Monocyte Chemotactic Protein-1 Production Research Articles

Osteochondral allograft and xenograft immunogenicity decrease following ex vivo tissue culture

Introduction/ObjectiveOsteochondral allografting can be an effective option for the treatment of focal cartilage injuries. However, wide scale applicability of this technique is limited by availability of donor tissue. To overcome this limitation, osteochondral xenografting has been proposed as a potential alternative, but immune-mediated rejection precludes its clinical application at this time. We hypothesized that removing much of the marrow cell content through tissue culture would result in a decrease in xenograft immunogenicity. We believed this could be tested in a preclinical animal model. MethodsFresh porcine femoral condyles were harvested and cultured for 0, 14, or 28 days before coculture with either allogeneic enriched porcine T-cells or xenogeneic enriched equine T-cells for 5 days. After 5 days, these cocultures were pulsed with 5-ethynyl-2′deoxyuridine then analyzed by flow cytometry to assess the level of cell proliferation. Coculture supernatant samples were also collected to evaluate the levels of prostaglandin-E2 and monocyte chemotactic protein-1 (MCP-1) as markers of inflammation. ResultsThe osteochondral tissue induced significantly less T-cell proliferation, prostaglandin-E2, and MCP-1 production when the tissue had been cultured for either 14 or 28 days prior to the coculture assay (P < .0001). ConclusionsWe conclude that T-cell responses to either allogeneic or xenogeneic osteochondral tissues were significantly reduced by 14 days of preculture prior to exposure to T-cells.

Triterpenoid CDDO-EA inhibits lipopolysaccharide-induced inflammatory responses in skeletal muscle cells through suppression of NF-κB.

Chronic inflammation is a major contributor to the development of obesity-induced insulin resistance, which then can lead to the development of type 2 diabetes (T2D). Skeletal muscle plays a pivotal role in insulin-stimulated whole-body glucose disposal. Therefore, dysregulation of glucose metabolism by inflammation in skeletal muscle can adversely affect skeletal muscle insulin sensitivity and contribute to the pathogenesis of T2D. The mechanism underlying insulin resistance is not well known; however, macrophages are important initiators in the development of the chronic inflammatory state leading to insulin resistance. Skeletal muscle consists of resident macrophages which can be activated by lipopolysaccharide (LPS). These activated macrophages affect myocytes via a paracrine action of pro-inflammatory mediators resulting in secretion of myokines that contribute to inflammation and ultimately skeletal muscle insulin resistance. Therefore, knowing that synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acids (CDDOs) can attenuate macrophage pro-inflammatory responses in chronic disorders, such as cancer and obesity, and that macrophage pro-inflammatory responses can modulate skeletal muscle inflammation, we first examined whether CDDO-ethyl amide (CDDO-EA) inhibited chemokine and cytokine production in macrophages since this had not been reported for CDDO-EA. CDDO-EA blocked LPS-induced tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukine-1beta (IL-1β), and interleukine-6 (IL-6) production in RAW 264.7 mouse and THP-1 human macrophages. Although many studies show that CDDOs have anti-inflammatory properties in several tissues and cells, little is known about the anti-inflammatory effects of CDDOs on skeletal muscle. We hypothesized that CDDO-EA protects skeletal muscle from LPS-induced inflammation by blocking nuclear factor kappa B (NF-κB) signaling. Our studies demonstrate that CDDO-EA prevented LPS-induced TNF-α and MCP-1 gene expression by inhibiting the NF-κB signaling pathway in L6-GLUT4myc rat myotubes. Our findings suggest that CDDO-EA suppresses LPS-induced inflammation in macrophages and myocytes and that CDDO-EA is a promising compound as a therapeutic agent for protecting skeletal muscle from inflammation.

