CD36/Src/ERK pathway is involved in process of monocyte differentiation into macrophages

AIM To investigate the role of fatty acid translocase (FAT/CD36) on differentiation of monocytes to macrophages. METHODS Human monocyte THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 0, 100 and 200 μg /L. Small interfering RNA (siRNA) targeting CD36 (siCD36) was employed to knock down the expression of CD36 in THP-1 cells. The CD36 over-expression (CD36OE) cell line was constructed by transfection with a recombinant lentivirus containing CD36 cDNA. Optical microscopy and crystal violet staining were used to detect the monocyte morphological changes and adhesion ability. The protein expression of CD36 was measured by flow cytometry and Western blot. The mRNA levels of CD36, CD11b and CD80 were detected by real-time PCR. The protein levels of extracellular signal-regulated kinase (ERK） and Src tyrosine kinase were determined by Western blot. RESULTS The cellular adhesiveness of THP-1 cells was elevated in the process of monocytes differentiation, and the expression of CD36 was increased in this process as well (P<0.01). siCD36 was transfected into the THP-1 cells (CD36i group) and the silencing efficiency was approximately 80%. The cell surface area and cellular adhesiveness were significantly decreased in CD36i group compared with scrambled siRNA (NCi) group (P<0.01). The mRNA levels of CD11b and CD80 were decreased in CD36i group compared with NCi group (P<0.01). The cell surface area and cellular adhesiveness were increased in CD36OE group compared with empty vector (vector) group (P<0.05). The mRNA levels of CD11b and CD80 were increased in CD36OE group compared with vector group (P<0.01). The phosphorylation levels of ERK and Src were decreased in CD36i group compared with NCi group (P<0.05). CONCLUSION CD36 promotes the differentiation of human monocyte THP-1 cells to macrophages by increasing the phosphorylation of Src and further activating ERK.     

Immunomodulatory effects of β-glucans on porcine alveolar macrophages and bone marrow haematopoietic cell-derived dendritic cells

The immunopharmacological activities of β-glucans with a backbone of β-1,3/β-1,6-linkages associated with anti-tumor, anti-viral, bacterial and fungal infections have been well documented. Dectin-1, a specific pattern recognition receptor for β-1,3/β-1,6-glucans, is expressed mainly on phagocytes, especially macrophages and dendritic cells (DCs). In this study, the encoding nucleotide for the carbohydrate-recognition domain (CRD) of porcine dectin-1 was sequenced for the first time, and the immunomodulatory functions of a synthetic particulate β-glucan (p-β-glucan) were examined. Results showed that p-β-glucan significantly enhanced cell activity and phagocytosis in porcine alveolar macrophages (AMs), immature DCs (imDCs) and mature DCs (mDCs), in a similar way to zymosan. Zymosan enhanced dectin-1/TLR2/TLR4 expression and TNF-α/IL-10 production in all of three types of cell, whereas p-β-glucan increased dectin-1/TLR4 and TNF-α/IL-12 production in AMs but inhibited IL-10 in mDCs. These results indicate that the complex collaborating interactions between dectin-1 and TLRs in the recognition of β-1,3/β-1,6-glucans with different structural features may direct different cellular responses.     

Toll-like receptor-mediated anti-inflammatory action of glaucine and oxoglaucine

Two isochinoline alkaloids, glaucine and oxoglaucine were investigated for their suggested anti-inflammatory influence concerning nitric oxide and cytokine production. Mouse peritoneal macrophages were stimulated with different Toll-like receptor (TLR) ligands such as LPS for TLR4, zymosan for TLR2 and CpG for TLR9. The alkaloids inhibited TNF-α and IL-6 production induced by these ligands. In regard to IL-12 suppressive effect was registered in the case of CpG stimulation. Glaucine succeeded to enhance LPS and zymosan-induced IL-10 production. The reduction of pro-inflammatory cytokines and increase of anti-inflammatory IL-10 are indicative for their use in different acute and chronic inflammatory diseases.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.

The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.

In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

LRRK2/NF-κB signaling modulates inflammatory responses in BCG-infected RAW264.7 macrophages

AIM: To investigate the biological function and potential mechanism of leucine-rich repeat kinase 2 (LRRK2) in RAW264.7 macrophages during Mycobacterium tuberculosis infection. METHODS: The bacillus Calmette-Guerin (BCG)-infected RAW264.7 cell model was established. Colony-forming unit (CFU) analysis was used to determine the mycobacterial viability. The releases of interleukin (IL)-1β, IL-6 and interferon-γ (IFN-γ) in the RAW264.7 cells were detected by ELISA. qPCR and Western blot were used to measure the mRNA and protein expression levels, respectively. RESULTS: LRRK2 was robustly enhanced in the RAW264.7 cells in response to BCG infection. Additionally, silencing of LRRK2 suppressed intracellular growth of mycobacteria during BCG challenge. Moreover, silencing of LRRK2 dramatically attenuated the accumulation of inflammatory cytokines IL-1β, IL-6 and IFN-γ induced by BCG infection. More importantly, LRRK2 modulated BCG-induced inflammatory responses by positively regulating the nuclear factor-κB (NF-κB) signaling pathway. CONCLUSION: LRRK2/NF-κB signaling pathway positively modulates inflammatory responses during BCG infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease.     

