Helicobacter pylori disrupts gastric mucosal homeostasis by stimulating macrophages to secrete CCL3.

BACKGROUND: Helicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis. METHODS: The expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry. RESULTS: After H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased. CONCLUSIONS: In summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.

Exploring causal correlations between inflammatory cytokines and knee osteoarthritis: a two-sample Mendelian randomization.

Objectives: Knee osteoarthritis (KOA) and certain inflammatory cytokines (such as interleukin 1 [IL-1] and tumor necrosis factor alpha [TNF-a]) are related; however, the causal relationship remains unclear. Here, we aimed to assess the causal relationship between 41 inflammatory cytokines and KOA using Mendelian randomization (MR). Methods: Two-sample bidirectional MR was performed using genetic variation data for 41 inflammatory cytokines that were obtained from European Genome-Wide Association Study (GWAS) data (n=8293). KOA-related genetic association data were also obtained from European GWAS data (n=40,3124). Inverse variance weighting (IVW), MR, heterogeneity, sensitivity, and multiple validation analyses were performed. Results: Granulocyte colony-stimulating factor (G-CSF) or colony-stimulating factor 3 (CSF-3) levels were negatively associated with the risk of developing KOA (OR: 0.93, 95%CI:0.89-0.99, P=0.015). Additionally, macrophage inflammatory protein-1 alpha (MIP-1A/CCL3) was a consequence of KOA (OR: 0.72, 95%CI:0.54-0.97, P=0.032). No causal relationship was evident between other inflammatory cytokines and KOA development. Conclusion: This study suggests that certain inflammatory cytokines may be associated with KOA etiology. G-CSF exerts an upstream influence on KOA development, whereas MIP-1A (CCL-3) acts as a downstream factor.

Anti-Neuroinflammatory Effects of a Macrocyclic Peptide-Peptoid Hybrid in Lipopolysaccharide-Stimulated BV2 Microglial Cells.

Inflammation processes of the central nervous system (CNS) play a vital role in the pathogenesis of several neurological and psychiatric disorders like depression. These processes are characterized by the activation of glia cells, such as microglia. Clinical studies showed a decrease in symptoms associated with the mentioned diseases after the treatment with anti-inflammatory drugs. Therefore, the investigation of novel anti-inflammatory drugs could hold substantial potential in the treatment of disorders with a neuroinflammatory background. In this in vitro study, we report the anti-inflammatory effects of a novel hexacyclic peptide-peptoid hybrid in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The macrocyclic compound X15856 significantly suppressed Interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), c-c motif chemokine ligand 2 (CCL2), CCL3, C-X-C motif chemokine ligand 2 (CXCL2), and CXCL10 expression and release in LPS-treated BV2 microglial cells. The anti-inflammatory effects of the compound are partially explained by the modulation of the phosphorylation of p38 mitogen-activated protein kinases (MAPK), p42/44 MAPK (ERK 1/2), protein kinase C (PKC), and the nuclear factor (NF)-κB, respectively. Due to its remarkable anti-inflammatory properties, this compound emerges as an encouraging option for additional research and potential utilization in disorders influenced by inflammation, such as depression.

Dual fluorescence reporter mice for Ccl3 transcription, translation, and intercellular communication.

Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.

Stressing the Role of CCL3 in Reversing the Immunosuppressive Microenvironment in Gliomas.

Patients with gliomas often experience mental health problems, such as depression and anxiety, that lead to worsening tumor progression and shortened survival. In this issue, Wang and colleagues report a novel mechanism underlying this, finding that chronic stress reduces secretion of the chemokine CCL3, which leads to an immunosuppressive glioma microenvironment. CCL3 administration enhances the infiltration of antitumor immune cells, providing rationale for a potential new therapeutic approach. See related article by Wang et al., p. 516 (4).

[The functional differences of macrophages derived from murine bone marrow and peritoneal cavity].

