TNF-α Production In Macrophages Research Articles

Omentin-1 inhibits the development of benign prostatic hyperplasia by attenuating local inflammation

BackgroundBenign prostatic hyperplasia (BPH) is a prevalent disease affecting elderly men, with chronic inflammation being a critical factor in its development. Omentin-1, also known as intelectin-1 (ITLN-1), is an anti-inflammatory protein primarily found in the epithelial cells of the small intestine. This study aimed to investigate the potential of ITLN-1 in mitigating BPH by modulating local inflammation in the prostate gland.MethodsOur investigation involved two in vivo experimental models. Firstly, ITLN-1 knockout mice (Itln-1−/−) were used to study the absence of ITLN-1 in BPH development. Secondly, a testosterone propionate (TP)-induced BPH mouse model was treated with an ITLN-1 overexpressing adenovirus. We assessed BPH severity using prostate weight index and histological analysis, including H&E staining, immunohistochemistry, and enzyme-linked immunosorbent assay. In vitro, the impact of ITLN-1 on BPH-1 cell proliferation and inflammatory response was evaluated using cell proliferation assays and enzyme-linked immunosorbent assay.ResultsIn vivo, Itln-1−/− mice exhibited elevated prostate weight index, enlarged lumen area, and higher TNF-α levels compared to wild-type littermates. In contrast, ITLN-1 overexpression in TP-induced BPH mice resulted in reduced prostate weight index, lumen area, and TNF-α levels. In vitro studies indicated that ITLN-1 suppressed the proliferation of prostate epithelial cells and reduced TNF-α production in macrophages, suggesting a mechanism involving the inhibition of macrophage-mediated inflammation.ConclusionThe study demonstrates that ITLN-1 plays a significant role in inhibiting the development of BPH by reducing local inflammation in the prostate gland. These findings highlight the potential of ITLN-1 as a therapeutic target in the management of BPH.

Butyrate Prevents the Pathogenic Anemia-Inflammation Circuit by Facilitating Macrophage Iron Export.

Most patients with inflammatory bowel disease (IBD) develop anemia, which is attributed to the dysregulation of iron metabolism. Reciprocally, impaired iron homeostasis also aggravates inflammation. How this iron-mediated, pathogenic anemia-inflammation crosstalk is regulated in the gut remains elusive. Herein, it is for the first time revealed that anemic IBD patients exhibit impaired production of short-chain fatty acids (SCFAs), particularly butyrate. Butyrate supplementation restores iron metabolism in multiple anemia models. Mechanistically, butyrate upregulates ferroportin (FPN) expression in macrophages by reducing the enrichment of histone deacetylase (HDAC) at the Slc40a1 promoter, thereby facilitating iron export. By preventing iron sequestration, butyrate not only mitigates colitis-induced anemia but also reduces TNF-α production in macrophages. Consistently, macrophage-conditional FPN knockout mice exhibit more severe anemia and inflammation. Finally, it is revealed that macrophage iron overload impairs the therapeutic effectiveness of anti-TNF-α antibodies in colitis, which can be reversed by butyrate supplementation. Hence, this study uncovers the pivotal role of butyrate in preventing the pathogenic circuit between anemia and inflammation.

In Vitro Biocompatibility of the Novel Ceramic Composite Baghdadite for Defect Augmentation in Revision Total Hip Arthroplasty.