Interleukin-37 ameliorates cigarette smoke-induced lung inflammation in mice

Cigarette smoking (CS) is the leading cause of chronic obstructive pulmonary disease, and its severity is closely related to lung inflammation. Interleukin (IL)-37 is a newly discovered member of the IL-1 family with anti-inflammatory activity. Our study aimed to elucidate the effect of IL-37 on CS-induced lung inflammation in mice. In this study, mice were exposed to six cigarettes for 1 h three times daily (4 h smoke-free intervals) for 10 consecutive days. Mice were treated intranasally with IL-37-expressing lentivirus and empty lentivirus particles 1 day before the first CS or sham exposure. Mice were sacrificed on day 11 to evaluate the effect of IL-37 on CS-induced pulmonary inflammation in mice. Administering IL-37-expressing lentivirus significantly reduced CS-induced weight loss in mice compared to empty lentivirus controls (P < 0.05). Histological analysis showed that IL-37 significantly alleviated inflammatory cell recruitment, alveolar septum enlargement, alveolar wall attenuation, mucus hypersecretion, and goblet cell metaplasia in mouse lungs (P < 0.001). IL-37 expression also significantly inhibited CS-induced increases in inflammatory cells (including lymphocytes, neutrophils, and macrophages) in mouse lungs (P < 0.05), as well as pro-inflammatory cytokines such as IL-1β, IL-6, IL-17, monocyte chemotactic protein-1 and tumor necrosis factor-α production (P < 0.05). IL-37 also significantly reduced myeloperoxidase activity in mouse serum (P < 0.01) and lung tissues (P < 0.001). Therefore, IL-37 can ameliorate CS-induced pulmonary inflammation in mice and IL-37 may be a potential therapeutic strategy for CS-induced lung inflammatory diseases.

SIRT6 inhibits inflammatory response through regulation of NRF2 in vascular endothelial cells

Emerging evidence suggests that inflammation plays a pivotal role in Atherosclerosis. Sirtuin 6 (SIRT6), a member of NAD+-dependent protein lysine deacylases of the sirtuin family, plays an important role in the regulation of metabolism, aging and stress resistance. However, the role of SIRT6 in vascular inflammation and its molecular mechanism is unknown. The present study showed that TNF-α significantly reduced the expression of SIRT6 protein and mRNA in a concentration- and time-dependent manner and increased the expression of monocyte chemotactic protein 1 (MCP-1), interleukin (IL) -6 and IL-1β in human umbilical vein endothelial cells (HUVECs). Overexpression of SIRT6 but not its catalytically inactive mutant inhibited TNF-α-induced expression of MCP-1, IL-6 and IL-1β. Knockdown of SIRT6 significantly enhanced TNF-α-induced expression of MCP-1, IL-6 and IL-1β. Moreover, knockdown of SIRT6 reduced TNF-α-induced nuclear factor erythroid 2 related factor 2 (NRF2) nucleus protein expression, whereas knockdown of NRF2 significantly enhanced TNF-α-induced expression of MCP-1, IL-6 and IL-1β. In addition, overexpression of SIRT6 increased NRF2 and its target genes expression, and knockdown of SIRT6 decreased NRF2 and its target genes expression. Meanwhile, knockdown of SIRT6 inhibited NRF2 nucleus protein expression. Further, knockdown of SIRT6 decreased phosphorylation of NRF2, overexpression of SIRT6 increased phosphorylation of NRF2. SIRT6 interacted with NRF2. In vivo, the levels of TNF-α and IL-1β were increased in the serum of hyperlipidemia mice. Hyperlipidemia-induced production of MCP-1, IL-6 and IL-1β was significantly augmented in the endothelium specific SIRT6 knockout mice. In contrast, the expression of NRF2 and its target genes was reduced. Taken together, these results indicate that SIRT6 protects against vascular inflammation via its deacetylase activity and the NRF2-dependent signaling pathway.

Regulation of Monocytes/Macrophages by the Renin-Angiotensin System in Diabetic Nephropathy: State of the Art and Results of a Pilot Study.

