulating blood exosomes leading to neuroinflammation during systemic inflammatory conditions was further characterized. Serum-derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice that were earlier treated with LPS and used to study SDEL effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as a control. In-vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the murine cell lines of microglia (BV-2), astrocytes (C8-D1A), and cerebral microvascular endothelial cells (bEnd.3). To further study their effects in-vivo, SDEL were intravenously injected into normal adult mice. Elevated mRNA expression of pro-inflammatory cytokines was observed in the brains of SDEL recipient mice. Proteomic analysis of the SDEL confirmed the increased expression of inflammatory cytokines in them. Together, these results further demonstrate and strengthen the novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions.

Innate immune responses are increased in children with acute asthma exacerbation.

Little is known about the immune responses during acute asthma exacerbation. In this study, we examined immune responses in children following an acute asthma exacerbation. We evaluated pro-inflammatory cytokine levels and gene expression profiles in blood samples from pediatric patients admitted for acute asthma exacerbation. Viral PCR was performed to differentiate between viral or non-viral-associated exacerbations. Following informed consent, clinical data were obtained from 20 children with asthma (median [interquartile range, IQR]: age 11.5 [8.0, 14.2]) years and 14 healthy age-matched controls (10.5 [7.0, 13.0]). Twelve had positive nasopharyngeal Polymerase chain reaction (PCR) for viral infection (11 rhinoviruses and 1 respiratory syncytial virus (RSV)). Nine were in the pediatric intensive care unit (PICU) and among them five required continuous positive airway pressure (CPAP). Mean (±SD) days on systemic steroids before drawing blood sample were 2.5 ± 1.6. Twelve had history of environmental allergies with 917 (274, 1396) IU/mL total IgE (median (IQR)). Compared with controls, IL-1RA and IL-10 levels were significantly increased and TNF-α significantly decreased in asthma subjects (p < .05 for all). RNA-seq analysis revealed 852 differentially expressed genes in subjects with asthma. Pathway analysis found upregulated genes and pathways involved in innate immune responses in subjects with asthma. Significantly reduced genes included pathways associated with T helper cell differentiation and activation. In acute asthma exacerbation, innate immune pathways remained increased while adaptive immune responses related to T helper cells are blunted and are independent of trigger or asthma severity. Our novel findings highlight the need to identify new therapies to target persistent innate immune responses to improve outcomes in acute asthma.

Effects of Fucoidans on Activated Retinal Microglia.

Sulfated marine polysaccharides, so-called fucoidans, have been shown to exhibit anti-inflammatory and immunomodulatory activities in retinal pigment epithelium (RPE). In this study, we tested the effects of different fucoidans (and of fucoidan-treated RPE cells) on retinal microglia to investigate whether its anti-inflammatory effect can be extrapolated to the innate immune cells of the retina. In addition, we tested whether fucoidan treatment influenced the anti-inflammatory effect of RPE cells on retinal microglia. Three fucoidans were tested (FVs from Fucus vesiculosus, Fuc1 and FucBB04 from Laminaria hyperborea) as well as the supernatant of primary porcine RPE treated with fucoidans for their effects on inflammatory activated (using lipopolysaccharide, LPS) microglia cell line SIM-A9 and primary porcine retinal microglia. Cell viability was detected with a tetrazolium assay (MTT), and morphology by Coomassie staining. Secretion of tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β) and interleukin 8 (IL8) was detected with ELISA, gene expression (NOS2 (Nitric oxide synthase 2), and CXCL8 (IL8)) with qPCR. Phagocytosis was detected with a fluorescence assay. FucBB04 and FVs slightly reduced the viability of SIM-A9 and primary microglia, respectively. Treatment with RPE supernatants increased the viability of LPS-treated primary microglia. FVs and FucBB04 reduced the size of LPS-activated primary microglia, indicating an anti-inflammatory phenotype. RPE supernatant reduced the size of LPS-activated SIM-A9 cells. Proinflammatory cytokine secretion and gene expression in SIM-A9, as well as primary microglia, were not significantly affected by fucoidans, but RPE supernatants reduced the secretion of LPS-induced proinflammatory cytokine secretion in SIM-A9 and primary microglia. The phagocytosis ability of primary microglia was reduced by FucBB04. In conclusion, fucoidans exhibited only modest effects on inflammatorily activated microglia by maintaining their cell size under stimulation, while the anti-inflammatory effect of RPE cells on microglia irrespective of fucoidan treatment could be confirmed, stressing the role of RPE in regulating innate immunity in the retina.