Biological augmentation of bony defects in weight-bearing areas of both the acetabulum and the femur remains challenging. The calcium-silicate-based ceramic Baghdadite is a very interesting material to be used in the field of revision total hip arthroplasty for the treatment of bony defects in weight-bearing and non-weight-bearing areas alike. The aim of this study was to investigate the biocompatibility of Baghdadite utilizing an osteoblast-like, human osteosarcoma cell line (MG-63) and the human monocytic leukemia-derived cell line (THP-1). THP-1-derived macrophages and MG-63 were indirectly exposed to Baghdadite for 7 days using a transwell system. Viability was assessed with MTT assay and pH analysis. To investigate proliferation rate, both cell lines were labelled using CFSE and flow cytometrically analyzed. ELISA was used to measure the secretion of IL-1ß, IL-6 and TNFα. The investigation of viability, while showing a slight difference in optical density for the MTT assays in MG-63 cells, did not present a meaningful difference between groups for both cell lines. The comparison of pH and the proportion of living cells between groups did not present with a significant difference for both THP-1 and MG-63. Baghdadite did not have a relevant impact on the proliferation rate of the investigated cell lines. Mean fluorescence intensity was calculated between groups with no significant difference. Baghdadite exerted a proinflammatory effect, which could be seen in an upregulated production of TNFα in macrophages. Production of IL-1ß and IL-6 was not statistically significant, but the IL-6 ELISA showed a trend to an upregulated production as well. A similar effect on MG-63 was not observed. No relevant cytotoxicity of Baghdadite ceramics was encountered. Baghdadite ceramics exhibit a proinflammatory potential by significantly increasing the secretion of TNFα in THP-1-derived macrophages. Whether this proinflammatory potential results in a clinically relevant effect on osteointegration is unclear and requires further investigation. Baghdadite ceramics provide an interesting alternative to conventional bone substitutes and should be further investigated in a biomechanical and in vivo setting.

Azilsartan inhibits inflammation-triggered bone resorption and osteoclastogenesis in vivo via suppression of TNF-α expression in macrophages.

Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

A TNFα/Miz1-positive feedback loop inhibits mitophagy in hepatocytes and propagates non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a chronic inflammatory disease that can further progress to cirrhosis and hepatocellular carcinoma. However, the key molecular mechanisms behind this process have not been clarified. We analyzed human NASH and normal liver tissue samples by RNA-sequencing and liquid chromatography-mass spectrometry, identifying hepatocyte cytosolic protein Myc-interacting zinc-finger protein 1 (Miz1) as a potential target in NASH progression. We established a Western diet+fructose-induced NASH model in hepatocyte-specific Miz1 knockout and adeno-associated virus type 8-overexpressing mice. Human NASH liver organoids were used to confirm the mechanism, and immunoprecipitation and mass spectrometry were used to detect proteins that could interact with Miz1. We demonstrate that Miz1 is reduced in hepatocytes in human NASH. Miz1 is shown to bind to peroxiredoxin 6 (PRDX6), retaining it in the cytosol, blocking its interaction with mitochondrial Parkin at Cys431, and inhibiting Parkin-mediated mitophagy. In NASH livers, loss of hepatocyte Miz1 results in PRDX6-mediated inhibition of mitophagy, increased dysfunctional mitochondria in hepatocytes, and production of proinflammatory cytokines, including TNFα, by hepatic macrophages. Crucially, the increased production of TNFα results in a further reduction in hepatocyte Miz1 by E3-ubiquitination. This produces a positive feedback loop of TNFα-mediated hepatocyte Miz1 degradation, resulting in PRDX6-mediated inhibition of hepatocyte mitophagy, with the accumulation of dysfunctional mitochondria in hepatocytes and increased macrophage TNFα production. Our study identified hepatocyte Miz1 as a suppressor of NASH progression via its role in mitophagy; we also identified a positive feedback loop by which TNFα production induces degradation of cytosolic Miz1, which inhibits mitophagy and thus leads to increased macrophage TNFα production. Interruption of this positive feedback loop could be a strategy to inhibit the progression of NASH. Non-alcoholic steatohepatitis (NASH) is a chronic inflammatory disease that can further develop into cirrhosis and hepatocellular carcinoma. However, the key molecular mechanism of this process has not been fully clarified. Herein, we identified a positive feedback loop of macrophage TNFα-mediated hepatocyte Miz1 degradation, resulting in PRDX6-mediated inhibition of hepatocyte mitophagy, aggravation of mitochondrial damage and increased macrophage TNFα production. Our findings not only provide mechanistic insight into NASH progression but also provide potential therapeutic targets for patients with NASH. Our human NASH liver organoid culture is therefore a useful platform for exploring treatment strategies for NASH development.