Diabetic nephropathy (DN) is characterized by albuminuria, loss of renal function, renal fibrosis and infiltration of macrophages originating from peripheral monocytes inside kidneys. DN is also associated with intrarenal overactivation of the renin–angiotensin system (RAS), an enzymatic cascade which is expressed and controlled at the cell and/or tissue levels. All members of the RAS are present in the kidneys and most of them are also expressed in monocytes/macrophages. This review focuses on the control of monocyte recruitment and the modulation of macrophage polarization by the RAS in the context of DN. The local RAS favors the adhesion of monocytes on renal endothelial cells and increases the production of monocyte chemotactic protein-1 and of osteopontin in tubular cells, driving monocytes into the kidneys. There, proinflammatory cytokines and the RAS promote the differentiation of macrophages into the M1 proinflammatory phenotype, largely contributing to renal lesions of DN. Finally, resolution of the inflammatory process is associated with a phenotype switch of macrophages into the M2 anti-inflammatory subset, which protects against DN. The pharmacologic interruption of the RAS reduces albuminuria, improves the trajectory of the renal function, decreases macrophage infiltration in the kidneys and promotes the switch of the macrophage phenotype from M1 to M2.

CD63+ extracellular vesicles from retinal pigment epithelial cells participate in crosstalk with macrophages in the innate inflammatory axis

The aim of the study is to clarify the participation of extracellular vesicles (EV) secreted by murine primary retinal pigment epithelial (mpRPE) cells in the cell to cell communication with macrophages (Mps), firstly described by the authors in 2016. In ocular inflammation, Mps act as sources of tumor necrosis factor-α (TNF-α), an activator of RPE cells. TNF-α stimulates the production of monocyte chemotactic protein (MCP-1) by RPE cells, thereby causing greater recruitment of Mps to the sub-RPE space. Murine RAW 264.7 Mps cells were co-cultured with C57BL/6 mouse mpRPE cells, either together or separated in transwells, vertically or horizontally connectable, with 0.40 or 0.03 μm membrane filters. The association of EV with mpRPE or RAW 264.7 was quantified by fluorescence cell sorting (FACS) using Qdot655 streptavidin-conjugated biotinylated EV. Increased levels of CD63+ EV were detected in co-cultures by western blotting or FACS analysis, in accordance with the increased production of nanoparticles (50–150 nm) detected by Nanosight tracking analysis. The gene expressions of cytokines, MCP-1, IL-6, IL-8, and VEGF in mpRPE cells and the corresponding proteins were increased in co-cultures even in transwells, vertically connected with 0.40 μm membrane filters, while the repressed TNF-α protein production was not affected. Most of the CD63+ EVs produced by mpRPE cells in co-cultures were associated with Raw264.7, but not with mpRPE cells. Semi-purified CD63+ EV secreted from mpRPE cells, increased the secretion of MCP-1, IL-6, and VEGF in co-cultures with RAW 264.7. Culture chamber separation horizontally connected with 0.03 μm membrane filters reduced this increased secretion. Collectively, mpRPE derived CD63+ EV partly participate in the sub-retinal innate inflammation. To evaluate the functional damage of RPE cells upon chronic exposure to here defined EVs will be the critical issue to uncover their roles in chronic retinal degenerative diseases.

Indoxyl Sulfate, a Tubular Toxin, Contributes to the Development of Chronic Kidney Disease.

Indoxyl sulfate (IS), a uremic toxin, causes chronic kidney disease (CKD) progression via its tubulotoxicity. After cellular uptake, IS directly induces apoptotic and necrotic cell death of tubular cells. Additionally, IS increases oxidative stress and decreases antioxidant capacity, which are associated with tubulointerstitial injury. Injured tubular cells are a major source of transforming growth factor-β1 (TGF-β1), which induces myofibroblast transition from residual renal cells in damaged kidney, recruits inflammatory cells and thereby promotes extracellular matrix deposition in renal fibrosis. Moreover, IS upregulates signal transducers and activators of transcription 3 phosphorylation, followed by increases in TGF-β1, monocyte chemotactic protein-1 and α-smooth muscle actin production, which participate in interstitial inflammation, renal fibrosis and, consequently, CKD progression. Clinically, higher serum IS levels are independently associated with renal function decline and predict all-cause mortality in CKD. The poor removal of serum IS in conventional hemodialysis is also significantly associated with all-cause mortality and heart failure incidence in end-stage renal disease patients. Scavenging the IS precursor by AST-120 can markedly reduce tubular IS staining that attenuates renal tubular injury, ameliorates IS-induced oxidative stress and rescues antioxidant glutathione activity in tubular epithelial cells, thereby providing a protective role against tubular injury and ultimately retarding renal function decline.

Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1

(1) Background: A detailed understanding of the pathophysiology of hemorrhagic stroke is still missing. We hypothesized that expression of heme oxygenase-1 (HO-1) in microglia functions as a protective signaling pathway. (2) Methods: Hippocampal HT22 neuronal cells were exposed to heme-containing blood components and cell death was determined. We evaluated HO-1-induction and cytokine release by wildtype compared to tissue-specific HO-1-deficient (LyzM-Cre.Hmox1 fl/fl) primary microglia (PMG). In a study involving 46 patients with subarachnoid hemorrhage (SAH), relative HO-1 mRNA level in the cerebrospinal fluid were correlated with hematoma size and functional outcome. (3) Results: Neuronal cell death was induced by exposure to whole blood and hemoglobin. HO-1 was induced in microglia following blood exposure. Neuronal cells were protected from cell death by microglia cell medium conditioned with blood. This was associated with a HO-1-dependent increase in monocyte chemotactic protein-1 (MCP-1) production. HO-1 mRNA level in the cerebrospinal fluid of SAH-patients correlated positively with hematoma size. High HO-1 mRNA level in relation to hematoma size were associated with improved functional outcome at hospital discharge. (4) Conclusions: Microglial HO-1 induction with endogenous CO production functions as a crucial signaling pathway in blood-induced inflammation, determining microglial MCP-1 production and the extent of neuronal cell death. These results give further insight into the pathophysiology of neuronal damage after SAH and the function of HO-1 in humans.

Sitagliptin inhibits vascular inflammation via the SIRT6-dependent signaling pathway

Sitagliptin has recently been shown to inhibit inflammatory response in cardiovascular disease. Sirtuin6 (SIRT6), a NAD+-dependent class III histone deacetylase, participates in the regulation of cellular inflammation. We hypothesized that sitagliptin could attenuate vascular inflammation via modulation of SIRT6 pathway. It was found that sitagliptin decreased the expression of monocyte chemotactic protein-1 (MCP-1), interleukin (IL)-6 and IL-1β, but up-regulated SIRT6 expression, both in mice and in TNF-α-stimulated endothelial cells. Moreover, knockdown of SIRT6 reversed the inhibitory effect of sitagliptin on MCP-1, IL-6 and IL-1β expression. Further study revealed that sitagliptin also decreased the expression of MCP-1, IL-6 and IL-1β partly through suppression of reactive oxygen species (ROS). In vivo, hypercholesterolemia in mice was induced by intraperitoneal administration of poloxamer 407 for 1 month. Hyperlipidemia-induced production of MCP-1, IL-6 and IL-1β was significantly suppressed in the sitagliptin-supplemented animals, but the effect of was abolished by SIRT6 knockout in endothelium. These results indicate that sitagliptin protects against vascular inflammation via the SIRT6/ROS-dependent signaling pathway.

Involvement of multiple transcription factors in regulation of IL-β-induced MCP-1 expression in alveolar type II epithelial cells

During acute lung injury, a large number of monocytes are recruited into the pulmonary tissue, which is mainly mediated by local production of monocyte chemotactic protein 1 (MCP-1). As an essential component of the lung tissues, alveolar type II epithelial cells are one of the major sources of MCP-1. Therefore, uncovering the mechanism whereby MCP-1 production is regulated in the alveolar type II cells will provide a pivotal theoretical basis for clinical intervention in acute lung injury. In the current study, we find that there is a κB binding site in the MCP-1 promoter region, and mutation of the site leads to reduced production of MCP-1 in alveolar type II epithelial cells. In contrast, overexpression of NF-κB p65 significantly increases MCP-1 expression. Furthermore, we elucidate that IKKα/β—NF-κB p65 signaling pathway and phosphorylation of serine 534 in NF-κB p65 are required for the maximal expression of MCP-1. Also, Activator protein 1 (AP-1) site in the promoter region and JNK1/2—c-Jun signaling are required for MCP-1 generation in alveolar type II epithelial cells. Moreover, a CCAAT/enhancer-binding protein (C/EBP) element is identified in the MCP-1 promoter region through the point mutation technique, and further experiments demonstrate that both C/EBPβ and C/EBPδ are involved in basic and IL-1β-mediated MCP-1 expression. Of note, specificity protein 1—Sp1 expression is not changed in alveolar type II epithelial cells incubated with IL-1β, but it still control MCP-1 production by binding to the consensus sequence in the promoter region. More importantly, we find that the results derived from the cell line—MLE-12 cells and primary cells are consistent. Taken together, our data provide insights into the molecular mechanism how MCP-1 expression in inflammatory alveolar type II epithelial cells is regulated at transcription level.

