Upregulation Of IL-6 Research Articles

Critical roles of PU.1/cathepsin S activation in regulating inflammatory responses of macrophages in periodontitis.

To determine the critical roles of PU.1/cathepsin S activation in regulating inflammatory responses of macrophages during periodontitis. Cathepsin S (CatS) is a cysteine protease and exerts important roles in the immune response. Elevated CatS has been found in the gingival tissues of periodontitis patients and is involved in alveolar bone destruction. However, the underlying mechanism of CatS-driven IL-6 production in periodontitis remains unclear. Western blot was applied to measure mature cathepsin S(mCatS) and IL-6 expression in gingival tissues from periodontitis patients and RAW264.7 cells exposed to lipopolysaccharide from Porphyromonas gingivalis (P.g. LPS). Immunofluorescence was applied to confirm the localization of PU.1, and CatS in the gingival tissues of periodontitis patients. ELISA was performed to determine IL-6 production by the P.g. LPS-exposed RAW264.7 cells. Knockdown by shRNA was used to determine the effects of PU.1 on p38/ nuclear factor (NF)-κB activation, mCatS expression and IL-6 production in RAW264.7 cells. The expressions mCatS and IL-6 were significantly upregulated in gingival macrophages. In cultured RAW264.7 cells, increased mCatS and IL-6 protein paralleled the activation of p38 and NF-κB after exposure to P.g. LPS. CatS knockdown by shRNA significantly decreased P.g. LPS-induced IL-6 expression and p38/NF-κB activation. PU.1 was significantly increased in P.g. LPS-exposed RAW264.7 cells, and PU.1 knockdown dramatically abolished the P.g. LPS-induced upregulation of mCatS and IL-6 and the activation of p38 and NF-κB. Furthermore, PU.1 and CatS colocalized in macrophages within the gingival tissues of periodontitis patients. PU.1-dependent CatS drives IL-6 production in macrophages by activating p38 and NF-κB in periodontitis.

#5413 FRONTIER-1: A PHASE 2B STUDY OF IL-33 BLOCKADE IN DIABETIC KIDNEY DISEASE

Abstract Background and Aims Several biomarkers of inflammation have been correlated with poor renal outcomes and mortality in DKD. However, an effective, well-tolerated treatment of progressive renal disease driven by inflammation has remained elusive. Inhibition of IL-33 may provide a novel opportunity to address this unmet medical need in high-risk patients. Here, we: 1) show that tozorakimab, a high-affinity IL-33-neutralizing mAb, promotes glomerular health in inflammation; 2) describe the clinical study to test tozorakimab in patients with DKD; 3) present participant baseline characteristics; and 4) evaluate inflammation-associated biomarker in FRONTIER-1 and four external validation cohorts. Method FRONTIER-1 (NCT04170543) is a phase 2b, randomized, double-blind, placebo-controlled, multicenter study to evaluate the efficacy, safety, pharmacokinetics (PK), and immunogenicity of tozorakimab in patients with DKD; defined as T2DM with an eGFR of 25-75 mL/min/1.73 m2 and a UACR in the range of 100-3,000 mg/g. All participants are expected to receive an ACEi/ARB for > 6 weeks before the treatment period. Approximately 565 patients from multiple countries will be randomized to four dose levels of tozorakimab, or volume-matched placebo dosed subcutaneously every 28 days over a 24-week treatment period. All participants will receive 10 mg dapagliflozin Day 85-168. The primary objective of the study is to evaluate the effect of tozorakimab on albuminuria. Secondary objectives are to assess the safety and tolerability of tozorakimab with and without SGLT2i, evaluate the effect of tozorakimab in combination with ACEi or ARB with and without SGLT2i on albuminuria, and describe the PK and immunogenicity of tozorakimab. Key exploratory objective is to assess baseline and treatment-response for urine and/or circulating biomarkers of inflammation, including hsCRP, IL-33/sIL1RL1, CCL2, and TNFR-1/2. Biomarker response was, furthermore, evaluated in pre-clinical models of DKD, and respective distributions and risk were assessed in four independent DKD cohorts (N > 200). Results Transcriptomic profiling of kidney biopsies from patients with DKD indicated a significant glomerular up-regulation of IL-33. Inhibition of IL-33 signalling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model of DKD. This was associated with improved glomerular endothelial health as indicated by decreased cellular inflammation and reduced release of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, TNFR-1, and CCL2. Hence, FRONTIER-1 was started in 2019. So far, all trial groups are well-balanced and in line with our expectations for this patient population (N = 574). At baseline, > 95% were receiving ACEi/ARBs and approximately 25% were receiving SGLT2i. The mean eGFR was 47.8 ml/min/1,73 m2 (+/- 14.7) and mean UACR was 765 mg/g (+/- 775). The inflammatory state of participants at baseline was assessed in circulation and urine by hsCRP, TNFR-1 and CCL2 (N > 146). Comparison to independent reference cohorts (N > 200) confirmed elevated risk in DKD patients with high urinary TNFR-1 and CCL2 and showed that 6-week treatment of dapagliflozin did not alter these biomarkers significantly. Conclusion Taken together, inhibition of IL-33 may be beneficial in patients with DKD on standard of care, who have elevated biomarkers of inflammation such as TNFR-1 and/or CCL2. This hypothesis is currently being tested in the FRONTIER-1 phase 2b clinical study. To identify the patients most likely to benefit from immunomodulatory treatment with tozorakimab, the study will conduct a retrospective analysis of albuminuria and inflammatory biomarkers. The primary analysis of the study is planned for the second half of 2023.