Objective To compare the functional differences between bone marrow derived macrophages and peritoneal macrophages, which may provide the basis for the selection of macrophages in immunological research and immunoregulatory drug evaluation. Methods Marophage colony-stimulating factor (M-CSF) was used to induce the differentiation of bone marrow monocytes into macrophages, and thioglycolate medium was used to induce peritonitis to obtain peritoneal macrophages. After both macrophages being stimulated by zymosan, LPS, R848 and CpG respectively, mRNA levels of tumor necrosis factor α(TNF-α), interleukin 6(IL-6), macrophage inflammatory protein 1α(MIP-1α), monocyte chemoattractant protein 1(MCP-1) were measured by Real-time fluorescent quantitative PCR and the concentrations of secreted TNF-α, IL-6, MIP-1α and MCP-1 were detected by ELISA. In addition, the expression of costimulatory molecules CD80, CD86, CD40 and histocompatibility complex II (MHC II) on the cell surface was analyzed by flow cytometry. Results After inducing by different TLR ligands, mRNA expression levels of inflammatory cytokines and chemokines were increased in both macrophages. The secretion of TNF-α, IL-6, MIP-1α and MCP-1 in peritoneal macrophages and the expression of CD86 and MHC II on the surface of peritoneal macrophages were significantly higher than those of bone marrow derived macrophages. Conclusion There are significant differences in the expression of inflammatory factors, chemokines, costimulatory molecules, and histocompatibility complex between bone marrow derived macrophages and peritoneal macrophages. Peritoneal macrophages have more complete macrophage function and is more suitable for immunological research and immunomodulatory drug evaluation.

Kaempferia parviflora Extracellular Vesicle Loaded with Clarithromycin for the Treatment of Helicobacter pylori Infection.

Purpose: Kaempferia parviflora extracellular vesicles (KPEVs) have been reported as promising nanovesicles for drug delivery. This study aimed to load clarithromycin (CLA) into KPEVs (KPEVS-CLA) and determine the physical properties, drug-releasing efficiency, gastric cell uptake, anti-H. pylori activities, and anti-inflammatory responses in comparison with free CLA and KPEVs. Methods: The size and surface charge of KPEVs-CLA were evaluated using dynamic light scattering and visualized using a transmission electron microscope. The encapsulation efficiency (EE%), loading capacity (LC%), and drug release of KPEVs-CLA were examined using HPLC. Anti-H. pylori growth and anti-adhesion were evaluated. IL-8 gene expression, NF-κB signaling proteins, and anti-inflammatory profiles were examined using qRT-PCR, Western blotting, and Bio-Plex immunoassay, respectively. Anti-chemotaxis was then examined using a Transwell assay. Results: KPEVs-CLA were intact and showed a negative surface charge similar to that of KPEVs. However, slightly enlarged KPEVs were observed. CLA was successfully loaded into KPEVs with EE of 93.45% ± 2.43%, LC of 9.3% ± 3.02%. CLA release in the PBS and gastric mimic buffer with Fickian diffusion (n ≤ 0.43) according to Korsmeyer-Peppas kinetic model (R2=0.98). KPEVs-CLA was localized in the gastric cells' cytoplasm and perinuclear region. Anti-H. pylori growth and anti-H. pylori adhesion of KPEVs-CLA were compared with those of free CLA with no cytotoxicity to adenocarcinoma gastric cells. KPEVs-CLA significantly reduced IL-8, G-CSF, MIP-1α, and MIP-1ß levels. Moreover, KPEVs-CLA showed a superior effect over CLA in reducing G-CSF, MIP-1α, and NF-κB phosphorylation and monocyte chemotactic activities. Conclusion: KPEVs serve as potential carriers of CLA. They exhibited a higher efficiency in inhibiting gastric cell inflammation mediated by H. pylori infection than free CLA. The establishment of KPEVs-CLA as a nanodrug delivery model for H. pylori treatment could be applied to other plant extracellular vesicles or loaded with other cancer drugs for gastric cancer treatment.

Th2-predominant immune response underlies the pathogenesis of Dengue.

