Cytokine Expression In Macrophages Research Articles

An antibody that targets cell-surface glucose-regulated protein-78 inhibits expression of inflammatory cytokines and plasminogen activator inhibitors by macrophages.

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.

LPS inhibits TRIM65 expression in macrophages and C57BL/6J mouse by activating the ERK1/2 signaling pathway.

Activated macrophages serve a key role in various inflammatory diseases, such as atherosclerosis and septic shock. Tripartite motif-containing protein 65 (TRIM65) has been previously reported to participate in tumor progression and lung inflammation. However, the molecular mechanisms that controls its expression under inflammatory conditions and its consequences in activated macrophages remain poorly understood. The present study first collected the tissues of C57BL/6J mice, smooth muscle cells, macrophages and endothelial cells to determine the expression and distribution of TRIM65 by reverse transcription-quantitative (RT-q) PCR and western blotting. Mouse and human macrophages were treated with LPS and C57BL/6J mice were intraperitoneally injected with LPS followed by isolation of spleen, lung, aorta and bone marrow. Following treatment, TRIM65 mRNA and protein level was examined by RT-qPCR and western blotting. The results showed that TRIM65 was highly expressed in organs of the immune system, such as the spleen, lymph node and thymus, but lowly expressed in heart, liver, brain and kidneys. TRIM65 was also highly expressed in macrophages and endothelial cells. TRIM65 mRNA and protein expression levels were found to be decreased in LPS-treated macrophages in vitro and in tissues isolated from C57BL/6J mice intraperitoneally injected with LPS in vivo. In addition, to identify the signaling pathways by which LPS regulates TRIM65 expression, inhibitors of MAPK and Akt signaling pathways were used to treat macrophages followed by examination the expression of TRIM65 by western blotting. The results demonstrated that LPS-inhibited TRIM65 expression was blocked by treatment with the ERK1/2 inhibitor U0126. Moreover, the RT-qPCR results showed that TRIM65 knockout potentiated LPS-induced expression of inflammatory cytokines in macrophages. Taken together, data from the present study suggest that LPS decreased TRIM65 expression in macrophages and C57BL/6J mouse by activating the ERK1/2 signaling pathway, whilst TRIM65 knockout promoted macrophage activation. This information may facilitate the development of potential therapeutic strategies for the prevention and treatment of inflammatory diseases, such as atherosclerosis.

Novel long non-coding RNAs associated with inflammation and macrophage activation in human

Inflammation plays a central role in immune response and macrophage activation. Emerging studies demonstrate that along with proteins and genomic factors, noncoding RNA are potentially involved in regulation of immune response and inflammation. Our recent study demonstrated that lncRNA HOTAIR plays key roles in cytokine expression and inflammation in macrophages. The primary goal of this study is to discover novel lncRNAs that are crucial players in inflammation, macrophage activation, and immune response in humans. Towards this, we have stimulated THP1-derived macrophages (THP1-MΦ) with lipopolysaccharides (LPS) and performed the whole transcriptome RNA-seq analysis. Based on this analysis, we discovered that along with well-known marker for inflammation (such as cytokines), a series of long noncoding RNAs (lncRNAs) expression were highly induced upon LPS-stimulation of macrophages, suggesting their potential roles in inflammation and macrophage activation. We termed these family of lncRNAs as Long-noncoding Inflammation Associated RNA (LinfRNA). Dose and time dependent analysis demonstrated that many human LinfRNA (hLinfRNAs) expressions follow similar patterns as cytokine expressions. Inhibition of NF-κB suppressed the expression of most hLinfRNAs suggesting their potential regulation via NF-κB activation during inflammation and macrophage activation. Antisense-mediated knockdown of hLinfRNA1 suppressed the LPS-induced expression of cytokines and pro-inflammatory genes such as IL6, IL1β, and TNFα expression, suggesting potential functionality of the hLinfRNAs in cytokine regulation and inflammation. Overall, we discovered a series of novel hLinfRNAs that are potential regulators of inflammation and macrophage activation and may be linked to inflammatory and metabolic diseases.

A metal-organic framework-based immunomodulatory nanoplatform for anti-atherosclerosis treatment.