Evaluation of protection and immunity induced by infectious bronchitis vaccines administered by oculonasal, spray or gel routes in commercial broiler chicks

Broiler chicks’ responses following combined IBV live attenuated Massachusetts and 793B strains through gel, spray or oculonasal (ON) vaccination routes were cross-compared. Subsequently, the responses following IBV M41 challenge of the unvaccinated and vaccinated groups were also assessed. Post-vaccination humoral and mucosal immune responses, alongside viral load kinetics in swabs and tissues, were determined using commercial ELISA assays, monoclonal antibody-based IgG and IgA ELISA assays and qRT-PCR respectively. After challenged with IBV-M41 strain, humoral and mucosal immune responses, ciliary protection, viral load kinetics, and immune gene mRNA transcriptions between the three vaccination methods were examined and compared. Findings showed that post-vaccinal humoral and mucosal immune responses were similar in all three vaccination methods. Post vaccinal viral load kinetics is influenced by method of administration. The viral load peaked in the ON group within the tissues and the OP/CL swabs in the first and third weeks respectively. Following M41 challenge, ciliary protection and mucosal immune responses were not influenced by vaccination methods as all three methods offered equal ciliary protection. Immune gene mRNA transcriptions varied by vaccination methods. Significant up-regulation of MDA5, TLR3, IL-6, IFN-α and IFN-β genes were recorded for ON method. For both spray and gel methods, significant up-regulation of only MDA5 and IL-6 genes were noted. The spray and gel-based vaccination methods gave equivalent levels of ciliary protection and mucosal immunity to M41 virulent challenge comparable to those provided by the ON vaccination. Analysis of viral load and patterns of immune gene transcription of the vaccinated-challenged groups revealed high similarity between turbinate and choanal cleft tissues compared to HG and trachea. With regards to immune gene mRNA transcription, for all the vaccinated-challenged groups, similar results were found except for IFN-α, IFN-β and TLR3, which were up-regulated only in ON compared to gel and spray vaccination methods.

Thymoquinone Treatment Enhances T Cell-Mediated Anti-Tumor Response In Vitro

Oil from Nigella sativa seeds (“black seed oil”) has been used for health care and medical purposes for over 2000 years. The major active compound thymoquinone (TQ) has been shown to have immune regulatory effects in various diseases. Tumor microenvironment is critical for tumor development. Immune cells, particularly T cells, are central players to kill tumor cells and affect tumor progression and metastasis. The impact of TQ on human T cell-mediated anti-tumor response was investigated. Using human PBMCs, we activated T cells with anti-CD3/CD28 in vitro, and treated cells with TQ. First, we tested the impact of TQ on T cell activation, proliferation and apoptosis; second, the anti-tumor effect of T cells after treatment with TQ; last, we determined alterations in T cells gene expression at the RNA level after TQ treatment. TQ treatment increased activation and proliferation of T cells at days 2-4 and more activation-induced cell death at day 3. After 7-11 days, TQ-treated cells showed less survival than cells treated with control medium. At low TQ concentration (10uM), treatment induced more CD3+ T cell proliferation; while at 20uM and 50uM TQ showed more cytotoxic effect to T cells, and more cell death. When co-cultured with multiple types of cancer cells, TQ-treated T cells exhibited significantly enhanced anti-tumor activity, indicated by more tumor cell apoptosis and less cell expansion. Gene expression study showed upregulation of NFAT signaling pathway, including 10+ fold up-regulation of IL-2, which might explain the enhanced anti-tumor effect after TQ treatment. In summary, TQ treatment can enhance human T cell-mediated anti-tumor effect in vitro. This immunoregulatory function of black seed oil could potentially benefit patients of different cancer types.

In vitro safety assessment of reduced graphene oxide in human monocytes and T cells.