Mannose metabolism normalizes gut homeostasis by blocking the TNF-α-mediated proinflammatory circuit.

Mannose is a naturally occurring sugar widely consumed in the daily diet; however, mechanistic insights into how mannose metabolism affects intestinal inflammation remain lacking. Herein, we reported that mannose supplementation ameliorated colitis development and promoted colitis recovery. Macrophage-secreted inflammatory cytokines, particularly TNF-α, induced pathological endoplasmic reticulum stress (ERS) in intestinal epithelial cells (IECs), which was prevented by mannose via normalization of protein N-glycosylation. By preserving epithelial integrity, mannose reduced the inflammatory activation of colonic macrophages. On the other hand, mannose directly suppressed macrophage TNF-α production translationally by reducing the glyceraldehyde 3-phosphate level, thus promoting GAPDH binding to TNF-α mRNA. Additionally, we found dysregulated mannose metabolism in the colonic mucosa of patients with inflammatory bowel disease. Finally, we revealed that activating PMM2 activity with epalrestat, a clinically approved drug for the treatment of diabetic neuropathy, elicited further sensitization to the therapeutic effect of mannose. Therefore, mannose metabolism prevents TNF-α-mediated pathogenic crosstalk between IECs and intestinal macrophages, thereby normalizing aberrant immunometabolism in the gut.

CX3CR1 deficiency exacerbates immune-mediated hepatitis by increasing NF-κB-mediated cytokine production in macrophage and T cell.

Immune-mediated hepatitis is marked by liver inflammation characterized by immune cell infiltration, chemokine/cytokine production, and hepatocyte injury. C-X3C motif receptor 1 (CX3CR1), as the receptor of chemokine C-X3C motif ligand 1 (CX3CL1)/fractalkine, is mainly expressed on immune cells including monocytes and T cells. Previous studies have shown that CX3CR1 protects against liver fibrosis, but the exact role of CX3CL1/CX3CR1 in acute immune-mediated hepatitis remains unknown. Here, we investigate the role of the CX3CL1/CX3CR1 axis in immune-mediated hepatitis using concanavalin A (ConA)-induced liver injury model in CX3CR1-deficient (Cx3cr1-/-) mice. We observed that Cx3cr1-/- mice had severe liver injury and increased pro-inflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interferon-gamma [IFN-γ], interleukin-1 beta [IL-1β], and IL-6) in serum and liver compared to wild-type (Cx3cr1+/+) mice after ConA injection. The deficiency of CX3CR1 did not affect ConA-induced immune cell infiltration in liver but led to elevated production of TNF-α in macrophages as well as IFN-γ in T cells after ConA treatment. On the contrary, exogenous CX3CL1 attenuated ConA-induced cytokine production in wild type, but not CX3CR1-deficient macrophages and T cells. Furthermore, in vitro results showed that CX3CR1 deficiency promoted the pro-inflammatory cytokine expression by increasing the phosphorylation of nuclear factor kappa B (NF-κB) p65 (p-NF-κB p65). Finally, pre-treatment of p-NF-κB p65 inhibitor, resveratrol, attenuated ConA-induced liver injury and inflammatory responses, especially in Cx3cr1-/- mice. In conclusion, our data show that the deficiency of CX3CR1 promotes pro-inflammatory cytokine production in macrophages and T cells by enhancing the phosphorylation of NF-κB p65, which exacerbates liver injury in ConA-induced hepatitis.

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses.

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro. The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.

Uterus globulin associated protein 1 (UGRP1) binds podoplanin (PDPN) to promote a novel inflammation pathway during Streptococcus pneumoniae infection.