BACKGROUND: Dengue is a rapidly emerging pandemic-prone disease, whose manifestations range from asymptomatic infection to life-threatening complications like Dengue Hemorrhagic Fever and Dengue Shock Syndrome. This study investigates and compares the immune response in clinically defined cohorts of Dengue with and without warning signs, with the aim of identifying immunological correlates of clinical disease and potential markers of disease severity. METHODS: Blood samples, collected from study participants fulfilling the WHO definition of Dengue with and without warning signs and healthy volunteers, were analyzed using flow cell-based fluorometric methods for cytokines and chemokines. Gene expression analysis, using RT-PCR, was conducted on T helper cell subset-specific transcription factors and cytokines. Demographic details, virological markers, serotype distribution, and hematological parameters were also investigated in all the subjects. RESULTS: The 35 participants recruited in the study, included 11 healthy volunteers and 12 patients each fulfilling the WHO criteria of Dengue with and without warning signs. While the demographic characteristics and serotype distribution was similar in Dengue with and without warning signs cohorts of the disease, platelet counts and Aspartate Aminotransferase (AST) levels changed significantly between Dengue with and without warning signs patients. Plasma cytokine analysis showed up-regulation of IL-4, IL-10, IP-10, and MCP-1 in Dengue patients compared to healthy volunteers. Disease severity was associated with elevated levels of IL-10, IP-10, IL-4, MCP-1, and MIP-1α. IL-8 and MIP-1α were significantly up-regulated in Dengue with warning sign compared to Dengue without warning signs cases. Transcription factor analysis indicated increased expression of RORα, FoxP3, and GATA3 in Dengue patients. mRNA expression of TGFß and IL-4 was also elevated in Dengue patients. A positive correlation between mRNA expression of IL-4 and plasma IL-4 was observed. CONCLUSION: The study reveals a Th2-predominant immune response in all Dengue patients, regardless of disease severity, with overexpression of IL-8 and MIP-1α being observed in patients with warning signs.

Chronic Stress Exacerbates the Immunosuppressive Microenvironment and Progression of Gliomas by Reducing Secretion of CCL3.

As understanding of cancer has deepened, increasing attention has been turned to the roles of psychological factors, especially chronic stress-induced depression, in the occurrence and development of tumors. However, whether and how depression affects the progression of gliomas are still unclear. In this study, we have revealed that chronic stress inhibited the recruitment of tumor-associated macrophages (TAM) and other immune cells, especially M1-type TAMs and CD8+ T cells, and decreased the level of proinflammatory cytokines in gliomas, leading to an immunosuppressive microenvironment and glioma progression. Mechanistically, by promoting the secretion of stress hormones, chronic stress inhibited the secretion of the chemokine CCL3 and the recruitment of M1-type TAMs in gliomas. Intratumoral administration of CCL3 reprogrammed the immune microenvironment of gliomas and abolished the progression of gliomas induced by chronic stress. Moreover, levels of CCL3 and M1-type TAMs were decreased in the tumor tissues of glioma patients with depression, and CCL3 administration enhanced the antitumor effect of anti-PD-1 therapy in orthotopic models of gliomas undergoing chronic stress. In conclusion, our study has revealed that chronic stress exacerbates the immunosuppressive microenvironment and progression of gliomas by reducing the secretion of CCL3. CCL3 alone or in combination with an anti-PD-1 may be an effective immunotherapy for the treatment of gliomas with depression. See related Spotlight by Cui and Kang, p. 514.

The interplay of Cxcl10+/Mmp14+ monocytes and Ccl3+ neutrophils proactively mediates silica-induced pulmonary fibrosis.

As a fatal occupational disease with limited therapeutic options, molecular mechanisms underpinning silicosis are still undefined. Herein, single-cell RNA sequencing of the lung tissue of silicosis mice identified two monocyte subsets, which were characterized by Cxcl10 and Mmp14 and enriched in fibrotic mouse lungs. Both Cxcl10+ and Mmp14+ monocyte subsets exhibited activation of inflammatory marker genes and positive regulation of cytokine production. Another fibrosis-unique neutrophil population characterized by Ccl3 appeared to be related to the pro-fibrotic process, specifically the "inflammatory response". Meanwhile, the proportion of monocytes and neutrophils was significantly higher in the serum of silicosis patients and slices of lung tissue from patients with silicosis further validated the over-expression of Cxcl10 and Mmp14 in monocytes, also Ccl3 in neutrophils, respectively. Mechanically, receptor-ligand interaction analysis identified the crosstalk of Cxcl10+/Mmp14+ monocytes with Ccl3+ neutrophils promoting fibrogenesis via coupling of HBEGF-CD44 and CSF1-CSF1R. In vivo, administration of clodronate liposomes, Cxcl10 or Mmp14 siRNA-loaded liposomes, Ccl3 receptor antagonist BX471, CD44 or CSF1R neutralizing antibodies significantly alleviated silica-induced lung fibrosis. Collectively, these results demonstrate that the newly defined Cxcl10+/Mmp14+ monocytes and Ccl3+ neutrophils participate in the silicosis process and highlight anti-receptor-ligand pair treatment as a potentially effective therapeutic strategy in managing silicosis.