SAT-197 Functional Cross-Talk Between MCP-1 and Integrin Linked Kinase Is Required for Endometriosis Disease Progression

Development of painful invasive endometriotic lesions is usually a combined function of the chronic inflammation and active sequential cellular events such as migration, cell adhesion, proliferation, colonization, and invasion in endometriosis. An elevated level of monocyte chemotactic protein-1 (MCP-1) in serum and peritoneal fluid has been reported in endometriosis. Further, Integrin-Linked Kinase (ILK) communicates with extracellular signals and regulates the sequential cellular events in uterine and ovarian cancer. The functional cross-talk of MCP-1 with ILK in the immune cells leads to the high MCP-1 production and increased ILK activity. However, the role of the elevated MCP-1 level and its functional interaction with ILK in the endometriosis development is not explored yet. In order to investigate this, we used the surgically induced heterologous mouse model of endometriosis and externally treated with MCP-1 intraperitoneally for two weeks. Our results demonstrated that MCP-1 boosted the growth of endometriotic lesions mediated through ILK, FAK, VAV and RAC1 signaling axis in endometriosis. In addition, in vivo pharmacological inhibition of ILK by CPD22 restricted the growth of endometriotic lesions in mice. Flow cytometric and inflammatory cytokines profiling in the peritoneal fluid demonstrated that ILK was involved in the MCP-1 mediated macrophageF4/80+ recruitment and inflammatory response in order to support the growth of endometriotic lesions. Inhibition of ILK induced the anti-inflammatory cytokine IL-10 and concomitantly suppressed the pro-inflammatory cytokines MCP-1, TNF-α and IL-10 levels in peritoneal fluid. In vitro studies on human ovarian cystic endometriosis cells (Hs832(C).T) and normal endometrial stromal cells (T-HESCs) revealed that MCP-1 accelerated the adhesion (on collagen and fibronectin substratum), colonization, migration and 3D-spheroids formation and invasion. As the basal level of CCR2, an MCP-1 receptor, expression was higher in ovarian endometriotic cells than the normal endometrial stromal cells giving the hint for the role of MCP-1 in endometriosis. Interestingly, the siRNA mediated knockdown of ILK renders the MCP-1 induced nuclear translocation of phosphorylated ILK and also reduced these accelerated cellular events. Taken together, our results provide compelling evidence that the functional cross-talk between MCP-1 and ILK drives endometriotic disease progression.

Effect of Welsh onion (Allium fistulosum L.) green leaf extract on immune response in healthy subjects: a randomized, double-blind, placebo-controlled study

Background: Welsh onion belongs to genus allium. Although its medicinal benefit such as cold prevention is well known by tradition, the underlying mechanism and active components have never been elucidated. The recent study revealed that Welsh onion mucus enhances tumor necrosis factor-α and monocyte chemotactic protein-1 production from RAW 264 cells and natural killer (NK) cell activity in murine spleen cells. This mucilage was abundant in the leaf blade of Welsh onion. Thus, we investigated the effect of Welsh onion green leaf extract (GLE) on immune competence in the human clinical trial. Methods: A randomized, double-blind, placebo-controlled trial was conducted. Fifty-five healthy subjects were enrolled after the screening based on NK cell activity, and each sample (placebo, low-dose GLE or high-dose GLE) was supplemented for four weeks. The immune competence activity was evaluated through the primary endpoints that were NK cell activity and the score of immunological vigor (SIV). The SIV was calculated from lymphocyte subset analysis by flow cytometry method.Results: NK cell activity was enhanced in all supplemented groups including placebo, especially, it was significant in both GLE groups. The result of immune score showed that the immunity of high-dose GLE group did not change although it was significantly lowered in placebo group (p&lt;0.01), suggesting that GLE ameliorated the immunity suppression. In addition, when SIV was compared between pre- and post-intake, the ratios of the improved subjects in two GLE groups were higher than that of placebo group while those of the aggravated subjects showed the opposite result. In particular, they were remarkable in high-dose GLE group.Conclusion: These results suggest that the intake of low- or high-dose GLE might positively regulate immune competence.Keywords: welsh onion, immunity, natural killer cell, clinical trial