Considering the increase in the use of graphene derivatives in different fields, the environmental and human exposure to these materials is likely, and the potential consequences are not fully elucidated. This study is focused on the human immune system, as this plays a key role in the organism's homeostasis. In this sense, the cytotoxicity response of reduced graphene oxide (rGO) was investigated in monocytes (THP-1) and human T cells (Jurkat). A mean effective concentration (EC50-24 h) of 121.45 ± 11.39 μg/mL and 207.51 ± 21.67 μg/mL for cytotoxicity was obtained in THP-1 and Jurkat cells, respectively. rGO decreased THP-1 monocytes differentiation at the highest concentration after 48 h of exposure. Regarding the inflammatory response at genetic level, rGO upregulated IL-6 in THP-1 and all cytokines tested in Jurkat cells after 4 h of exposure. At 24 h, IL-6 upregulation was maintained, and a significant decrease of TNF-α gene expression was observed in THP-1 cells. Moreover, TNF-α, and INF-γ upregulation were maintained in Jurkat cells. With respect to the apoptosis/necrosis, gene expression was not altered in THP-1 cells, but a down regulation of BAX and BCL-2 was observed in Jurkat cells after 4 h of exposure. These genes showed values closer to negative control after 24 h. Finally, rGO did not trigger a significant release of any cytokine at any exposure time assayed. In conclusion, our data contributes to the risk assessment of this material and suggest that rGO has an impact on the immune system whose final consequences should be further investigated.

Innate immune response in patients with acute Chikungunya disease.

Chikungunya disease (CHIKD) is an arbovirose that presents with high morbidity, mainly due to arthralgia. Inflammatory mediators including IL-6, IL-1β, GM-CSF and others have been implicated in the pathogenesis of CHIKD, whilst type I interferons can be associated with better outcomes. The role of pattern recognition receptors has been studied incompletely. Here, we evaluated the expression of RNA-specific PRRs, their adaptor molecules and downstream cytokines in acute CHIKD patients. Twenty-eight patients were recruited during the 3rd-5th day after the symptoms onset for clinical examination, peripheral blood collection and qRT-PCR analysis of PBMC to compare to the healthy control group (n = 20). We observed common symptoms of acute CHIKD, with fever, arthralgia, headache and myalgia being the most frequent. Compared with uninfected controls, acute CHIKV infection upregulates the expression of the receptors TLR3, RIG-I and MDA5, and also the adaptor molecule TRIF. Regarding cytokine expression, we found an upregulation of IL-6, IL-12, IFN-α, IFN-β and IFN-γ, which are related directly to the inflammatory or antiviral response. The TLR3-TRIF axis correlated with high expression of IL-6 and IFN-α. Interestingly, greater expression of MDA5, IL-12 and IFN-α was related to lower viral loads in CHIKD acute patients. Together, these findings help to complete the picture of innate immune activation during acute CHIKD, while confirming the induction of strong antiviral responses. Drawing the next steps in the understanding of the immunopathology and virus clearance mechanisms of CHIKD should be of utter importance in the aid of the development of effective treatment to reduce the severity of this debilitating disease.

Harnessing the probiotic properties and immunomodulatory effects of fermented food-derived Limosilactobacillus fermentum strains: implications for environmental enteropathy.

Environmental enteropathy (EE), a chronic small intestine disease characterized by gut inflammation, is widely prevalent in low-income countries and is hypothesized to be caused by continuous exposure to fecal contamination. Targeted nutritional interventions using potential probiotic strains from fermented foods can be an effective strategy to inhibit enteric pathogens and prevent chronic gut inflammation. We isolated potential strains from fermented rice water and lemon pickle and investigated their cell surface properties, antagonistic properties, adhesion to HT-29 cells, and inhibition of pathogen adherence to HT-29 cells. Bacteriocin-like inhibitory substances (BLIS) were purified, and in vivo, survival studies in Caenorhabditis elegans infected with Salmonella enterica MW116733 were performed. We further checked the expression pattern of pro and anti-inflammatory cytokines (IL-6, IL8, and IL-10) in HT-29 cells supplemented with strains. The strains isolated from rice water (RS) and lemon pickle (T1) were identified as Limosilactobacillus fermentum MN410703 and MN410702, respectively. Strains showed probiotic properties like tolerance to low pH (pH 3.0), bile salts up to 0.5%, simulated gastric juice at low pH, and binding to extracellular matrix molecules. Auto-aggregation of T1 was in the range of 85% and significantly co-aggregated with Klebsiella pneumoniae, S. enterica, and Escherichia coli at 48, 79, and 65%, respectively. Both strains had a higher binding affinity to gelatin and heparin compared to Bacillus clausii. Susceptibility to most aminoglycoside, cephalosporin, and macrolide classes of antibiotics was also observed. RS showed BLIS activity against K. pneumoniae, S. aureus, and S. enterica at 60, 48, and 30%, respectively, and the protective effects of BLIS from RS in the C. elegans infection model demonstrated a 70% survival rate of the worms infected with S. enterica. RS and T1 demonstrated binding efficiency to HT-29 cell lines in the 38-46% range, and both strains inhibited the adhesion of E. coli MDR and S. enterica. Upregulation of IL-6 and IL-10 and the downregulation of IL-8 were observed when HT-29 cells were treated with RS, indicating the immunomodulatory effects of the strain. The potential strains identified could effectively inhibit enteric pathogens and prevent environmental enteropathy.