Inflammatory cytokines and risk of allergic rhinitis: A Mendelian randomization study.

BACKGROUND: Epidemiological and experimental evidences have implicated chronic inflammation in the association with allergic rhinitis (AR). However, it remains unclear whether specific circulating cytokines are the cause of AR or the consequence of bias. To examine whether genetic-predicted changes in circulating cytokine concentrations are related to the occurrence of AR, we conducted a two-sample Mendelian randomization (MR) analysis. METHODS: We investigated the causal effects of 26 circulating inflammatory cytokines on AR through MR analysis. The primary method employed in this study was the inverse variance-weighted (IVW) method. Sensitivity analyses were conducted using simple median, weighted median, penalized weighted median, and MR-Egger regression. RESULTS: Our study revealed suggestive evidence that higher levels of circulating IL-18 (OR per one standard deviation [SD] increase: 1.006; 95 % CI, 1.002 to 1.011; P = 0.006, PFDR = 0.067, random-effects IVW method) and Macrophage inflammatory protein-1α (MIP-1α) (OR per one SD increase: 1.015; 95 % CI, 1.004 to 1.026; P = 0.009, PFDR = 0.048, random-effects IVW method) were associated with an increased risk of AR. Conversely, higher levels of circulating TRAIL were associated with a decreased risk of AR (OR per one SD increase: 0.993; 95 % CI, 0.989 to 0.997; P = 4.58E-4, PFDR = 0.004, random-effects IVW method). Only the results of TRAIL exist after Bonferroni-correction (the p-value < 0.0019). Sensitivity analysis yielded directionally consistent results. No significant associations were observed between other circulating inflammatory cytokines and AR. CONCLUSION: Genetically predicted levels of IL-18, and MIP-1α are likely to associated with an increased risk of AR occurrence. Genetically predicted levels of TRAIL are statistically significant in reducing the risk of AR occurrence. However, the current research evidence does not support an impact of other inflammatory cytokines on the risk of AR. Future studies are needed to provide additional evidence to support the current conclusions.

M1 cholinergic signaling in the brain modulates cytokine levels and splenic cell sub-phenotypes following cecal ligation and puncture.

BACKGROUND: The contribution of the central nervous system to sepsis pathobiology is incompletely understood. In previous studies, administration of endotoxin to mice decreased activity of the vagus anti-inflammatory reflex. Treatment with the centrally-acting M1 muscarinic acetylcholine (ACh) receptor (M1AChR) attenuated this endotoxin-mediated change. We hypothesize that decreased M1AChR-mediated activity contributes to inflammation following cecal ligation and puncture (CLP), a mouse model of sepsis. METHODS: In male C57Bl/6 mice, we quantified basal forebrain cholinergic activity (immunostaining), hippocampal neuronal activity, serum cytokine/chemokine levels (ELISA) and splenic cell subtypes (flow cytometry) at baseline, following CLP and following CLP in mice also treated with the M1AChR agonist xanomeline. RESULTS: At 48 h. post-CLP, activity in basal forebrain cells expressing choline acetyltransferase (ChAT) was half of that observed at baseline. Lower activity was also noted in the hippocampus, which contains projections from ChAT-expressing basal forebrain neurons. Serum levels of TNFα, IL-1ß, MIP-1α, IL-6, KC and G-CSF were higher post-CLP than at baseline. Post-CLP numbers of splenic macrophages and inflammatory monocytes, TNFα+ and ILß+ neutrophils and ILß+ monocytes were higher than baseline while numbers of central Dendritic Cells (cDCs), CD4+ and CD8+ T cells were lower. When, following CLP, mice were treated with xanomeline activity in basal forebrain ChAT-expressing neurons and in the hippocampus was significantly higher than in untreated animals. Post-CLP serum concentrations of TNFα, IL-1ß, and MIP-1α, but not of IL-6, KC and G-CSF, were significantly lower in xanomeline-treated mice than in untreated mice. Post-CLP numbers of splenic neutrophils, macrophages, inflammatory monocytes and TNFα+ neutrophils also were lower in xanomeline-treated mice than in untreated animals. Percentages of IL-1ß+ neutrophils, IL-1ß+ monocytes, cDCs, CD4+ T cells and CD8+ T cells were similar in xanomeline-treated and untreated post-CLP mice. CONCLUSION: Our findings indicate that M1AChR-mediated responses modulate CLP-induced alterations in serum levels of some, but not all, cytokines/chemokines and affected splenic immune response phenotypes.