Immunomodulatory therapy has become a promising method for the clinical treatment of many diseases. Recently, pilot studies revealed that immunomodulatory therapy exhibited good effects on the treatment of cardiovascular diseases, but many problems remain to be solved, such as useful platforms for drug co-delivery and combination therapies. In this study, we designed and constructed the multifunctional nanoparticle Rapa@UiO-66-NH-FAM-IL-1Ra (RUFI) for the treatment of atherosclerotic cardiovascular disease. This nanoplatform combined the advantages of metal-organic frameworks (MOFs) for drug co-delivery, rapamycin and IL-1Ra for immunomodulation, IL-1Ra for cellular targeting, and 5-FAM for fluorescence imaging. RUFI exhibited good drug release of rapamycin and IL-1Ra and specific cytotoxicity for inflammatory macrophages in vitro. In an atherosclerotic model of diet-fed ApoE-/- mice, RUFI significantly targeted and reduced atherosclerosis plaques in coronary arteries, carotid arteries, and aortas. Mechanistic studies indicated that RUFI modulated macrophage phenotype, cytokine expression, and autophagy. This study demonstrated that combination therapy with rapamycin and IL-1Ra via MOF carriers enhanced the immunoregulatory effects against atherosclerosis. This drug co-delivery system suggests that MOF carriers loaded with immunomodulators are promising treatments for atherosclerosis or other inflammatory diseases.

Suppressive modulation of host immune responses by Dermanyssus gallinae infestation

The poultry red mite (Dermanyssus gallinae, PRM) is a blood-sucking ectoparasite in chickens and is one of the most serious threats to poultry farms. Mass infestation with PRMs causes various health problems in chickens, resulting in significant productivity reduction in the poultry industry. Infestation with hematophagous ectoparasites, such as ticks, induces host inflammatory and hemostatic reactions. On the other hand, several studies have reported that hematophagous ectoparasites secrete various immunosuppressants from their saliva to suppress host immune responses to maintain blood sucking. Here, we examined the expression of cytokines in peripheral blood cells to investigate whether PRM infestation affects immunological states in chickens. In PRM-infested chickens, anti-inflammatory cytokines, IL-10 and TGF-β1, and immune checkpoint molecules, CTLA-4 and PD-1, were highly expressed compared to noninfested chickens. PRM-derived soluble mite extracts (SME) upregulated the gene expression of IL-10 in peripheral blood cells and HD-11 chicken macrophages. In addition, SME suppressed the expression of interferons and inflammatory cytokines in HD-11 chicken macrophages. Moreover, SME induces the polarization of macrophages into anti-inflammatory phenotypes. Collectively, PRM infestation could affect host immune responses, especially suppress the inflammatory responses. Further studies are warranted to fully understand the influence of PRM infestation on host immunity.

MRNA expression profile of cytokines in rat primary alveolar macrophages treated with multiwalled carbon nanotube (MWCNT)

Multiwalled carbon nanotubes (MWCNT) are fiber-shaped nanomaterials that have a potential risk for cancer due to properties that are similar to asbestos. One type of nanotube called MWCNT-7 was categorized in Group 2B as possibly carcinogenic to humans by the International Agency for Research on Cancer. MWCNT-N, which is similar to MWCNT-7, is carcinogenic to the lung and pleura when administered to rats via the respiratory tract using intra-tracheal intra‐pulmonary spraying. Macrophages have an important role in the MWCNT induced pulmonary carcinogenicity. In this study, rat primary alveolar macrophages were employed to examine possible mechanism of carcinogenic effects of the MWCNT-N. MWCNT-N was fractionated into flow-through and retained fraction by passing it through a sieve with a pore size of 25 μm. Microarray analysis showed up-regulation of various cytokines in macrophages exposed to MWCNT-N. MWCNT-N strongly induced the expression of cytokines such as interleukin-6 (IL-6) in macrophages. In contrast, few cytokines were prominently down-regulated in macrophages treated with MWCNT-N. The sieve fractions did not have a significant effect on mRNA expression of cytokines in macrophages. These results provide useful information for understanding MWCNT-induced carcinogenicity via cytokine expression by macrophages.

Multifunctional nanofibrous mats: toward antibacterial and anti-inflammatory applications, and visual bacterial diagnosis.