Background Streptococcus pneumoniae is the major cause of life‐threatening infections. Toll‐like receptors (TLRs) and NOD‐like receptors (NLRs) could recognise S. pneumoniae and regulate the production of pro‐inflammatory cytokines. UGRP1, highly expressed in lung, is predominantly secreted in airways. However, the function of UGRP1 in pneumonia is mainly unknown.Methods and resultsWe showed that upon TLR2/TLR4/NOD2 agonists stimulation or S. pneumoniae infection, treatment with UGRP1 could promote phosphorylation of p65 and enhance IL‐6, IL‐1β and TNFα production in macrophages. We further elucidated that after binding with cell‐surface receptor PDPN, UGRP1 could activate RhoA to enhance interaction of IKKγ and IKKβ, which slightly activated NF‐κB to improve expression of TLR2, MyD88, NOD2 and NLRP3. Deletion of UGRP1 or blocking UGRP1 interaction with PDPN protected mice against S. pneumoniae‐induced severe pneumococcal pneumonia, and activating RhoA with agonist in UGRP1‐deficient mice restored the reduced IL‐6 production.ConclusionWe demonstrated that UGRP1–PDPN–RhoA signaling could activate NF‐κB to promote expression of TLR2, MyD88, NOD2 and NLRP3, which enhanced inflammatory cytokines secretion during S. pneumoniae infection. Antibodies, which could interrupt interaction of UGRP1 and PDPN, are potential therapeutics against S. pneumoniae.

Differential regulation of lipopolysaccharide-induced IL-1β and TNF-α production in macrophages by palmitate via modulating TLR4 downstream signaling

Diabetic patients are susceptible to infectious diseases. Bacterial invasion activates immune cells such as macrophages through interaction between LPS and TLR4, and induces the expression of inflammatory mediators, including IL-1β and TNF-α, which play key roles in the elimination of infections. Unregulated overproduction or underproduction of these cytokines has been reported as a major factor in the development of septic shock, immune deficiency, and autoimmunity. Recent studies found that metabolic abnormalities of diabetes, such as hyperglycemia and dyslipidemia, played a major role in modulating the immune response. In this study, we studied the effects of palmitic acid (PA) pretreatment on LPS-induced IL-1β and TNF-α production and LPS-TLR4 signaling in macrophages. Compared with control, PA pretreatment significantly increased LPS-induced TNF-α production and secretion in macrophages. In contrast, LPS-induced IL-1β production and secretion was significantly suppressed by PA pretreatment. PA pretreatment did not affect the expression levels of TLR4 or Myd88, or the endocytosis of TLR4 in macrophages. However, PA pretreatment significantly suppressed the phosphorylation level and nuclear translocation of NF-κB, and the phosphorylation level of ERK1/2, whereas increased the phosphorylation levels of p38 and JNK. The activation of IKK which was upstream of NF-κB and ERK1/2 was attenuated, while the activation of TAK1 which was upstream of JNK and p38 was augmented by PA pretreatment. Inhibitors of NF-κB, MEK1/2, and p38 significantly decreased IL-1β expression, while JNK and p38 pathway inhibitors significantly inhibited TNF-α expression. The differential regulation of LPS-induced TNF-α and IL-1β production by PA was associated with cellular metabolism of PA, because inhibiting metabolism of PA with etomoxir or pretreatment with Br-PA which cannot be metabolized reversed these effects. We also showed that PA treatment increased acetylated IKK level which might contribute to the suppressed activation of IKK. The present study showed that LPS-induced production of TNF-α and IL-1β was regulated by different TLR4 downstream pathways in macrophages. PA differentially affected LPS-induced production of TNF-α and IL-1β in macrophages through differentially modulating these pathways. Further experiments will be needed to determine how these phenomena lead to the impaired immune response in patients with diabetes.

C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages.

Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme β-glucocerebrosidase (GCase). GCase catalyzes the conversion of glucosylceramide (GluCer), a ubiquitous glycosphingolipid, to glucose and ceramide. GCase deficiency causes the accumulation of GluCer and its metabolite glucosylsphingosine (GluSph) in a number of tissues and organs. In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives systemic macrophage-mediated inflammation remains incompletely understood. To help understand the mechanisms involved, we used human GD-induced pluripotent stem cell (iPSC)-derived macrophages. We found that GD macrophages exhibit exacerbated production of inflammatory cytokines via an innate immune response mediated by receptor 1 for complement component C5a (C5aR1). Quantitative RT-PCR and ELISA assays showed that in the presence of recombinant C5a (rC5a), GD macrophages secreted 8–10-fold higher levels of TNF-α compared to rC5a-stimulated control macrophages. PMX53, a C5aR1 blocker, reversed the enhanced GD macrophage TNF-α production, indicating that the observed effect was predominantly C5aR1-mediated. To further analyze the extent of changes induced by rC5a stimulation, we performed gene array analysis of the rC5a-treated macrophage transcriptomes. We found that rC5a-stimulated GD macrophages exhibit increased expression of genes involved in TNF-α inflammatory responses compared to rC5a-stimulated controls. Our results suggest that rC5a-induced inflammation in GD macrophages activates a unique immune response, supporting the potential use of inhibitors of the C5a-C5aR1 receptor axis to mitigate the chronic inflammatory abnormalities associated with GD.

PM2.5 promotes β cell damage by increasing inflammatory factors in mice with streptozotocin.

Emerging evidence indicates that exposure to fine particulate matter contributes to the onset of diabetes. The present study aimed to investigate the mechanism of particulate matters (PM)2.5 affecting glucose homeostasis in mice with type 1 diabetes mellitus. Male C57BL/6 mice were housed under filtered air (FA) or PM2.5 for 12 weeks and then received intraperitoneal injection of streptozotocin (STZ; 40 mg/kg) or acetic buffer daily for 5 days. At 4 weeks after the last injection, fasting glucose was tested. In the plasma and liver, cholesterol levels were determined by cholesterol oxidase-peroxidase and triglyceride levels were determined by triglycerophosphate oxidase-peroxidase. Homeostasis model assessment of β cell function (Homa-β) was computed based on fasting insulin and glucose levels. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) levels in plasma, visceral adipose tissues, RAW264.7 macrophages and MIN6 pancreatic β cells treated with PM2.5 (0-50 µg/ml) were quantified via ELISA. Before STZ injection, fasting blood glucose (FBG) levels were similar between FA and PM2.5 groups. After STZ injection, FBG levels were higher in mice pre-exposed to PM2.5 compared with those pre-exposed to FA. When taking FBG levels ≥7 mmol/l as the criteria for impaired glucose level, its incidence was 53.3% and 77.8% in FA and PM2.5 groups, respectively. Independent of STZ injection, IL-1β levels in the adipose tissue were upregulated in mice pre-exposed to PM2.5 compared with FA. The addition of PM2.5 stimulated IL-1β and TNFα production in macrophages and pancreatic β cells, and inhibited the secretion of insulin from MIN6 cells in a dose-dependent manner. In conclusion, pre-exposure of PM2.5 impaired pancreatic β cells in mice upon STZ injection, partially via enhanced inflammation, and suppressed the secretion of insulin.

MO330ACTIVATION OF Β2 ADRENERGIC RECEPTOR SIGNALING IN MACROPHAGES BLOCKS SYSTEMIC INFLAMMATION AND PROTECTS AGAINST RENAL ISCHEMIA/REPERFUSION INJURY