Evaluation of the serum levels of CCL2, CCL3, and IL-29 after first and second administrations of the COVID-19 vaccine (Oxford-AstraZeneca).

BACKGROUND: Previous studies show that chemokines and cytokines play a very important role in eliciting an appropriate response against viruses. Vaccination causes inflammation in the person receiving the vaccine, accompanied with production of inflammatory molecules by immune cells. The more and better the production and expression of chemokines and cytokines by immune cells, the better the response of the acquired immune system. Chemokines and cytokines are critical in promoting the innate immune response against the COVID-19. Here we intended to assess serum levels of CCL2, CCL3, and interleukin (IL)-29 in patients received COVID-19 vaccine. METHODS: In this study, 40 subjects vaccinated with the Oxford-AstraZeneca COVID-19 vaccine were selected. Blood samples were collected before injection of the vaccine, 3-5 days after the first dose injection, and 3-5 days subsequent to the second vaccination. To check the serum level of CCL2, CCL3, and IL-29, ELISA technique was used. RESULTS: Our results indicated that the serum levels of CCL2, CCL3, and IL-29 were significantly higher after first and second dose of vaccination compared to before vaccine administration. Furthermore, serum levels of all these mediators were higher after second dose of vaccine compared to the first vaccine administration. CONCLUSIONS: Oxford-AstraZeneca COVID-19 vaccine is able to induce inflammatory CCL2 and CCL3 chemokines as well as protective interferon lambda (IL-29).

Targeted checkpoint control of B cells undergoing positive selection in germinal centers by follicular regulatory T cells.

Follicular regulatory T cells (Tfr) can play opposite roles in the regulation of germinal center (GC) responses. Depending on the studies, Tfr suppress or support GC and B cell affinity maturation. However, which factors determine positive vs. negative effects of Tfr on the GC B cell is unclear. In this study, we show that GC centrocytes that express MYC up-regulate expression of CCL3 chemokine that is needed for both the positive and negative regulation of GC B cells by Tfr. B cell-intrinsic expression of CCL3 contributes to Tfr-dependent positive selection of foreign Ag-specific GC B cells. At the same time, expression of CCL3 is critical for direct Tfr-mediated suppression of GC B cells that acquire cognate to Tfr nuclear proteins. Our study suggests that CCR5 and CCR1 receptors promote Tfr migration to CCL3 and highlights Ccr5 expression on the Tfr subset that expresses Il10. Based on our findings and previous studies, we suggest a model of chemotactically targeted checkpoint control of B cells undergoing positive selection in GCs by Tfr, where Tfr directly probe and license foreign antigen-specific B cells to complete their positive selection in GCs but, at the same time, suppress GC B cells that present self-antigens cognate to Tfr.

DPP-4 inhibition by linagliptin ameliorates age-related mild cognitive impairment by regulating microglia polarization in mice.

Extensive preclinical evidence demonstrates a causative link between insulin signaling dysfunction and the pathogenesis of Alzheimer's disease (AD), and diabetic drugs may represent a promising approach to fighting AD. However, it remains to be determined which antidiabetic drugs are more effective in preventing cognitive impairment. Thus, the present study investigated the effect of dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin on cognitive impairment in middle-aged mice by comparing it with the effect of metformin. We found that DPP-4 activity increased in the hippocampus of middle-aged mice, and DPP-4 was mainly expressed by microglia rather than astrocytes and oligodendrocytes. DPP-4 directly regulated M1/M2 microglia polarization following LPS or IL-4 stimulation, while DPP-4 inhibitor, linagliptin, suppressed M1-polarized activation and induced M2-polarized activation. Both linagliptin and metformin enhanced cognitive ability, increased hippocampal synaptic plasticity and neurogenesis, and decreased age-related oxidative stress and inflammation by regulating microglia polarization in the hippocampus of middle-aged mice. The combination of linagliptin and metformin showed a maximum protective effect compared to the individual drugs alone. Loss of macrophage inflammatory protein-1α (MIP-1α), a DPP-4 substrate, abrogated the cognitive protection and anti-inflammation effects of linagliptin. Therefore, the current investigation exhibits a potential utility for DPP-4 inhibition in attenuating microglia-mediated inflammation and preventing mild cognitive impairment (MCI) in middle-aged mice, and the effect was partly mediated by MIP-1α.