Comparative Assessment of the Anti-Helicobacter pylori Activity and Gastroprotective Effects of Three Herbal Formulas for Functional Dyspepsia In Vitro.

Helicobacter pylori has been implicated in various gastrointestinal disorders, including functional dyspepsia. This study aimed to compare the anti-H. pylori activity and gastroprotective effects of three typical herbal formulas used for gastrointestinal disorders in Korea: Shihosogan-tang (ST), Yijung-tang (YT), and Pyeongwi-san (PS). Firstly, we assessed the total phenolic and flavonoid contents, as well as the antioxidative capacity. Additionally, we evaluated the antibacterial effect on H. pylori using an ammonia assay, minimum inhibitory concentration (MIC) test, and the disk agar diffusion method. Furthermore, we examined alterations in the gene expression of tight junction proteins, pro-inflammatory cytokines, and cellular vacuolation using an AGS cell model infected with H. pylori. While ST exhibited a higher total phenolic content, superior free radical scavenging, and inhibition of H. pylori compared to YT and PS, YT more evidently inhibited gastric cellular morphological changes such as vacuolation. All formulations significantly ameliorated changes in inflammatory and gastric inflammation-related genes and cellular morphological alterations induced by H. pylori infection. Overall, the present in vitro study suggests that all three herbal formulas possess potential for ameliorating gastrointestinal disorders, with ST relatively excelling in inhibiting H. pylori infection and inflammation, while YT potentially shows greater efficacy in directly protecting the gastric mucosa.

Roflumilast ameliorates GAN diet-induced non-alcoholic fatty liver disease by reducing hepatic steatosis and fibrosis in ob/ob mice

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent progressive liver disease. Currently, there is only one drug for NAFLD treatment, and the options are limited. Phosphodiesterase-4 (PDE-4) inhibitors have potential in treating NAFLD. Therefore, this study aims to investigate the effect of roflumilast on NAFLD. Here, we fed ob/ob mice to induce the NAFLD model by GAN diet. Roflumilast (1 mg/kg) was administered orally once daily. Semaglutide (20 nmol/kg), used as a positive control, was injected subcutaneously once daily. Our findings showed that roflumilast has beneficial effects on NAFLD. Roflumilast prevented body weight gain and improved lipid metabolism in ob/ob-GAN NAFLD mice. In addition, roflumilast decreased hepatic steatosis by down-regulating the expression of hepatic fatty acid synthesis genes (SREBP1c, FASN, and CD36) and improving oxidative stress. Roflumilast not only reduced liver injury by decreasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, but also ameliorated hepatic inflammation by reducing the gene expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). Roflumilast lessened liver fibrosis by inhibiting the expression of fibrosis mRNA (TGFβ1, α-SMA, COL1a1, and TIMP-1). Collectively, roflumilast could ameliorate NAFLD, especially in reducing hepatic steatosis and fibrosis. Our findings suggested a PDE-4 inhibitor roflumilast could be a potential drug for NAFLD.

Stryphnodendron adstringens have a modulatory effect on inflammatory cytokines markers of in vitro activated macrophages

Aim: The purpose of this study is to conduct a comprehensive investigation into the modulatory effects of Stryphnodendron adstringens (Mart.; S. adstringens), a Brazilian wound-healing plant, on the expression of inflammatory cytokines. This will be achieved using an in vitro protocol with the commercial macrophage cell line RAW 264.7. Methods: The macrophage inflammatory response was induced by the natural antigen phytohemagglutinin (PHA), with and without supplementation of different concentrations of S. adstringens extract. The effects on cell proliferation rate and the concentration and production of transcripts of pro-inflammatory cytokines interleukin 1β (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), as well as the anti-inflammatory cytokine IL-10, were assessed using spectrophotometric, immunoassay, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques. Results: S. adstringens extract at all concentrations tested here reduced the cellular proliferation rate of activated macrophages. Extracts at concentrations of 0.49 mg/mL and 0.99 mg/mL decreased the protein and gene expression of pro-inflammatory cytokines, exhibiting the opposite effect concerning IL-10. Conclusions: The findings suggest that the wound-healing action of S. adstringens may encompass differential modulation of inflammation associated with tissue injury.