Cell-specific elevation of Runx2 promotes hepatic infiltration of macrophages by upregulating MCP-1 in high-fat diet-induced mice NAFLD.

We have demonstrated runt-related transcription factor 2 (Runx2) plays important role in atherosclerosis. It has been indicated that atherosclerosis shares the similar histopathology with nonalcoholic steatohepatitis (NASH), a progressive stage of nonalcoholic fatty liver disease (NAFLD), on macrophages infiltration. However, the function of Runx2 in NAFLD is completely unknown. Here, we investigated the underlying mechanism of Runx2 triggering macrophages infiltration in the development of NAFLD. Mice were fed with high-fat diet (HFD) for a long time. Histopathologic features, macrophages infiltration, expression of monocyte chemotactic protein 1 (MCP-1), and Runx2 were, respectively, analyzed in vivo. Lentivirus or short interfering RNA were transfected in murine hepatic stellate cells (HSCs) and the transwell assay was performed to verify the contribution of Runx2 for macrophages migration in vitro. Long-term treatment with HFD induced the progression of NAFLD, and NASH was initiated from 8 months on diet. HFD increased the expression of F4/80 upon HFD feeding, indicated HFD promotes hepatic infiltration of macrophages in NAFLD. In addition, HFD upregulated the expression of MCP-1 and Runx2 during NAFLD development. Unexpectedly, Runx2 upregulation is cell-type depended in NAFLD, and only abundantly elevated in activated HSCs. Furthermore, we found that Runx2 could increase or decrease the expression of MCP-1 in HSCs, and regulate macrophages migration by influencing MCP-1 production in vitro. Our results give evidence that the upregulation of Runx2 specific in activated HSCs promotes hepatic infiltration of macrophages by increasing MCP-1 expression in NAFLD, which reveals a novel mechanism and provides a cell-specific therapeutic target for NAFLD.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) in diabetic retinopathy: latest evidence and clinical considerations.

Diabetic retinopathy (DR) is considered as a diabetes-related complication that can render severe visual impairments and is also a risk factor for acquired blindness in both developed as well as developing countries. Through fibrovascular epiretinal membranes (ERMs), this condition can similarly lead to tractional retinal detachment. Laboratory efforts evaluating the DR pathogenesis can be provided by ocular vitreous fluid and ERMs resulting from vitrectomy. The clinical stages of DR are significantly associated with expression levels of certain chemokines, including monocyte chemotactic protein-1 (MCP-1) in the intraocular fluid. The MCP-1 is also a known potent chemotactic factor for monocytes and macrophages that can stimulate them to produce superoxide and other mediators. Following hyperglycemia, retinal pigmented epithelial (RPE) cells, endothelial cells, and Müller's glial cells are of utmost importance for MCP-1 production, and vitreous MCP-1 levels rise in patients with DR. Increased expression of the MCP-1 in the eyes can also play a significant role in the pathogenesis of DR. In this review, current clinical and laboratory progress achieved on the MCP-1 and the DR concerning neovascularization and inflammatory responses in vitreous and/or aqueous humor of DR patients was summarized. It was suggested that further exploration of the MCP-1/CCR2 axis association between clinical stages of DR and expression levels of inflammatory and angiogenic cytokines and chemokines, principally the MCP-1 might lead to potential therapies aiming at neutralizing antibodies and viral vectors.