Myeloid-specific deletion of group VIA calcium-independent phospholipase A2 induces pro-inflammatory LPS response predominantly in male mice via MIP-1α activation.

Polymorphisms of group VIA calcium-independent phospholipase A2 (PLA2G6) are associated with blood C-reactive protein suggesting its role in inflammation. We showed that myeloid-specific Pla2g6-deficiency in Pla2g6M-/- mice led to exaggerated inflammation and fibrosis in a lean fatty liver model. We here investigated whether these mutants display alteration in immune response after treatment with E. coli lipopolysaccharides (LPS) under acute (a single dose) and persistent (four doses) conditions. Without LPS treatment, male Pla2g6M-/- (but not Flox) mice at 12 months of age exhibited splenomegaly and hepatic necrosis, and ~ 30 % of them exhibited autoimmune hepatitis showing lymphoplasma cells with CD3(+) and CD45R(+) staining. Under acute LPS, male mutants showed an elevation of plasma MIP-1α and immunoglobulinA as well as upregulation of hepatic apoptosis and fibrosis PARP-1, Bax, MCP-1, α-SMA, and collagen I proteins. Their bone-marrow-derived macrophages also showed an elevation of MIP-1α release upon LPS stimulation in vitro. Female mutants under acute LPS showed a moderate increase in plasma KC/CXCL1, MCP-1, and IL10, and they showed no remarkable increase in hepatic fibrosis under acute or persistent LPS. Male mutants under persistent LPS displayed an elevation of aspartate aminotransferase, blood eosinophils, and hepatic apoptosis. Moreover, ~30 % of these mutants exhibited eosinophilic sclerosing portal hepatitis associated with an upregulated protein expression of hepatic CD8α, CD68, eosinophilic cationic protein, and Ly6G. Thus, myeloid-PLA2G6 deficiency led to an autoimmune and LPS-induced inflammatory liver disease via MIP-1α in a male-predominant manner. Our results may be applicable to patients with PLA2G6 mutations who undergo bacterial infection and sepsis.

Biostimulating fillers and induction of inflammatory pathways: A preclinical investigation of macrophage response to calcium hydroxylapatite and poly-L lactic acid.

INTRODUCTION: Initial macrophage response to biostimulatory substances is key in determining the subsequent behavior of fibroblasts and the organization of newly synthesized collagen. Though histological studies suggest that calcium hydroxylapatite (CaHA) filler initiates a regenerative healing response with collagen and elastin deposition similar to natural, healthy tissue rather than an inflammatory response with fibrosis, the relative activity of macrophages stimulated by CaHA, as well as how this activity compares to that induced by other biostimulatory fillers, has not been explored. The aim of the study is to characterize the in vitro macrophage response to two biostimulory fillers, CaHA and PLLA (poly-L lactic acid), and to evaluate their inflammatory potential. METHODS: Primary human macrophages were incubated with two dilutions (1:50 and 1:100) of commercially available CaHA or PLLA. After 24 h incubation, an inflammation array was used to screen for the expression of 40 cytokines, released by macrophages. ELISA was used to confirm array results. RESULTS: Four cytokines were significantly upregulated in M1 macrophages incubated with PLLA compared to both unstimulated controls and CaHA: CCL1 (p < 0.001), TNFRII (p < 0.01), MIP-1α (p < 0.05), and IL-8 (p < 0.001). In M2 macrophages, MIP-1α (p < 0.01) and MIP-1ß (p < 0.01) were significantly upregulated by PLLA compared to CaHA and unstimulated controls. CONCLUSION: Together, these findings indicate that the CaHA mode of action is a non-inflammatory response while PLLA initiates expression of several cytokines known to play a role in inflammation. Our study supports the concept that these two "biostimulatory" fillers follow distinct pathways and should be considered individually with regard to mechanism of action.

The PPARα agonist fenofibrate reduces the cytokine imbalance in a maternal immune activation model of schizophrenia.