In most circumstances, wounds face the challenges of bacterial invasions and inappropriate inflammatory responses when they lack proper wound management. Endowing dressings with both antibacterial and anti-inflammatory functions is a compelling strategy for resolving the above issues. However, seizing the right moment to change the dressings and providing satisfactory management of wounds are still urgently required. Herein, an antibacterial and anti-inflammatory nanofibrous mat is proposed by encapsulating antibiotic gentamicin sulfate (GS) and anti-inflammatory drug ibuprofen (IB) into nanofibers via a coaxial electrospinning technique and is further decorated with Prussian blue nanocrystals (PBNCs) to enhance anti-inflammatory activity and, more importantly, to monitor bacterial infections and guide dressing changes in a timely manner. Such a nanofibrous mat releases most of the therapeutic drugs within 120 min and reveals excellent antibacterial activity and anti-inflammatory ability. Specifically, it can destroy both Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), as well as conspicuously reduce the production of reactive oxygen species (ROS) and the expression of pro-inflammatory cytokines in macrophages. In addition, the nanofibrous mat can be used for point-of-use diagnosis of living bacteria relying on the naked eye or color analysis, which exhibits the potential of monitoring wound infection and guiding dressing changes promptly. This finding demonstrates the theranostic applications of multifunctional nanofibrous mats in wound healing.

Dissecting the multifaceted impact of statin use on fatty liver disease: a multidimensional study.

Statin use could benefit patients with non-alcoholic fatty liver disease (NAFLD), but the evidence is segmented and inconclusive. This multidimensional study comprehensively investigated the potential benefits and mechanism-of-action of statins in NAFLD. A cross-sectional investigation was performed within the Rotterdam Study (general population; n=4.576) and the PERSONS cohort (biopsy-proven NAFLD patients; n=569). Exclusion criteria were secondary causes for steatosis and insufficient data on alcohol, dyslipidemia or statin use. Associations of statin use with NAFLD (among entire general population), fibrosis and NASH (among NAFLD individuals and patients) were quantified. These results were pooled with available literature in meta-analysis. Last, we assessed statins' anti-lipid and anti-inflammatory effects in 3D cultured human liver organoids and THP-1 macrophages, respectively. Statin use was inversely associated with NAFLD in the Rotterdam study compared to participants with untreated dyslipidemia. In the PERSONS cohort, statin use was inversely associated with NASH, but not with fibrosis. The meta-analysis included 7 studies and indicated a not significant inverse association for statin use withNAFLD (pooled-Odds Ratio: 0.69, 95% Confidence Interval: 0.46-1.01) and significant inverse associations with NASH (pooled-OR: 0.59, 95% CI: 0.44-0.79) and fibrosis (pooled-OR: 0.48, 95% CI: 0.33-0.70). Invitro, statins significantly reduced lipid droplet accumulation in human liver organoids and downregulated expression of pro-inflammatory cytokines in macrophages. Pooled results demonstrated that statin use was associated with a lower prevalence of NASH and fibrosis and might prevent NAFLD. This may be partially attributed to the anti-lipid and anti-inflammatory characteristics of statins. Given their under-prescription, adequate prescription of statins may limit the disease burden of NAFLD. ZonMw, KWF, NWO, SLO, DGXII, RIDE, National and regional government, Erasmus MC and Erasmus University.

AnteAGE MDX Exosome Characterization and Functional Testing

Introduction: The skincare industry is witnessing a remarkable integration of cutting-edge scientific advancements with beauty and wellness, with exosomes emerging as a promising trend in cosmetics. Exosomes, small extracellular vesicles, have garnered attention for their potential in enhancing skin health and rejuvenation. They possess unique abilities, including deep skin penetration and sophisticated delivery of bioactive compounds, setting them apart from traditional skincare products. Exosomes can stimulate regenerative processes, modulate inflammation, and regulate cellular functions, attracting scientific and cosmetic interest. Robust scientific investigation and thorough characterization are imperative before incorporating exosomes into cosmetic formulations. This involves isolating and purifying exosomes from reliable sources and assessing their size, shape, surface markers, and cargo contents. Adherence to the International Society of Extracellular Vesicles (ISEV) guidelines ensures rigorous scientific standards. Materials and Methods: The study involved the isolation and purification of exosomes from stem cells, their characterization through techniques including Cryo-TEM, NTA, ELISA, and functional testing. Functional assays evaluated exosomes' ability to deliver active ingredients and stimulate cellular responses. These experiments confirmed that AnteAge MDX exosomes met the ISEV criteria for characterization as sEVs. Results: Exosomes were characterized as sEVs with a modal size of 103nm, and Cryo-TEM confirmed their size and structure. They expressed tetraspannins CD9, CD81, and CD63 on their surface, and endosomal protein Alix. The negative marker, calnexin, was not present. Fluorescently labeled exosomes were taken up by dermal fibroblasts and keratinocytes. Functional testing demonstrated anti-inflammatory properties, influencing macrophage polarization and cytokine expression. Exosomes also enhanced skin cell proliferation. Exosomes were found to be stable after lyophilization and storage for up to 9 months at 4℃, maintaining their structure and lipid bilayer integrity. In vivo application of AnteAge MDX exosomes improved skin quality, reducing fine lines, wrinkles, and redness. Conclusion: Exosomes hold substantial promise in skincare, providing deep penetration and cellular rejuvenation. Comprehensive scientific research, characterization, and functional testing are essential for product safety and effectiveness. This study adhered to ISEV guidelines, offering valuable insights into exosome potential. Collaboration between researchers, regulatory bodies, and cosmetic manufacturers is vital in establishing industry standards. Exosomes are poised to redefine beauty and wellness, offering innovative and effective products while maintaining scientific rigor and consumer safety.