Palmitoleic Acid has Stronger Anti‐Inflammatory Potential in Human Endothelial Cells Compared to Oleic and Palmitic Acids

Fatty acids (FAs) may affect endothelial cell (EC) function, influencing atherogenesis and inflammatory processes. Palmitoleic acid (POA) has been described as an anti-inflammatory FA. However, its effects on ECs are underexplored. This study compares the effects of POA with those of palmitic acid (PA) and oleic acid (OA) on EC inflammatory responses. EAHy926 cells (EC lineage) are exposed to PA, OA, or POA, and stimulated with tumor necrosis factor (TNF)-α. Associated with the FA's own incorporation, PA induces a twofold increase in arachidonic acid, while POA increases the amount of cis-vaccenic acid. PA, but not OA, enhances the production of IL-6 and IL-8 in response to TNF-α. In contrast, POA decreases production of monocyte chemotactic protein (MCP)-1, IL-6, and IL-8 compared to PA. TNF-α increases surface intercellular adhesion molecule-1 expression previously decreased by POA. TNF-α stimulation increases the expression of NFκB, cyclooxygenase (COX)-2, MCP-1, and IL-6 genes and reduces the expression of peroxisome proliferator-activated receptor (PPAR)-α gene. PA enhances the expression of MCP-1, IL-6, and COX-2 genes, while POA downregulates these genes, decreases expression of NFκB, and upregulates PPAR-α gene expression. POA has anti-inflammatory effects on ECs stimulated with TNF-α and may counter endothelial dysfunction.

Lactose Induces Phenotypic and Functional Changes of Neutrophils and Macrophages to Alleviate Acute Pancreatitis in Mice.

Acute pancreatitis (AP) is one common clinical acute abdominal disease, for which specific pharmacological or nutritional therapies remain elusive. Lactose, a macronutrient and an inducer of host innate immune responses, possesses immune modulatory functions. The current study aimed to investigate potential modulatory effects of lactose and the interplay between the nutrient and pancreatic immunity during experimentally induced AP in mice. We found that either prophylactic or therapeutic treatment of lactose time-dependently reduced the severity of AP, as evidenced by reduced pancreatic edema, serum amylase levels, and pancreatic myeloperoxidase activities, as well as by histological examination of pancreatic damage. Overall, lactose promoted a regulatory cytokine milieu in the pancreas and reduced infiltration of inflammatory neutrophils and macrophages. On acinar cells, lactose was able to suppress caerulein-induced inflammatory signaling pathways and to suppress chemoattractant tumor necrosis factor (TNF)-α and monocyte chemotactic protein-1 production. Additionally, lactose acted on pancreas-infiltrated macrophages, increasing interleukin-10 and decreasing tumor necrosis factor alpha production. Notably, lactose treatment reversed AP-associated infiltration of activated neutrophils. Last, the effect of lactose on neutrophil infiltration was mimicked by a galectin-3 antagonist, suggesting a potential endogenous target of lactose. Together, the current study demonstrates an immune regulatory effect of lactose to alleviate AP and suggests its potential as a convenient, value-added therapeutic macronutrient to control AP, and lower the risk of its systemic complications.

HBP induces the expression of monocyte chemoattractant protein-1 via the FAK/PI3K/AKT and p38 MAPK/NF-κB pathways in vascular endothelial cells

Inflammation is characterized by early influx of polymorphonuclear neutrophils (PMNs), followed by a second wave of monocyte recruitment. PMNs mediate monocyte recruitment via their release of heparin binding protein (HBP), which activates CCR2 (CC-chemokine receptor 2) on monocytes. However, the pathways for such signal transmission remain unknown. Accumulating evidences have highlighted the importance of leukocyte-endothelial cell interactions in the initiation of inflammation. In this study, an interesting finding is that HBP enhances the secretion of monocyte chemotactic protein 1(MCP-1), ligand of CCR2, from a third party, the endothelial cells (ECs). HBP-induced increase in MCP-1 production was demonstrated at the protein, mRNA and secretion levels. Exposure of ECs to HBP elicited rapid phosphorylation of FAK/PI3K/AKT and p38 MAPK/NF-κB signaling. MCP-1 levels were attenuated during the response to HBP stimulation by pretreatment with a FAK inhibitor (or siRNA), a PI3K inhibitor, an AKT inhibitor, a p38 inhibitor (or siRNA) and two NF-κB inhibitors. Additionally, pretreatment with inhibitors to FAK, PI3K and AKT led to a decrease in HBP-induced phosphorylation of p38/NF-κB axis. These results showed that HBP induced MCP-1 expression via a sequential activation of the FAK/PI3K/AKT pathway and p38 MAPK/NF-κB axis. Interestingly, the patterns of HBP regulation of the expression of the adhesion molecular VCAM-1 were similar to those seen in MCP-1 after pretreatment with inhibitors (or not). These findings may help to determine key pharmacological points of intervention, thus slowing the progress of inflammatory-mediated responses in certain diseases where inflammation is detrimental to the host.