The effect of gut microbiota-derived carnosine on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima

In the first study, an in vitro culture system was developed to investigate the effects of carnosine on macrophage proinflammatory cytokine response using an established chicken macrophage cell line (CMC), gut integrity using a chicken intestinal epithelial cell line (IEC), muscle differentiation in quail muscle cells (QMCs) and primary chicken embryonic muscle cells (PMCs), and direct anti-parasitic effect against Eimeria maxima sporozoites. Cells to be tested were seeded in 24-well plates and treated with carnosine at 4 different concentrations (0.1, 1.0, and 10.0 µg). After 18 h of incubation, cells were harvested to measure gene expression of proinflammatory cytokines in CMC, tight junction (TJ) proteins in IECs, and muscle cell growth markers in QMCs and PMCs. In vivo trials were conducted to investigate the effect of dietary carnosine on disease parameters in broiler chickens challenged with E. maxima. One hundred and twenty male broiler chickens (0-day-old) were allocated into four treatment groups: (1) basal diet without infection (NC), (2) basal diet with E. maxima infection (PC), (3) carnosine at 10.0 mg/kg feed with PC (HCS), and (4) carnosine at 1.0 mg/kg feed with PC (LCS). All groups except NC were orally infected with E. maxima on day 14. Jejunal samples were collected for lesion scoring and jejunum gut tissues were used for transcriptomic analysis of cytokines and TJ proteins. In vitro, carnosine treatment significantly decreased IL-1β gene expression in CMC following LPS stimulation. In vivo feeding studies showed that dietary carnosine increased BW and ADG of chickens in E. maxima-infected groups and reduced the jejunal lesion score and fecal oocyst shedding in HCS group. Jejunal IL-1β, IL-8, and IFN-γ expression were suppressed in the HCS group compared to PC. The expression levels of claudin-1 and occludin in IECs were also increased in HCS following carnosine treatment. In conclusion, these findings highlight the beneficial effects of dietary carnosine supplementation on intestinal immune responses and gut barrier function in broiler chickens exposed to E. maxima infection.

IRE1α regulates ROS and immune responses after UVB irradiation

Objective UV irradiation of the skin induces photo damage and generates cytotoxic intracellular reactive oxygen species (ROS), activating the unfolded protein response (UPR) to adapt or reduce these UVB-mediated damages. This study was designed to understand the role of the UPR mediator IRE1α in the antioxidant response following UVB irradiation of mouse skin and keratinocytes. Methods We used mice with an epidermal deletion of IRE1α and primary mouse keratinocytes to examine effects of UV on different parameters of the antioxidant response in the presence and absence of functional IRE1α. Results In the absence of IRE1α, PERK activity and protein levels are significantly compromised following UVB irradiation. Additionally, the loss of IRE1α suppressed phosphorylation of the PERK target, nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2-dependent antioxidant gene expression after UVB irradiation. Interestingly, IRE1α-deficient keratinocytes exhibit elevated basal ROS levels, while a robust ROS induction upon UVB exposure is abolished. Because UVB-induced ROS plays an essential role in regulating skin inflammation, we analyzed recruited immune cell populations and the expression of pro-inflammatory cytokines, Il-6 and Tnfα, in mice with epidermally targeted deletion of Ire1α. Following UVB irradiation, there was significantly less recruitment of neutrophils and leukocytes and reduced expression of pro-inflammatory cytokine genes in the skin of mice lacking IRE1α. Furthermore, keratinocyte proliferation was also significantly reduced after chronic UVB exposure in the skin of these mice. Conclusion Collectively, our findings indicate that IRE1α is essential for basal and UVB-induced oxidative stress response, UV-induced skin immune responses, and keratinocyte proliferation. Significance statement These findings shed new light on the protective function of IRE1α in the response to UV. IRE1α plays an important role in the regulation of ROS, PERK stability, and antioxidant gene expression in response to UVB in mouse keratinocytes and epidermis.