Effects of GW1929 on macrophage TLR expression and inflammation induced by ox-LDL

AIM: To investigate the effects of ox-LDL on TLR2 and TLR4 expression and production of TNF-α, IL-10, IL-12, NO and MDA in macrophages and to observe intervention effect of GW1929 in above procedure. METHODS: The mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L, 100 mg/L) and GW1929 (20 μmol/L) respectively for 24 h. The concentrations of MDA, NO-2/NO-3, TNF-α, IL-10 and IL-12 in the culture fluid were detected. Flow cytometry was used to observe TLR2 and TLR4 expressions after the mouse peritoneal macrophages were pretreated with ox-LDL (50 mg/L) and GW1929 (20 μmol/L) respectively for 6 h, 12 h, and 24 h. RESULTS: The concentrations of MDA, NO-2/NO-3, TNF-α and IL-10 in ox-LDL (50 mg/L, 100 mg/L) group were higher than those in control and GW1929 group obviously, but the concentrations of above index in ox-LDL (50 mg/L, 100 mg/L)+GW1929 group were lower than those in ox-LDL (50 mg/L, 100 mg/L) group apparently. No IL-12 in every group was detected. Expressions of TLR-2 in ox-LDL+GW1929 (6 h, 12 h, 24 h) group were lower than those in ox-LDL (6 h, 12 h, 24 h) group respectively. TLR-4 expressions in ox-LDL+GW1929 (12 h) were lower than those in ox-LDL (12 h) apparently. CONCLUSION: ox-LDL up-regulates TLR2 and TLR4 expressions and promotes the production of ROX, NO, TNF-α and IL-10 in macrophages. GW1929 is capable of inhibiting the above ox-LDL effects.     

Regulatory effects of spirolactone on SiO2-induced lung macrophage subtype switching

AIM: To investigate the influence of spirolactone (SPI) on mouse pulmonary macrophage subtype switching induced by silicon dioxide (SiO₂). METHODS: C57BL/6 mice were divided into normal saline (NS) group, SiO₂ group, SiO₂+SPI group and SiO₂+NS group. A mouse model of silicosis was developed with crystalline SiO₂ particles (40 mg/kg, via oropharyngeal instillation). SPI (20 mg/kg) or (NS) was delivered daily by oral gavage after SiO₂ administration. The mice were sacrificed on days 3, 14 and 28. Alveolar washing, total cell counting, differential cell counting and flow cytometry analysis were performed. The right lower lobe of lavaged lungs was collected to prepare single-cell suspension for flow cytometry analysis. RESULTS: Compared with SiO₂ group, SPI treatment significantly alleviated SiO₂-induced inflammatory cell accumulation in brochoalveolar lavage fluid on day 3 and 14. Compared with NS group, the M1 alveolar macrophages and M1 pulmonary interstitial macrophages in SiO₂ group switched to M2 subtype dramatically, while SPI treatment reversed the switching effectively.CONCLUSION: SPI treatment alleviates SiO₂-induced inflammatory cell accumulation, which may be related to reversing macrophage subtype switching.     

Trichinella: differing effects of antigens from encapsulated and non-encapsulated species on in vitro nitric oxide production

Trichinellosis is a cosmopolitan zoonotic disease affecting a wide variety of animals, including man. Non-encapsulated and encapsulated species diverge with respect to their developmental strategies. Little is known at the molecular level about parasite-derived mediators responsible for host muscle cell transformation occurring during trichinellosis. In this context, host-parasite relationships in Trichinella-infected animals could be related to different host-immune and cell mediators, e.g. nitric oxide (NO). Here, we investigate the stimulatory/inhibitory role of L1 antigens from four encapsulated (T. spiralis, T. britovi, T. nelsoni and T. nativa) and one non-encapsulated (T. pseudospiralis) Trichinella species on NO production from rat macrophages in vitro. Our results demonstrate that encapsulated and non-encapsulated Trichinella species differ in their capacity to stimulate the secretion of NO from host macrophages. Biological significance of these differences should be further assessed in the available experimental models.     

An experimental exploration of focal inflammatory response in cerebral ischemic foci in the rat

AIM: To study the pathological relationship of vascular cell adhesion molecule-1 (VCAM-1) expression and monocyte/macrophage infiltration with focal brain ischemia. METHODS: Immunohistochemical technique and focal brain ischemia/reperfusion model were used in the study in order to explore profiles and time-course of VCAM-1 expression and monocyte macrophage (ED2 positive cell) infiltration in ischemic rat brain. RESULTS: VCAM-1 was up-regulated in microvascular endothelial cells in ischemic cortex at 1h postischemia, and continuously expressed during the time of reperfusion. ED2 positive cells infiltrated into ischemic cortex at 1h iscehmia/ 2h reperfusion and then ED2 positive cells increased gradually with the time of reperfusion, ED2 positive cell infiltration showed apparently relationship with VCAM-1 expression, and both of them exhibited the some changes of time-dependence. CONCLUSION: Cerebral ischemia induced VCAM-1 expression and ED2 positive cell infiltration and VCAM-1 may regulate the recruitment of ED2 positive cells in the ischemic brain region. The results suggested that VCAM-1 and ED2 positive cells may be participated in the pathogenesis of cerebral ischemic injury.