Dietary-Polysaccharide-Modified Fish-Oil-Based Double Emulsion as a Functional Colloidal Formulation for Oral Drug Delivery.

Oral delivery is the most convenient drug administration route. However, oral delivery of peptides is extremely challenging due to the physical and chemical barriers within the gastrointestinal tract. Polysaccharides are often utilized as polymeric biomaterials in drug delivery. Among these, dietary polysaccharides extracted from okra, yam, and spirulina have been reported to stimulate innate immunity with well-known nutritional benefits. In this study, we developed a dietary-polysaccharide-modified fish-oil-based emulsion for oral co-delivery of a hydrophilic PD-L1 blocking peptide and the hydrophobic small molecule simvastatin. The optimal emulsion was nano-sized and exhibited a negative surface charge, high drug encapsulation efficiency of over 97%, low viscosity, and sustained drug release manner. The formulation could significantly increase the uptake of peptides by intestinal Caco-2 cells, which demonstrated the great potential of the formulation for promoting the oral absorption of peptides. Additionally, these dietary polysaccharides could promote dendritic cell maturation and cytokine expression in macrophages, demonstrating that these nutraceutical polysaccharides had dual roles of functioning as promising colloidal delivery systems and as potential immune modulators or adjuvants. Thus, this food-based colloidal delivery system shows promise for the oral delivery of peptide drugs and lays a great platform for future applications in immunotherapy.

SULFORAPHANE ADMINISTRATION AFTER HEMORRHAGIC SHOCK/RESUSCITATION IN MICE REDUCES THE SECRETION OF INFLAMMATORY CYTOKINES AND INCREASES THE IMMUNOCOMPETENCE OF SPLENIC MACROPHAGES.

Objective : The purpose of this study was to investigate the immunomodulatory effects of sulforaphane (SFN), a nuclear factor erythroid 2-related factor (Nrf2) pathway activator, on splenic macrophages' immunocompetence after hemorrhagic shock/resuscitation (HS/R). Methods : Male C57/BL6 wild-type mice (n = 6 per group) were subjected to either pressure-controlled HS (MAP, 35-45 mm Hg) or a sham procedure surgery (without HS). After 90 minutes of HS, fluid resuscitation with withdrawn blood and 0.9% NaCl was performed. Sulforaphane (50 mg/kg of body weight) was applied intraperitoneally immediately after the resuscitation phase as well as 24 and 48 h thereafter, depending on group allocation. The mice were killed at 6, 24, and 72 h after resuscitation. After killing, spleens were harvested to perform Nrf2 immunofluorescence histology. Splenic macrophages were isolated and cultured to measure cytokine secretion in the cell culture supernatant. Furthermore, macrophages isolated after 24-hour resuscitation were treated with 100 ng/mL of bacterial LPS to measure immunocompetence. Matrix-assisted laser desorption/ionization mass spectrometry imaging was performed to verify the distribution of SFN in the spleen after intraperitoneal injection. Results : We showed that administered SFN reached the spleen within the first hour after administration. Furthermore, we identified that SFN increased splenic Nrf2 activation and decreased cytokine expression in splenic macrophages after HS/R. In addition, we showed that SFN exhibited splenic anti-inflammatory properties of macrophages in vitro (IL-6/IL-10-ratio of the HS/R group: 51.79 ± 9.99 [at 6 h] and 15.70 ± 3.35 [at 24 h] vs. HS/R + SFN group: 20.54 ± 5.35 [at 6 h] and 8.60 ± 2.37 [at 24 h], P < 0.05). Furthermore, SFN improved in vitro splenic macrophage immunocompetence after HS/R, as evidenced by the increased secretion of inflammatory cytokines in response to LPS stimulation in vitro . Conclusions : Our study shows that SFN can reduce inflammatory cytokines secreted by splenic macrophages after HS/R and increase their immunocompetence toward a more anti-inflammatory profile.