Circular RNA circTRAPPC6B Enhances IL-6 and IL-1β Expression and Repolarizes Mycobacteria Induced Macrophages from M2- to M1-Like Phenotype by Targeting miR-892c-3p.

Mycobacterium tuberculosis (Mtb) infection elicits macrophage polarization into M2 phenotype to block the host's protective immune response. However, it remains unclear how Mtb regulates macrophage polarization. Recent studies have suggested that noncoding RNA may play a role in macrophage polarization. In this study, we investigated the potential involvement of circTRAPPC6B, a circular RNA that is downregulated in tuberculosis (TB) patients, in regulating macrophage polarization. We found that Mtb infection downregulated M1-related IL-6 and IL-1β while highly expressed M2-related CCL22 and CD163. Overexpressed circTRAPPC6B had switched Mtb-infected macrophages from M2- to M1-like phenotype, accompanied by upregulation of IL-6 and IL-1β. Meanwhile overexpressed circTRAPPC6B significantly inhibited Mtb growth in macrophages. Our findings suggest that circTRAPPC6B may regulate macrophage polarization by targeting miR-892c-3p, which is highly expressed in TB patients and M2-like macrophages. And miR-892c-3p inhibitor decreased intracellular Mtb growth in macrophages. Thus, TB-inhibited circTRAPPC6B could specifically induce IL-6 and IL-1β expression to switch/antagonize Mtb-induced macrophage polarization from M2- to M1-like phenotype by targeting miR-892c-3p, leading to enhanced host clearance of Mtb. Our results reveal a potential role for circTRAPPC6B in regulating macrophage polarization during Mtb infection and provide new insights into the molecular mechanisms underlying host defense against Mtb.

Prior exposure to stress exacerbates neuroinflammation and causes long-term behavior changes in sepsis

BackgroundNeuroinflammation can occur during sepsis and is now regarded as the main mechanism underlying various related central nervous system (CNS) disorders. Another well-known factor causing neuroinflammation is psychological stress. In the current study, we examined the effects of prior exposure to stress on sepsis-induced neuroinflammation and CNS symptoms. Experimental procedureBalb/c mice were subjected to wet bedding stress for 2 days, then lipopolysaccharide (LPS) was intraperitoneally administered. For examining the neuroinflammation, the expression of proinflammatory cytokines and NF-κB activity in the brain was analyzed by RT-PCR and ELISA-based assay. Additionally, immunohistochemical study using Iba-1 was performed. Finally, behavior tests were examined one month after LPS treatment. Result and conclusionStress exposure induced the upregulation of IL-1β, IL-6 and TNFα mRNA in the cerebral cortex 4 h after LPS administration. Suggesting an underlying mechanism, LPS-induced NF-κB activation was significantly upregulated with stress in the brain. Histologically, microglia in the cerebral cortex were reactive and became more abundant with stress, while these effects were further increased with LPS injection. Behavioral analysis conducted showed a significant increase of anxiety-like behaviors in the stressed mice. These results suggest that prior exposure to stress exacerbates neuroinflammation during sepsis and induces long-term behavior changes.

Silica nanocarrier-mediated intracellular delivery of rapamycin promotes autophagy-mediated M2 macrophage polarization to regulate bone regeneration

Targeting macrophages to regulate the immune microenvironment is a new strategy for bone regeneration with nano-drugs. Nano-drugs have achieved surprising anti-inflammatory and bone-regenerative effects, however, their underlying mechanisms in macrophages remain to be clarified. Macrophage polarization, immunomodulation, and osteogenesis are governed by autophagy. Rapamycin, an autophagy inducer, has shown promising results in bone regeneration, but high dose-mediated cytotoxicity and low bioavailability hinder its clinical application. This study aimed to develop rapamycin-loaded virus-like hollow silica nanoparticles (R@HSNs) which are easily phagocytosed by macrophages and translocated to lysosomes. R@HSNs induced macrophage autophagy, promoted M2 polarization, and alleviated the degree of M1 polarization as indicated by the downregulation of inflammatory factors IL-6, IL-1β, TNF-α, and iNOS, and upregulation of anti-inflammatory factors CD163, CD206, IL-1ra, IL-10, and TGF-β. These effects were nullified by cytochalasin B-induced inhibition of R@HSNs uptake in macrophages. The conditioned medium (CM) collected from R@HSNs-treated macrophages promoted osteogenic differentiation of mouse bone marrow mesenchymal stromal cells (mBMSCs). In a mouse calvaria defect model, free rapamycin treatment was inhibited, but R@HSNs robustly promoted bone defect healing. In conclusion, silica nanocarrier-mediated intracellular rapamycin delivery to macrophages effectively triggers autophagy-mediated M2 macrophage polarization, further enhancing bone regeneration by triggering osteogenic differentiation of mBMSCs.