Coarse particulate matter (PM10) induce an inflammatory response through the NLRP3 activation

IntroductionPM exposure can induce inflammatory and oxidative responses; however, differences in these adverse effects have been reported depending on the chemical composition and size. Moreover, inflammatory mechanisms such as NLRP3 activation by PM10 have yet to be explored.ObjectiveTo assess the impact of PM10 on cell cytotoxicity and the inflammatory response through in vitro and in vivo models.MethodologyPeripheral blood mononuclear cells (PBMCs) from healthy donors were exposed to PM10. Cytotoxicity was determined using the LDH assay; the expression of inflammasome components and the production of pro-inflammatory cytokines were quantified through qPCR and ELISA, respectively; and the formation of ASC complexes was examined using confocal microscopy. For in vivo analysis, male C57BL6 mice were intranasally challenged with PM10 and bronchoalveolar lavage fluid was collected to determine cell counts and quantification of pro-inflammatory cytokines by ELISA. RNA was extracted from lung tissue, and the gene expression of inflammatory mediators was quantified.ResultsPM10 exposure induced significant cytotoxicity at concentrations over 100 µg/mL. Moreover, PM10 enhances the gene expression and release of pro-inflammatory cytokines in PBMCs, particularly IL-1β; and induces the formation of ASC complexes in a dose-dependent manner. In vivo, PM10 exposure led to cell recruitment to the lungs, which was characterized by a significant increase in polymorphonuclear cells compared to control animals. Furthermore, PM10 induces the expression of several inflammatory response-related genes, such as NLRP3, IL-1β and IL-18, within lung tissue.ConclusionBriefly, PM10 exposure reduced the viability of primary cells and triggered an inflammatory response, involving NLRP3 inflammasome activation and the subsequent production of IL-1β. Moreover, PM10 induces the recruitment of cells to the lung and the expression of multiple cytokines; this phenomenon could contribute to epithelial damage and, thus to the development and exacerbation of respiratory diseases such as viral infections.

Immunization of turkeys with Clostridium septicum alpha toxin-based recombinant subunit proteins can confer protection against experimental Clostridial dermatitis.

Clostridial dermatitis (CD), caused by Clostridium septicum, is an emerging disease of increasing economic importance in turkeys. Currently, there are no effective vaccines for CD control. Here, two non-toxic domains of C. septicum alpha toxin, namely ntATX-D1 and ntATX-D2, were identified, cloned, and expressed in Escherichia coli as recombinant subunit proteins to investigate their use as potential vaccine candidates. Experimental groups consisted of a Negative control (NCx) that did not receive C. septicum challenge, while the adjuvant-only Positive control (PCx), ntATX-D1 immunization (D1) and ntATX-D2 immunization (D2) groups received C. septicum challenge. Turkeys were immunized subcutaneously with 100 μg of protein at 7, 8 and 9 weeks of age along with an oil-in-water nano-emulsion adjuvant, followed by C. septicum challenge at 11 weeks of age. Results showed that while 46.2% of birds in the PCx group died post-challenge, the rate of mortality in D1- or D2-immunization groups was 13.3%. The gross and histopathological lesions in the skin, muscle and spleen showed that the disease severity was highest in PCx group, while the D2-immunized birds had significantly lower lesion scores when compared to PCx. Gene expression analysis revealed that PCx birds had significantly higher expression of pro-inflammatory cytokine genes in the skin, muscle and spleen than the NCx group, while the D2 group had significantly lower expression of these genes compared to PCx. Peripheral blood cellular analysis showed increased frequencies of activated CD4+ and/or CD8+ cells in the D1 and D2-immunized groups. Additionally, the immunized turkeys developed antigen-specific serum IgY antibodies. Collectively, these findings indicate that ntATX proteins, specifically the ntATX-D2 can be a promising vaccine candidate for protecting turkeys against CD and that the protection mechanisms may include downregulation of C. septicum-induced inflammation and increased CD4+ and CD8+ cellular activation.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties

BackgroundEsculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention.ResultsIn the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1β) expression in RAW264.7 cells.ConclusionsThis novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.

Black soldier fly larvae oil (Hermetia illucens L.) calcium salt enhances intestinal morphology and barrier function in laying hens