Cyclic stretch regulates immune responses via tank-binding kinase 1 expression in macrophages.

Macrophages distributed in tissues throughout the body contribute to homeostasis. In the inflammatory state, macrophages undergo mechanical stress that regulates the signal transduction of immune responses and various cellular functions. However, the effects of the inflammatory response on macrophages under physiological cyclic stretch are unclear. We found that physiological cyclic stretch suppresses inflammatory cytokine expression in macrophages by regulating NF-κB activity. NF-κB phosphorylation at Ser536 in macrophages was inhibited, suggesting that tank-binding kinase (TBK1) regulates NF-κB activity during physiological stress. Moreover, TBK1 expression was suppressed by physiological stretch, and TBK1 knockdown by siRNA induced the suppression of NF-κB phosphorylation at Ser536. In conclusion, physiological stretch triggers suppression of a TBK1-dependent excessive inflammatory response, which may be necessary to maintain tissue homeostasis.

Protective effects of S100A8 on sepsis mortality: Links to sepsis risk in obesity and diabetes.

Obesity and diabetes are independent risk factors for death during sepsis. S100A8, an alarmin, is related to inflammation, obesity, and diabetes. Here, we examine the role of S100A8 in sepsis of obesity and diabetes models. Injection of S100A8 prolongs the survival of septic mice induced by lethal endotoxemia, Escherichia coli injection, or cecal ligation and puncture. S100A8 decrease the LPS-induced expression of proinflammatory cytokines in peritoneal macrophages by inhibiting TLR4-mediated signals in an autocrine manner. db/db, ob/ob, and western diet-fed mice demonstrate reduced upregulation of S100A8 induced by LPS treatment in both serum and peritoneal cells. These mice also show shorter survival after LPS injection, and S100A8 supplementation prolonged the survival. While myelomonocytic cells-specific S100A8-deficient mice (Lyz2 cre :S100A8 floxed/floxed ) exhibit shorter survival after LPS treatment, S100A8 supplementation prolonged the survival. Thus, myelomonocytic cell-derived S100A8 is crucial for protection from sepsis, and S100A8 supplementation improves sepsis, particularly in mice with obesity and diabetes.

IL-18 deficiency ameliorates the progression from AKI to CKD

Inflammation is an important factor in the progression from acute kidney injury (AKI) to chronic kidney disease (CKD). The role of interleukin (IL)-18 in this progression has not been examined. We aimed to clarify whether and how IL-18 limits this progression. In a folic acid induced renal injury mouse model, we studied the time course of kidney injury and renal IL-18 expression. In wild-type mice following injection, renal IL-18 expression increased. In parallel, we characterized other processes, including at day 2, renal tubular necroptosis assessed by receptor-interacting serine/threonine-protein kinase1 (RIPK1) and RIPK3; at day 14, transdifferentiation (assessed by transforming growth factor β1, vimentin and E-cadherin); and at day 30, fibrosis (assessed by collagen 1). In IL-18 knockout mice given folate, compared to wild-type mice, tubular damage and necroptosis, transdifferentiation, and renal fibrosis were attenuated. Importantly, IL-18 deletion decreased numbers of renal M1 macrophages and M1 macrophage cytokine levels at day 14, and reduced M2 macrophages numbers and macrophage cytokine expression at day 30. In HK-2 cells, IL-18 knockdown attenuated necroptosis, transdifferentiating and fibrosis.In patients with tubulointerstitial nephritis, IL-18 protein expression was increased on renal biopsies using immunohistochemistry. We conclude that genetic IL-18 deficiency ameliorates renal tubular damage, necroptosis, cell transdifferentiation, and fibrosis. The renoprotective role of IL-18 deletion in the progression from AKI to fibrosis may be mediated by reducing a switch in predominance from M1 to profibrotic M2 macrophages during the process of kidney repair.