Neutrophil extracellular traps and extracellular histones potentiate IL-17 inflammation in periodontitis.

Neutrophil infiltration is a hallmark of periodontitis, a prevalent oral inflammatory condition in which Th17-driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model, we demonstrate that neutrophils infiltrate the gingival oral mucosa at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanisms by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL-17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document significantly increased levels of NET complexes and extracellular histones bearing classic NET-associated posttranslational modifications, in blood and local lesions of severe periodontitis patients, in the absence of confounding disease. Our findings suggest a feed-forward loop in which NETs trigger IL-17 immunity to promote immunopathology in a prevalent human inflammatory disease.

A peripheral signature of Alzheimer’s disease featuring microbiota-gut-brain axis markers

BackgroundIncreasing evidence links the gut microbiota (GM) to Alzheimer’s disease (AD) but the mechanisms through which gut bacteria influence the brain are still unclear. This study tests the hypothesis that GM and mediators of the microbiota-gut-brain axis (MGBA) are associated with the amyloid cascade in sporadic AD.MethodsWe included 34 patients with cognitive impairment due to AD (CI-AD), 37 patients with cognitive impairment not due to AD (CI-NAD), and 13 cognitively unimpaired persons (CU). We studied the following systems: (1) fecal GM, with 16S rRNA sequencing; (2) a panel of putative MGBA mediators in the blood including immune and endothelial markers as bacterial products (i.e., lipopolysaccharide, LPS), cell adhesion molecules (CAMs) indicative of endothelial dysfunction (VCAM-1, PECAM-1), vascular changes (P-, E-Selectin), and upregulated after infections (NCAM, ICAM-1), as well as pro- (IL1β, IL6, TNFα, IL18) and anti- (IL10) inflammatory cytokines; (3) the amyloid cascade with amyloid PET, plasma phosphorylated tau (pTau-181, for tau pathology), neurofilament light chain (NfL, for neurodegeneration), and global cognition measured using MMSE and ADAScog. We performed 3-group comparisons of markers in the 3 systems and calculated correlation matrices for the pooled group of CI-AD and CU as well as CI-NAD and CU. Patterns of associations based on Spearman’s rho were used to validate the study hypothesis.ResultsCI-AD were characterized by (1) higher abundance of Clostridia_UCG-014 and decreased abundance of Moryella and Blautia (p < .04); (2) elevated levels of LPS (p < .03), upregulation of CAMs, Il1β, IL6, and TNFα, and downregulation of IL10 (p < .05); (3) increased brain amyloid, plasma pTau-181, and NfL (p < 0.004) compared with the other groups. CI-NAD showed (1) higher abundance of [Eubacterium] coprostanoligenes group and Collinsella and decreased abundance of Lachnospiraceae_ND3007_group, [Ruminococcus]_gnavus_group and Oscillibacter (p < .03); (2) upregulation of PECAM-1 and TNFα (p < .03); (4) increased plasma levels of NfL (p < .02) compared with CU. Different GM genera were associated with immune and endothelial markers in both CI-NAD and CI-AD but these mediators were widely related to amyloid cascade markers only in CI-AD.ConclusionsSpecific bacterial genera are associated with immune and endothelial MGBA mediators, and these are associated with amyloid cascade markers in sporadic AD. The physiological mechanisms linking the GM to the amyloid cascade should be further investigated to elucidate their potential therapeutic implications.

AXL-Induced Autophagy mitigates experimental autoimmune encephalomyelitis by suppressing microglial inflammation via the PI3K/AKT/mTOR signaling pathway

Microglia, being the primary immune cells of the central nervous system (CNS), are responsible for pathological inflammatory demyelination in multiple sclerosis (MS). It has been demonstrated that AXL, one of the receptor tyrosine kinases, could alleviate the inflammatory response of microglia. However, the specific mechanism remains unclear. Herein, we explored the role of AXL in the autophagy of microglia and its effect on the experimental autoimmune encephalomyelitis (EAE) model. We revealed that knockout of AXL in BV2 microglia significantly promoted the expression of phosphorylated-PI3K/p-AKT/p-mTOR while significantly inhibiting LC3-Ⅱ/Beclin1. Similarly, autophagy was significantly inhibited in the AXL-/- mice. Knockout of AXL induced serious symptoms, infiltration of inflammatory cells, and demyelination changes, manifesting as the upregulation of pro-inflammatory factors TNF-α and IL-6 and downregulation of anti-inflammatory factors TGF-β and IL-10. In conclusion, this study substantiated that autophagy induced by AXL inhibited the inflammatory response of microglia and alleviated symptoms of EAE. Autophagy activation was mediated by the PI3K/AKT/mTOR signaling pathway.