This study aimed to determine the influence of black soldier fly larvae oil calcium salt (BSFLO-SCa) supplementation on performance, jejunal histomorphology and gene expression of tight junctions and inflammatory cytokines in laying hens. A total of 60 ISA Brown laying hens (40 wk of age) were divided into 3 treatment groups, including a control group fed a basal diet (T0) and basal diets supplemented with 1% (T1) and 2% (T2) of BSFLO-SCa. Each treatment group consisted of 5 replicates with 4 laying hens each. Results showed that 1% and 2% BSFLO-SCa supplementation significantly reduced (P < 0.05) feed conversion ratio (FCR), while egg weight (EW) increased (P < 0.05). The inclusion with 2% increased (P < 0.05) both egg production (HDA) and mass (EM). The addition of 1% and 2% BSFLO-SCa significantly increased (P < 0.05) villus height (VH) and villus width (VW), while crypt depth (CD) significantly increased (P < 0.05) with 2% BSFLO-SCa. The tight junction and gene expression of claudin-1 (CLDN-1), junctional adhesion molecules-2 (JAM-2), and occludin (OCLN) were significantly upregulated (P < 0.05) with 2% BSFLO-SCa. The pro-inflammatory cytokines and gene expression of interleukin-6 (IL-6) was significantly downregulated (P < 0.05) with the addition of BSFLO-SCa, while gene expression of interleukin-18 (IL-18), toll-like receptor 4 (TLR-4), and tumor necrosis factor-α (TNF-α) were downregulated with 2% BSFLO-SCa. On the other hand, the anti-inflammatory cytokines and gene expression of interleukin-13 (IL-13) and interleukin-10 (IL-10) were significantly upregulated (P < 0.05) at 2% BSFLO-SCa. In conclusion, dietary supplementation with 2% BSFLO-SCa improved productivity, intestinal morphology and integrity by upregulating tight junction-related protein of gene expression of laying hens. In addition, supplementation with BSFLO-SCa enhanced intestinal immune responses by upregulating anti-inflammatory and downregulating pro-inflammatory cytokine gene expression.

Lack of mTORC2 signaling in CD11c+ myeloid cells inhibits their migration and ameliorates experimental colitis.

The mammalian target of rapamycin (mTOR) pathway plays a key role in determining immune cells function through modulation of their metabolic status. By specific deletion of Rictor in CD11c+ myeloid cells (referred to here as CD11cRicΔ/Δ), we investigated the role of mTOR complex 2 (mTORC2) signaling in dendritic cells (DCs) function in mice. We showed that upon dextran sulfate sodium-induced colitis, the lack of mTORC2 signaling CD11c+ cells diminishes the colitis score and abrogates DC migration to the mesenteric lymph nodes, thereby diminishing the infiltration of T helper 17 cells in the lamina propria and subsequent inflammation. These findings corroborate with the abrogation of cytoskeleton organization and the decreased activation of Rac1 and Cdc42 GTPases observed in CD11c+-mTORC2-deficient cells. Meta-analysis on colonic samples from ulcerative colitis patients revealed increased gene expression of proinflammatory cytokines, which coincided with augmented expression of the mTOR pathway, a positive correlation between the DC marker ITGAX and interleukin-6, the expression of RICTOR, and CDC42. Together, this work proposes that targeting mTORC2 on DCs offers a key to hamper inflammatory responses, and this way, ameliorates the progression and severity of intestinal inflammatory diseases.

Effect of PCV-2 Vaccination on Cytokines Gene Expression Profile in Wild Boar Peripheral Blood Mononuclear Cells after Stimulation with Mycobacteria Antigens

Wild boar (Sus scrofa) is a common wild ungulate known as the most important reservoir of tuberculosis (TB) in Spain. The severity of TB lesions in this species and the high prevalence of porcine circovirus type 2 (PCV-2) have been related. PCV-2 is ubiquitous in swine populations, being usual for the free-living ones the contact with this agent. Recent studies found a correlation between a decrease of generalised TB prevalence in wild boar populations and the PCV-2-vaccination. The aim of this study was to find out if PCV-2 vaccination modulates the gene expression of cytokines from immune cells after its exposition with mycobacterial antigens using an in vitro methodology. A total of 46 wild boars from a PCV-2 infection endemic area were blood-sampled before and after the PCV-2 vaccination of 22 of them. Peripheral blood mononuclear cells (PBMC) were obtained and isolated from these samples. Aliquots of the cells were in vitro cultured and respectively stimulated with PPDa, PPDb, and a mitogen. A complete analysis of the gene expression of cytokines from the cultured PBMC was carried out. Also, Mycobacterium bovis and PCV-2 contacts were revealed by ELISA and/or qPCR. The results demonstrated that the animals which have had contact with PCV-2 and had been vaccinated, manifested a significant decrease in gene expression of proinflammatory cytokines, like interleukin 1 beta, interleukin 6, and tumour necrosis factor-alpha, possibly related with the severity of TB lesions, and also a significant decrease of interleukin 10, a key cytokine. In conclusion, in case of possible infection or contact events with the virus, PCV-2 vaccination could be an effective measure to reduce the TB severity in wild boar populations, which could decrease the intra and interspecies transmission of TB.