Activation of macrophages by a laccase-polymerized polyphenol is dependent on phosphorylation of Rac1

Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.

The Anti-Inflammatory Effect of Ripasudil (K-115), a Rho Kinase (ROCK) Inhibitor, on Endotoxin-Induced Uveitis in Rats.

To investigate the anti-inflammatory properties of ripasudil, a Rho kinase (ROCK) inhibitor, using endotoxin-induced uveitis (EIU) in rats. Endotoxin-induced uveitis was induced by footpad injection of lipopolysaccharide (LPS). Ripasudil was administered intraperitoneally 1 hour before and after LPS injection. The aqueous humor was collected 24 hours after injection, and the infiltrating cells, protein concentration, and levels of monocyte chemotactic protein-1 (MCP-1) were determined. Infiltrating cells in the iris ciliary body (ICB) and adherent leukocytes in retinal vessels were evaluated. The mRNA levels of IL-1β, IL-6, TNF-α, and MCP-1 in the retina and ICB were determined. A mouse macrophage cell line, RAW264.7, was stimulated with LPS in the presence or absence of ripasudil, and the expression of MCP-1 and nuclear translocation of nuclear factor (NF)-κB was analyzed. Ripasudil significantly reduced infiltrating cells and protein exudation in the aqueous humor, as well as the number of infiltrating cells in the ICB and adherent leukocytes in retinal vessels in EIU. Additionally, the protein level of MCP-1 in the aqueous humor and mRNA levels of IL-1β, IL-6, TNF-α, MCP-1, and intercellular adhesion molecule-1 in the ICB and retina were suppressed by ripasudil. The production of MCP-1 and nuclear translocation of NF-κB in RAW264.7 cells were also suppressed by ripasudil. The Rho/ROCK pathway plays a role in adhesion molecule expression and inflammatory cell infiltration in EIU, and ripasudil is a potent anti-inflammatory agent against ocular inflammatory diseases, including acute uveitis and possibly uveitic glaucoma.

Response of human periodontal ligament stem cells to IFN-\u03b3 and TLR-agonists

Periodontal ligament stem cells similarly to the mesenchymal stem cells of other tissues possess immunomodulatory properties, which are regulated by different cytokines, particularly by interferon-γ (IFN-γ). In contrast, less information is provided about the effect of toll-like receptors ligand on immunomodulatory properties of these cells. In the present study we investigated the response of human periodontal ligament stem cells (hPDLSCs) in response to simultaneous stimulation with IFN-γ and toll-like receptor (TLR) agonists. The resulting expression of indoleamine-2,3-dioxygenase-1 (IDO-1), interleukin (IL)-6, IL-8 and monocyte chemotactic protein 1 (MCP-1) was investigated. The expression of IDO-1 was upregulated by IFN-γ in both gene and protein levels. TLR2 agonists Pam3CSK4 induced gene expression of IDO-1, but had no effect on protein expression. IFN-γ induced IDO-1 protein expression was further enhanced by Pam3CSK4. TLR-4 agonist E. coli LPS has no significant effect on neither basal nor IFN-γ induced IDO-1 protein expression. The production of IL-6, IL-8, and MCP-1 was induced by TLR agonists. Neither basal nor TLR agonists induced production of these proteins was affected by IFN-γ. Our data shows potential interaction between IFN-γ and TLR2 responses in hPDLSCs, which might be involved in regulation of immune response in inflammatory diseases, and particularly periodontitis.