Analysis of the microglia transcriptome across the human lifespan using single cell RNA sequencing

BackgroundMicroglia are tissue resident macrophages with a wide range of critically important functions in central nervous system development and homeostasis.MethodIn this study, we aimed to characterize the transcriptional landscape of ex vivo human microglia across different developmental ages using cells derived from pre-natal, pediatric, adolescent, and adult brain samples. We further confirmed our transcriptional observations using ELISA and RNAscope.ResultsWe showed that pre-natal microglia have a distinct transcriptional and regulatory signature relative to their post-natal counterparts that includes an upregulation of phagocytic pathways. We confirmed upregulation of CD36, a positive regulator of phagocytosis, in pre-natal samples compared to adult samples in situ. Moreover, we showed adult microglia have more pro-inflammatory signature compared to microglia from other developmental ages. We indicated that adult microglia are more immune responsive by secreting increased levels of pro-inflammatory cytokines in response to LPS treatment compared to the pre-natal microglia. We further validated in situ up-regulation of IL18 and CXCR4 in human adult brain section compared to the pre-natal brain section. Finally, trajectory analysis indicated that the transcriptional signatures adopted by microglia throughout development are in response to a changing brain microenvironment and do not reflect predetermined developmental states.ConclusionIn all, this study provides unique insight into the development of human microglia and a useful reference for understanding microglial contribution to developmental and age-related human disease.

Potential mechanisms mediating PM2.5-induced alterations of H3N2 influenza virus infection and cytokine production in human bronchial epithelial cells

Exposure to particulate matter (PM) has been associated with increased hospital admissions for influenza. Airway epithelial cells are a primary target for inhaled environmental insults including fine PM (PM2.5) and influenza viruses. The potentiation of PM2.5 exposure on the effects of influenza virus on airway epithelial cells has not been adequately elucidated. In this study, the effects of PM2.5 exposure on influenza virus (H3N2) infection and downstream modulation of inflammation and antiviral immune response were investigated using a human bronchial epithelial cell line, BEAS-2B. The results showed that PM2.5 exposure alone increased the production of pro-inflammatory cytokines including interleukin-6 (IL-6) and IL-8 but decreased the production of the antiviral cytokine interferon-β (IFN-β) in BEAS-2B cells while H3N2 exposure alone increased the production of IL-6, IL-8, and IFN-β. Importantly, prior exposure to PM2.5 enhanced subsequent H3N2 infectivity, expression of viral hemagglutinin protein, as well as upregulation of IL-6 and IL-8, but reduced H3N2-induced IFN-β production. Pre-treatment with a pharmacological inhibitor of nuclear factor-κB (NF-κB) suppressed pro-inflammatory cytokine production induced by PM2.5, H3N2, as well as PM2.5-primed H3N2 infection. Moreover, antibody-mediated neutralization of Toll-like receptor 4 (TLR4) blocked cytokine production triggered by PM2.5 or PM2.5-primed H3N2 infection, but not H3N2 alone. Taken together, exposure to PM2.5 alters H3N2-induced cytokine production and markers of replication in BEAS-2B cells, which in turn are regulated by NF-κB and TLR4.

An in vivo model of ligamentum flavum hypertrophy from early-stage inflammation to fibrosis.

Multi-joint disease pathologies in the lumbar spine, including ligamentum flavum (LF) hypertrophy and intervertebral disc (IVD) bulging or herniation contribute to lumbar spinal stenosis (LSS), a highly prevalent condition characterized by symptomatic narrowing of the spinal canal. Clinical hypertrophic LF is characterized by a loss of elastic fibers and increase in collagen fibers, resulting in fibrotic thickening and scar formation. In this study, we created an injury model to test the hypothesis that LF needle scrape injury in the rat will result in hypertrophy of the LF characterized by altered tissue geometry, matrix organization, composition and inflammation. An initial pilot study was conducted to evaluate effect of needle size. Results indicate that LF needle scrape injury using a 22G needle produced upregulation of the pro-inflammatory cytokine Il6 at 1 week post injury, and increased expression of Ctgf and Tgfb1 at 8 weeks post injury, along with persistent presence of infiltrating macrophages at 1, 3, and 8 weeks post injury. LF integrity was also altered, evidenced by increases in LF tissue thickness and loss of elastic tissue by 8 weeks post injury. Persistent LF injury also produced multi-joint effects in the lumbar IVD, including disc height loss at the injury and adjacent to injury level, with degenerative IVD changes observed in the adjacent level. These results demonstrate that LF scrape injury in the rat produces structural and molecular features of LF hypertrophy and IVD height and histological changes, dependent on level. This model may be useful for testing of therapeutic interventions for treatment of LSS and IVD degeneration associated with LF hypertrophy.