Abstract Background and Aims The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. Here, we focused on sympathetic signaling in macrophages and sought to determine its detailed roles in lipopolysaccharide (LPS)-induced systemic inflammation and renal ischemia/reperfusion injury (IRI). Method In vitro, RAW 264.7 cells and murine peritoneal macrophages were used to determine the effects of β2 adrenergic receptor (Adrb2) signaling on LPS-induced proinflammatory cytokine (tumor necrosis factor-α; TNF-α) production. We also identified the critical gene that mediates the anti-inflammatory effect of Adrb2 signaling by RNA-sequencing. In vivo, we examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. The involvement of macrophage Adrb2 signaling was confirmed by macrophage-specific Adrb2 conditional knockout (cKO) mice and adoptive transfer of salbutamol-treated macrophages. We also performed single-cell RNA sequencing of renal tissue to analyze the renoprotective role of salbutamol-treated macrophages in detail. Results In vitro, norepinephrine, a sympathetic neurotransmitter, suppressed LPS-induced TNF-α production in macrophages. This anti-inflammatory effect was also induced by salbutamol and reversed by butoxamine (a selective Adrb2 antagonist) in a dose-dependent manner, indicating the importance of Adrb2 in this process. RNA sequencing of these macrophages revealed that T-cell immunoglobulin and mucin-3 (Tim3) expressions were upregulated by the activation of Adrb2 signaling, which partially mediated the anti-inflammatory phenotypic alteration in macrophages. In vivo, salbutamol administration mitigated LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific Adrb2 cKO mice. Adoptive transfer of salbutamol-treated macrophages also protected against renal IRI (Figure 1). Single-cell RNA sequencing revealed that this protection was associated with the accumulation of Tim3-expressing macrophages in the renal tissue. Conclusion The activation of β2 adrenergic receptor signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of Tim3 expressions, which blocks LPS-induced systemic inflammation and protects against renal IRI.

POS-074 ACTIVATION OF SYMPATHETIC SIGNALING IN MACROPHAGES MITIGATES SYSTEMIC INFLAMMATION AND PROTECTS AGAINST RENAL ISCHEMIA/REPERFUSION INJURY

The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. Here, we focused on sympathetic signaling in macrophages and sought to determine its detailed roles in lipopolysaccharide (LPS)-induced systemic inflammation and renal ischemia/reperfusion injury (IRI). In vitro, we used RAW 264.7 cells and murine peritoneal macrophages to determine the effects of β2 adrenergic receptor (ADRB2) signaling on LPS-induced proinflammatory cytokine (TNF-α) production. We also identified the critical gene that mediates the anti-inflammatory effect of ADRB2 signaling by RNA-sequencing (RNA-seq). In vivo, we examined the effects of salbutamol (a selective ADRB2 agonist) in LPS-induced systemic inflammation and renal IRI. The involvement of macrophage ADRB2 signaling was confirmed by macrophage-specific ADRB2 conditional knockout (cKO) mice and adoptive transfer of salbutamol-treated macrophages. We also performed single-cell RNA-seq of renal tissue to analyze the renoprotective role of salbutamol-treated macrophages in detail. In vitro Norepinephrine, a main sympathetic neurotransmitter, suppressed LPS-induced TNF-α production in macrophages. This anti-inflammatory effect was also induced by salbutamol and reversed by butoxamine (a selective ADRB2 antagonist) in a dose-dependent manner, indicating the importance of ADRB2 in this process. RNA-seq of these macrophages revealed that T-cell immunoglobulin and mucin-3 (TIM-3) expressions were upregulated by the activation of ADRB2 signaling, which partially mediated the anti-inflammatory phenotypic alteration in macrophages.<In vivo>Salbutamol administration mitigated LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific ADRB2 cKO mice. Adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA-seq revealed that this protection was associated with the accumulation of TIM-3-expressing macrophages in the renal tissue. The activation of sympathetic signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of TIM-3 expressions, which blocks LPS-induced systemic inflammation and protects against renal IRI.