Pim1 promotes IFN-β production by interacting with IRF3

The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-β by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-β and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-β signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1−/− mice produced less serum IFN-β and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-β production, thus revealing a novel target for controlling antiviral innate immune responses.

The Impact of Sphingosine Kinases on Inflammation-Induced Cytokine Release and Vascular Endothelial Barrier Integrity.

Sphingosine kinases type 1 and 2 (SphK1/2) are required for the production of the immune modulator sphingosine 1-phosphate (S1P). SphK1 deficient mice (SphK1-/-) revealed 50% reduced S1P in plasma, while SphK2-/- mice demonstrated 2-3 times increased S1P levels in plasma. Since plasma S1P is a potent inducer of vascular endothelial cell (VEC) barrier stability, we hypothesized that higher and lower levels of S1P in SphK2-/- and SphK1-/- mice, respectively, compared to wild type (wt) mice should translate into decreased and increased severity of induced systemic inflammation due to improved or damaged VEC barrier maintenance. To our surprise, both SphK1-/- and SphK2-/- mice showed improved survival rate and earlier recovery from inflammation-induced weight loss compared to wt mice. While no difference was observed in VEC barrier stability by monitoring Evans blue leakage into peripheral tissues, SphK1-/- mice demonstrated a distinct delay and SphK2-/- mice an improved resolution of early pro-inflammatory cytokine release in plasma. Ex vivo cell culture experiments demonstrated that bone marrow-derived dendritic cells (BMDC) generated from SphK1-/- and SphK2-/- mice responded with decreased interferon-γ (IFN-γ) production upon stimulation with lipopolysaccharides (LPS) compared to wt BMDC, while activation-induced cytokine expression of lymphocytes and macrophages was not majorly altered. Ex vivo stimulation of macrophages with IFN-γ resulted in increased cytokine release. These results suggest that SphK1/2 are involved in production and secretion of IFN-γ by DC. DC-derived IFN-γ subsequently stimulates the production and secretion of a large panel of inflammatory cytokines by macrophages, which belong to the main cytokine-releasing cells of the early innate immune response. Inhibitors of SphK1/2 may therefore be attractive targets to dampen the early cytokine response of macrophages as part of the innate immune response.

Mouse Ocilrp2/Clec2i negatively regulates LPS-mediated IL-6 production by blocking Dap12-Syk interaction in macrophage.

C-type lectin Ocilrp2/Clec2i is widely expressed in dendritic cells, lymphokine-activated killer cells and activated T cells. Previous studies have shown that Ocilrp2 is an important regulator in the activation of T cells and NK cells. However, the role of Ocilrp2 in the inflammatory responses by activated macrophages is currently unknown. This study investigated the expression of inflammatory cytokines in LPS-induced macrophages from primary peritoneal macrophages silenced by specific siRNA target Ocilrp2. Ocilrp2 was significantly downregulated in macrophages via NF-κB and pathways upon LPS stimuli or VSV infection. Silencing Ocilrp2 resulted in the increased expression of IL-6 in LPS-stimulated peritoneal macrophages and mice. Moreover, IL-6 expression was reduced in LPS-induced Ocilrp2 over-expressing iBMDM cells. Furthermore, we found that Ocilrp2-related Syk activation is responsible for expressing inflammatory cytokines in LPS-stimulated macrophages. Silencing Ocilrp2 significantly promotes the binding of Syk to Dap12. Altogether, we identified the Ocilrp2 as a critical role in the TLR4 signaling pathway and inflammatory macrophages’ immune regulation, and added mechanistic insights into the crosstalk between TLR and Syk signaling.

Type II collagen facilitates gouty arthritis by regulating MSU crystallisation and inflammatory cell recruitment

ObjectiveIncreasing evidence suggests that impaired cartilage is a substantial risk factor for the progression from hyperuricaemia to gout. Since the relationship between cartilage matrix protein and gout flares remains unclear,...

Impairment of a NIK-SIX Feedback Axis Results in Dysregulation of Intestinal Immune Homeostasis and Promotes Early-onset Fatal Spontaneous Colitis.