Aerosol vaccination of chicken pullets with irradiated avian pathogenic Escherichia coli induces a local immunostimulatory effect.

The present study investigated the expression of cytokines and cellular changes in chickens following vaccination with irradiated avian pathogenic Escherichia coli (APEC) and/or challenge. Four groups of 11-week-old pullets, each consisting of 16 birds were kept separately in isolators before they were sham inoculated (N), challenged only (C), vaccinated (V) or vaccinated and challenged (V+C). Vaccination was performed using irradiated APEC applied via aerosol. For challenge, the homologous strain was administered intratracheally. Birds were sacrificed on 3, 7, 14 and 21 days post challenge (dpc) to examine lesions, organ to body weight ratios and bacterial colonization. Lung and spleen were sampled for investigating gene expression of cytokines mediating inflammation by RT-qPCR and changes in the phenotype of subsets of mononuclear cells by flow cytometry. After re-stimulation of immune cells by co-cultivation with the pathogen, APEC-specific IFN-γ producing cells were determined. Challenged only birds showed more severe pathological and histopathological lesions, a higher probability of bacterial re-isolation and higher organ to body weight ratios compared to vaccinated and challenged birds. In the lung, an upregulation of IL-1β and IL-6 following vaccination and/or challenge at 3 dpc was observed, whereas in the spleen IL-1β was elevated. Changes were observed in macrophages and TCR-γδ+ cells within 7 dpc in spleen and lung of challenged birds. Furthermore, an increase of CD4+ cells in spleen and a rise of Bu-1+ cells in lung were present in vaccinated and challenged birds at 3 dpc. APEC re-stimulated lung and spleen mononuclear cells from only challenged pullets showed a significant increase of IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. Vaccinated and challenged chickens responded with a significant increase of IFN-γ+CD8α+ T cells in the lung and IFN-γ+TCR-γδ+ cells in the spleen. Re-stimulation of lung mononuclear cells from vaccinated birds resulted in a significant increase of both IFN-γ+CD8α+ and IFN-γ+TCR-γδ+ cells. In conclusion, vaccination with irradiated APEC caused enhanced pro-inflammatory response as well as the production of APEC-specific IFN-γ-producing γδ and CD8α T cells, which underlines the immunostimulatory effect of the vaccine in the lung. Hence, our study provides insights into the underlying immune mechanisms that account for the defense against APEC.

Copper in airborne fine particulate matter (PM2.5) from urban sites causes the upregulation of pro-inflammatory cytokine IL-8 in human lung epithelial A549 cells.

Fine atmospheric particles, such as PM2.5, are strongly related to the onset and exacerbation of inflammatory responses leading to the development of respiratory and cardiovascular diseases. PM2.5 is a complex mixture of tiny particles with different properties (i.e., size, morphology, and chemical components). Moreover, the mechanism by which PM2.5 induces inflammatory responses has not been fully elucidated. Therefore, it is necessary to determine the composition of PM2.5 to identify the main factors causing PM2.5-associated inflammation and diseases. In the present study, we investigated PM2.5 from two sites (Fukue, a remote monitoring site, and Kawasaki, an urban monitoring site) with greatly different environments and PM2.5 compositions. The results of ICP-MS and EDX-SEM indicated that PM2.5 from Kawasaki contained more metals and significantly induced the expression of the pro-inflammatory cytokine gene IL-8 compared to the PM2.5 from Fukue. We also verified the increased secretion of IL-8 protein from exposure to PM2.5 from Kawasaki. We further investigated their effects on inflammatory response and cytotoxicity using metal nanoparticles (Cu, Zn, and Ni) and ions and found that the Cu nanoparticles caused a dose-dependent increase in IL-8 expression together with significant cell death. We also found that Cu nanoparticles enhanced the secretion of IL-8 protein. These results suggest that Cu in PM2.5 is involved in lung inflammation.