ЭКСПРЕССИЯ ПОВЕРХНОСТНОГО АНТИГЕНА BRUCELLA ABORTUS ОМР16 В РАСТЕНИИ NICOTIANA BENTHAMIANA

Brucellosis is one of the most contagious and infectious diseases with high incidence rates of cattle and humans in Kazakhstan. Using modern biotechnology techniques to develop vaccines that are reliable and affordable for farmers is an alternative solution to the problem. Plant viruses are often used as a vector for obtaining the expression of antigens of the pathogen. The grape virus A (BAB) is widely used among plant viruses. Brucella membrane proteins are the main objects of this research for futher development of vaccines or diagnostic texts against brucellosis, Membrane proteins (OMPs) are cell specific surface antigens that are immunogenic. OMPs are ideal candidates for the production of recombinant brucellosis vaccines. The object of the study was the outer membrane protein (Omp16), which plays an important role in the suppression of TNF-α production in macrophages. In this study, molecular cloning and analysis of the expression of the Omp16 gene, which was used to express the recombinant protein in plants, was carried out. We selected brucella from the vaccine strain of Brucella abortus 19, and the plant Nicotiana benthamiana, as the subjects for our research, since they widely used for the production of recombinant proteins, and they both appropriate for molecular genetic research. A viral vector was constructed to express the brucellosis antigen Omp16 in Nicotiana benthamiana plants. Nineteen explants were used for the regeneration of transgenic plants. As a result of this studies, the introduced gene of Omp16 was under the subgenomic promoter control of the ORF4 and was successfully expressed while maintaining the efficiency of expression in transgenic plants. The efficiency of viral vectors was evaluated at the level of transcription during expression of the protein Omp16 with viral proteins. The entire leaf blade was infiltrated; the density of Agrobacteria was 0.7. We were able to obtained transgenic plants Nicotiana benthamiana carrying the gene of capsid protein BAB, and the expression of the membrane antigen Omp16 in the viral vector was achieved by replacing the ORF4 with the Omp16 gene. The development of transgenic plants was carried out using agrobacterial transformation.

The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage.

Objective: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. Methods: The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated plate. Results: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 knockout decreased the protein levels of citrullinated nuclear factor κB (NF-κB) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1β and TNF-α in the U937 cells and IL-1β and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1β, IL-6, and TNF-α were all dramatically decreased in the PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1. Conclusion: This study showed that PADI2 could promote IL-1β, IL-6, and TNF-α production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages.

2322-PUB: PM2.5 Accelerated Impaired Pancreatic a-Cells by Increasing Expression of Inflammatory Factors in Mice with Streptozotocin

We investigated how PM2.5 exposure could affect glucose homeostasis in mice with streptozotocin (STZ). Male C57BL/6 mice were housed under filtered air (FA) or PM2.5 for 12 weeks and then received intraperitoneal injection of STZ (40 mg/kg of bodyweight) or acetic buffer daily for consecutive five days. Four weeks after the last injection, fasting glucose test was performed. Cholesterol and triglyceride levels were determined in the plasma and liver. Homeostasis Model Assessment of β cell function (Homa-β) were computed using fasting insulin and glucose. Interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) levels in plasma, visceral adipose tissues, macrophage RAW264.7 and pancreatic β-cells MIN6 treated with PM2.5 (0-50 μg/ml) were quantified by ELISA. Before STZ injection, fasting blood glucose (FBG) levels were similar between FA and PM2.5 groups (p=0.92). After STZ injection, FBG levels were 1.2- fold higher in mice pre-exposed to PM2.5 than those pre-exposed to FA (p=0.17). When taking FBG levels ≥7 mmol/L as the criteria for impaired glucose tolerance, its incidence was 53.3% and 77.8% in FA and PM2.5 groups, respectively (p=0.08). IL-1β and TNF-α levels in the adipose tissues were greater in diabetic mice pre-exposed to PM2.5 than diabetic mice pre-exposed to FA (p&amp;lt;0.05 for all). The addition of PM2.5 stimulated IL-1β and TNF-α production in macrophages and pancreatic β-cells, and impaired the incretion of insulin in MIN6 cells in a dose-dependent manner. In conclusion, pre-exposure of PM2.5 impaired pancreatic β-cells in mice upon STZ injection, partially via enhanced inflammation and suppressed the incretion of insulin. Disclosure R. Yin: None. L. Yang: None. J. Lang: None. D. Zhao: None. Funding National Natural Science Foundation of China (81800768)

Cholesterol-dependent cytolysins promote immune evasion by clustering host membrane lipids