The negative feedback circuit NIK-SIN could inhibit the systemic inflammation and protect mouse from endotoxic shock. However, the physiological significance of NIK-SIX feedback circuit in the maintenance of intestinal immune homeostasis and prevention of early-onset spontaneous colitis is not known. To explore the role of NIK-SIX axis in the maintenance of intestinal immune homeostasis. The conditional knockout of NIK encoding gene, Map3k14, in the Cd11c+ dendritic cells were generated by crossing Map3k14-flox mice with Cd11c-Cre mice. DSS was used for colitis models. The expression of cytokines in the intestinal immune cells, isolated from Map3k14-cKO mice were detected by qPCR. The siRNA molecules were used for the silencing of SIN-proteins. Then luciferase assays and chromatin immunoprecipitation combined with qPCR were applied for mechanism investigations. The expression of SIX1 and SIX2 protein in BMDMs from WT were significantly lower than in the Map3k14-cKO mice. In vitro, the NIK-/- human-derived circulating monocytes also failed to express SIX-proteins under the stimulation of non-canonical NF-κB agonists. The expression of cytokines was significantly decreased in human circulating monocytes with overexpression SIN-proteins. The expression of cytokines in macrophages, DCs and T cells isolated from Map3k14-cKO mice were significantly increased in the DSS-induced models. Higher expression of cytokines was observed in the SIN1-/- and SIN2-/- cells including human circulating monocytes, mouse-derived BMDMs, intestinal macrophages and DCs. SIN-proteins directly bound the promoter region of inflammatory genes. NIK-SIX axis down-regulated inflammatory gene expression and plays a pivotal role in the maintenance of intestinal immune homeostasis.

P17.06: Escape From Macrophage-Mediated Rejection by Human Surfactant Protein (SP)-A

Introduction: Macrophage-mediated xenogeneic rejection is one of the important immunological obstructions that need to be overcome. Because macrophage is one of the main sources of proinflammatory cytokines and the fact that proinflammatory cytokines orchestrate a variety of inflammatory responses, the regulation of macrophages could result in solving the problem of xenogeneic rejection. We recently reported that membrane-type surfactant D (SP-D) on swine endothelial cells (SECs) suppresses macrophage-mediated rejection. Similar to SP-D, the carbohydrate recognition domain of surfactant protein-A (SP-A) induces inhibitory signals in effector cells. In this study, we examined the suppressive effect of SP-A on macrophage-mediated xenogeneic rejection. Methods: Naïve SEC and SP-A-transfected SEC (SEC/SP-A) were co-cultured with THP-1 cells and cytotoxicity was evaluated. To investigate the effect on phagocytosis, human macrophages were co-cultured with SEC or SEC/SP-A, and the extent of phagocytosis and production of reactive oxygen species (ROS) were assessed by flow cytometry. The mRNA expression of inflammatory cytokines in macrophages was determined using RT-PCR. In addition, THP-1 cell with a luciferase reporter gene driven by an NF-kB response element (THP-1 Luc NF-kB) cells was used to verify their effects on the NF-κB transcription factors. Results: The cytotoxicity of SEC/SP-A was significantly reduced compared to those of naïve SEC (40.7 vs 22.9%, p=0.025). The phagocytosis by macrophages against SEC was also significantly suppressed by SP-A on SEC (75.8 vs 33.0%, n=5, p=0.0010). Co-culture of human macrophages with SEC/SP-A decreased ROS production (%MFI: 99.9 vs 79.5, p=0.013). The mRNA expression of TNFα was reduced in macrophages (1.160 vs 0.009, p=0.0022), whereas that of IL-10 was increased (1.357 vs 10.20, p=0.0007). The balance between iNOS and Arg-1 was significantly suppressed by CL-SPA (1.002 vs 0.4792, p = 0.0027), indicating that CL-SPA on porcine cells inhibits the differentiation of peripheral blood monocytes into inflammatory M1 macrophages. The NF-κB transcription was decreased in THP-1 Luc NF-kB which was co-cultured with SEC/SP-A compared to that in THP-1 Luc NF-kB with SEC (RLU: 1160 vs 983.8, p=0.0258). Conclusion: The ectopic expression of human SP-A in porcine cells represents an attractive method for suppressing macrophage-mediated cytotoxicity. To further investigate the effects of SP-A on xenogeneic innate immune response in more detail, in vivo studies should be performed in the future.