Maternal infections during pregnancy may increase the risk of psychiatric disorders in offspring. We recently demonstrated that activation of peroxisome proliferator-activate receptor-α (PPARα), with the clinically available agonist fenofibrate (FEN), attenuates the neurodevelopmental disturbances induced by maternal immune activation (MIA) in rat offspring. We hypothesized that fenofibrate might reduce MIA-induced cytokine imbalance using a MIA model based on the viral mimetic polyriboinosinic-polyribocytidilic acid [poly (I:C)]. By using the Bio-Plex Multiplex-Immunoassay-System, we measured cytokine/chemokine/growth factor levels in maternal serum and in the fetal brain of rats treated with fenofibrate, at 6 and 24 h after poly (I:C). We found that MIA induced time-dependent changes in the levels of several cytokines/chemokines/colony-stimulating factors (CSFs). Specifically, the maternal serum of the poly (I:C)/control (CTRL) group showed increased levels of (i) proinflammatory chemokine macrophage inflammatory protein 1-alpha (MIP-1α), (ii) tumor necrosis factor-alpha (TNF-α), the monocyte chemoattractant protein-1 (MCP-1), the macrophage (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Conversely, in the fetal brain of the poly (I:C)/CTRL group, interleukin 12p70 and MIP-1α levels were lower than in vehicle (veh)/CTRL group. Notably, MIP-1α, TNF-α, keratinocyte derived chemokine (GRO/KC), GM-CSF, and M-CSF levels were lower in the poly (I:C)/FEN than in poly (I:C)/CTRL rats, suggesting the protective role of the PPARα agonist. PPARα might represent a therapeutic target to attenuate MIA-induced inflammation.

Exploring causal relationships between inflammatory cytokines and allergic rhinitis, chronic rhinosinusitis, and nasal polyps: a Mendelian randomization study.

Objectives: Previous research has suggested connections between specific inflammatory cytokines and nasal conditions, including Allergic Rhinitis (AR), Chronic Rhinosinusitis (CRS), and Nasal Polyps (NP). However, a lack of robust research establishing the causal underpinnings of them. This Mendelian Randomization (MR) study aims to evaluate the causal relationships between 41 inflammatory cytokines and the incidence of AR, CRS and NP. Methods: This study employed a two-sample MR design, harnessing genetic variations derived from publicly accessible genome-wide association studies (GWAS) datasets. AR data was sourced from a GWAS with 25,486 cases and 87,097 controls (identifier: ukb-b-7178). CRS data originated from a GWAS encompassing 1,179 cases and 360,015 controls (identifier: ukb-d-J32). NP data was extracted from a GWAS involving 1,637 cases and 335,562 controls (identifier: ukb-a-541). The data for 41 inflammatory cytokines were obtained from an independent GWAS encompassing 8,293 participants. Inverse variance weighted (IVW), MR Egger regression and Weighted median were used to evaluate the causalities of exposures and outcomes. A range of sensitivity analyses were implemented to assess the robustness of the results. Results: The results revealed significant associations between elevated circulating levels of MIP-1α (odds ratio, OR: 1.01798, 95% confidence interval, CI: 1.00217-1.03404, p = 0.02570) and TNF-α (OR: 1.01478, 95% CI: 1.00225-1.02746, p = 0.02067) with an augmented risk of AR in the IVW approach. Heightened levels of circulating IL-2 exhibited a positive correlation with an increased susceptibility to NP in the IVW approach (OR: 1.00129, 95% CI: 1.00017-1.00242, p = 0.02434), whereas elevated levels of circulating PDGF-BB demonstrated a decreased risk of NP (OR: 0.99920, 95% CI: 0.99841-0.99999, p = 0.047610). The MR analysis between levels of 41 inflammatory cytokines and the incidence of CRS yielded no positive outcomes. Conclusion: This investigation proposes a potential causal association between elevated levels of MIP-1α and TNF-α with an elevated risk of AR, as well as an increased risk of NP linked to elevated IL-2 levels. Furthermore, there appears to be a potential association between increased levels of circulating PDGF-BB and a reduced risk of NP.

C-C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model.

BACKGROUND: Previous studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure. AIM: This study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis  virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism. RESULTS: By gene array analysis, chemokine (C-C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1ß and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1ß or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model. CONCLUSION: The CCR5-MIP-1ß/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.