Abstract Pathogens causing necrotizing soft tissue infections, such as Streptococcus pyogenes and Clostridium perfringens, use cholesterol dependent cytolysins (CDCs) to evade immune responses by blocking pro-inflammatory TNFα production in macrophages. This evasion may be an unintended consequence of membrane repair responses to the CDC. CDCs trigger lipid-dependent sequestration and shedding of CDC pores on microvesicles. However, the molecular mechanisms by which CDCs exploit membrane repair to disrupt immune signaling is unknown. One target may be cholesterol-rich microdomains (CRMs) because they are focal points for toxin binding and immune signaling. We hypothesized that when CDCs oligomerize, they cluster membrane lipids in CRMs, along with immune receptors, into microscale assemblies that are spontaneously shed as microvesicles. We tested this hypothesis by artificially clustering sphingolipids using the sphingolipid-binding protein Ostreolysin A (OlyA), fused with tandem FKBP dimerization domains. We challenged murine bone marrow derived macrophages with and without FKBP homodimerizer and OlyA prior to LPS challenge and measured intracellular TNFα by flow cytometry. We found that oligomerization reduced OlyA binding, consistent with shedding. OlyA oligomerization further suppressed TNFα production, suggesting that microscale lipid assemblies interfere with cellular signaling. Overall, these results suggest that one cost to repair by shedding toxins on microvesicles is the loss of cellular signaling needed to promote inflammation. This may represent one strategy CDC-producing pathogens use to promote immune evasion and cause disease.

B cells in rheumatoid arthritis synovial tissues encode focused antibody repertoires that include antibodies that stimulate macrophage TNF-α production

Rheumatoid arthritis (RA) is characterized by the production of anti-citrullinated protein antibodies (ACPAs). To gain insights into the relationship between ACPA-expressing B cells in peripheral blood (PB) and synovial tissue (ST), we sequenced the B cell repertoire in paired PB and ST samples from five individuals with established, ACPA+ RA. Bioinformatics analysis of paired heavy- and light-chain sequences revealed clonally-related family members shared between PB and ST. ST-derived antibody repertoires exhibited reduced diversity and increased normalized clonal family size compared to PB-derived repertoires. Functional characterization showed that seven recombinant antibodies (rAbs) expressed from subject-derived sequences from both compartments bound citrullinated antigens and immune complexes (ICs) formed using one ST-derived rAb stimulated macrophage TNF-α production. Our findings demonstrate B cell trafficking between PB and ST in subjects with RA and ST repertoires include B cells that encode ACPA capable of forming ICs that stimulate cellular responses implicated in RA pathogenesis.

A Comparison of Characterization and Its Actions on Immunocompetent Cells of Polysaccharides from Sijunzi Decoction

Sijunzi decoction (SJZD) is a classic recipe in Traditional Chinese Medicine (TCM) with strong immune-enhancement activity. To further understand the characterization and immunomodulatory effect of polysaccharides from SJZD, the monosaccharide compositions of crude polysaccharide (SJZDP), polysaccharide fraction (S-3), and homogeneous polysaccharide (S-3-AG) from SJZD were compared by GC analysis, as well as their immunomodulatory effects on Peyer's patch cells, splenocytes, and macrophages which are related to intestinal immunity, specific immunity, and nonspecific immunity. The results showed that S-3-AG mainly contained Ara with a proportion of 38.9%, while Glc accounted for the largest proportion in S-3 (55.6%) and SJZDP (87.6%). The SJZDP, S-3, and S-3-AG all showed strong capability to stimulate Peyer's patch cells to proliferate and produce IgA and promoted the proliferation and IFN-γ production of splenocytes and increased the NO production and TNF-α production of macrophages. However, S-3 and S-3-AG were able to stimulate splenocytes to secret IL-4, SJZDP had no effect on IL-4 production of splenocytes in the tested concentrations. In addition, S-3 could stimulate the phagocytic activity of macrophages, and S-3-AG restrained the proliferation of macrophages at the concentration of 50–200 µg/mL. These results suggested that SJZDP, S-3, and S-3-AG might have different immunomodulatory effects on intestinal immunity, specific immunity, and nonspecific immunity due to their different monosaccharide compositions. It will provide references for the material basis and mechanism of SJZD immunomodulation activity.