CXCL13 contributes to chronic pain of a mouse model of CRPS-I via CXCR5-mediated NF-κB activation and pro-inflammatory cytokine production in spinal cord dorsal horn

BackgroundComplex regional pain syndrome type-I (CRPS-I) causes excruciating pain that affect patients’ life quality. However, the mechanisms underlying CRPS-I are incompletely understood, which hampers the development of target specific therapeutics.MethodsThe mouse chronic post-ischemic pain (CPIP) model was established to mimic CRPS-I. qPCR, Western blot, immunostaining, behavioral assay and pharmacological methods were used to study mechanisms underlying neuroinflammation and chronic pain in spinal cord dorsal horn (SCDH) of CPIP mice.ResultsCPIP mice developed robust and long-lasting mechanical allodynia in bilateral hindpaws. The expression of inflammatory chemokine CXCL13 and its receptor CXCR5 was significantly upregulated in ipsilateral SCDH of CPIP mice. Immunostaining revealed CXCL13 and CXCR5 was predominantly expressed in spinal neurons. Neutralization of spinal CXCL13 or genetic deletion of Cxcr5 (Cxcr5−/−) significantly reduced mechanical allodynia, as well as spinal glial cell overactivation and c-Fos activation in SCDH of CPIP mice. Mechanical pain causes affective disorder in CPIP mice, which was attenuated in Cxcr5−/− mice. Phosphorylated STAT3 co-expressed with CXCL13 in SCDH neurons and contributed to CXCL13 upregulation and mechanical allodynia in CPIP mice. CXCR5 coupled with NF-κB signaling in SCDH neurons to trigger pro-inflammatory cytokine gene Il6 upregulation, contributing to mechanical allodynia. Intrathecal CXCL13 injection produced mechanical allodynia via CXCR5-dependent NF-κB activation. Specific overexpression of CXCL13 in SCDH neurons is sufficient to induce persistent mechanical allodynia in naïve mice.ConclusionsThese results demonstrated a previously unidentified role of CXCL13/CXCR5 signaling in mediating spinal neuroinflammation and mechanical pain in an animal model of CRPS-I. Our work suggests that targeting CXCL13/CXCR5 pathway may lead to novel therapeutic approaches for CRPS-I.

Ethanol extract of Piper wallichii ameliorates DSS-induced ulcerative colitis in mice: Involvement of TLR4/NF-κB/COX-2 signaling pathway.

Piper wallichii (family: Piperaceae), a folk herbal medicine with anti-inflammatory and anti-thrombotic properties, has been traditionally used to treat rheumatic arthralgia, lumbocrural pain, gastrointestinal flatulence, and other intestinal diseases in China, Thailand, and India. However, there is no scientific report on the efficacy and potential mechanisms of Piper wallichii for ulcerative colitis (UC). The study aims to investigate the therapeutic effect and possible molecular mechanisms of the ethanol extract of Piper wallichii (EEPW) on DSS-induced UC in BALB/c mice. The main components in EEPW were characterized by UPLC-QE-Orbitrap-MS. Subsequently, the anti-inflammatory effect of EEPW in vitro was preliminarily evaluated in RAW264.7cells stimulated with LPS. UC model mice were triggered by free access to 4% DSS aqueous solution for 12 consecutive days, and simultaneously, EEPW (25, 50, and 100mg/kg) and tofacitinib (positive control, 30mg/kg) were orally administrated, respectively. The therapeutic efficacy of EEPW on UC was assessed by body weight, DAI, colon length, and pathological morphology. Besides, we investigated the effects of EEPW on intestinal barrier function, inflammatory factors, and immune systems of UC mice through immunohistochemistry (IHC), flow cytometry, and other techniques. Moreover, the expression of related proteins in the TLR4/NF-κB/COX-2 pathway was analyzed by Western blot. A total of 14 components were identified in the positive and negative modes, including isofutoquinol A (11), hancinone C (12), and futoquinol (14) which characterized by references. In the RAW264.7cells experiments, the extract significantly suppressed the levels of TNF-α and IL-6. More importantly, EEPW distinctly improved the symptoms of DSS-induced UC mice as reflected by a significant recovery from body weight, colon length, pathological injuries of the colon, and so on. Further research found that EEPW remarkably restored the levels of occludin, promoted proliferation, and inhibited apoptosis in colon to maintain the integrity of intestinal barrier. In addition, the down-regulation of TNF-α and IL-1β in colon, Th1 and Th17cells in spleen, as well as the up-regulation of IL-10 in colon and Th2 cells in spleen were distinctly observed in EEPW-treated groups. Furthermore, the protein expression of TLR4, p-IκB-α, p-p65, and COX-2 were significantly inhibited by EEPW. This study confirmed for the first time that EEPW effectively ameliorated DSS-induced UC in mice, which might be related to improving intestinal barrier function, maintaining the levels of inflammatory factors, and regulating the immune system. In addition, we found that the anti-inflammatory effect of EEPW on UC mice was involved in the TLR4/NF-κB/COX-2 signaling pathway. In conclusion, Piper wallichii can be used as a candidate for the treatment of UC.