Rat Macrophages Research Articles

Laparoscopic Cholecystectomy Intervention in Rats with Severe Acute Pancreatitis Delays Apoptosis of Myocardial Cells

In order to study the mechanism of laparoscopic cholecystectomy intervention in macrophages of rats with severe acute pancreatitis to delay apoptosis of cardiac myocytes, Wistar rats were chosen and divided into 3 groups. In addition, the related indices and protein expression in rats were observed at 2, 8, 16 and 24 h for each component and caspase-3 and bcl-2 levels in myocardium are detected to study the effect of laparoscopic cholecystectomy intervention. Results indicated that there were no obvious symptoms in control rats, while the rats in intervention group have the same symptoms as the experimental group. White blood cells of the 3 groups of rats, when compared it was found that the white blood cells in the experimental group decrease, followed by the intervention group but there is no significant change in the control group. Morphological examination of myocardial tissue from the 3 groups of rats indicated that there was no significant change in myocardial cells in the control group, while piles of blood cells were found between myocardial cells in the experimental group with obvious nuclear deformation. There were patches of blood between myocardial cells in the intervention group, without any significant changes in nucleus. Among the changes in the content of tumour necrosis factor-α and apoptosis of myocardial tissue, it is found that the tumour necrosis factor-α content and apoptotic phenomena were most significant in the experimental group and there was a significant correlation between the experimental group and the control group. After laparoscopic cholecystectomy, it was found that the content of tumour necrosis factor-α in myocardial tissue decreased and apoptotic phenomena were alleviated, which was significantly different from the experimental group (p<0.01). Western blot results indicated that expression of caspase-3 and bcl-2 in myocardial tissue were the highest in the experimental group, followed by the intervention group and the lowest in the control group. It was also found that the change of tumour necrosis factor-α was synchronous and positively correlated with the change of apoptotic index of myocardial cells. Therefore, it could be concluded that laparoscopic cholecystectomy could be used to intervene in severe acute pancreatitis to delay the apoptosis of myocardial cells in rats.

Influence of isoniazid on T lymphocytes, cytokines, and macrophages in rats

T lymphocytes, cytokines, and macrophages play important roles in the clearance of Mycobacterium tuberculosis (Mtb) by the immune system. This study aimed to investigate the effects of isoniazid on the functions of both innate and adaptive immune cells. Healthy rats were randomly divided into experimental and control groups. Each group was randomly divided into three subgroups and named according to the duration of drug feeding, 1, 3, and 3 months followed by drug withdrawal for 1 month. The experimental groups were fed with isoniazid (12 mg/mL) and the control groups with normal saline. The percentage of CD4+ and CD8+ T lymphocytes, level of interleukin (IL)-12 and interferon (IFN)-γ, and function of macrophages were determined at these three time points. Isoniazid significantly increased the percentage of CD4+ T lymphocytes and the CD4+/CD8+ T lymphocyte cell ratio (P < 0.05). It transiently (<1 month) enhanced the functions of rat macrophages significantly (P < 0.05). In summary, isoniazid could increase the percentage of CD4+ T lymphocytes, CD4+/CD8+ T lymphocyte cell ratio, and enhance macrophage function in healthy rats.

Immunomodulatory effect of rutin, catechin, and hesperidin on macrophage function

The inflammation is observed by a sequence of comprehensive, correlated prevalence which causes enrollment of macrophages along with by removal of foreign particles and instigation of tissue repair. Though, massive release of chemokine and cytokines from macrophages can cause the progression of auto-inflammation and auto-immune diseases. The control overproduction of these mediators without damaging macrophages seems to be a novel strategy to control inflammation. Plants have been an integral part of the health-care system since ancient times. Many modern-day pharmaceuticals find their origin from phytochemicals. Rutin, catechin, and hesperidin are well-recognized for analgesic, anti-inflammatory and antiarthritic effects. Scientific reports rationalize their protective effect on humoral and cell-mediated immunity. The present study is focused to determine the immunomodulatory effect of rutin, catechin, and hesperidin on macrophage function. Rat peritoneal macrophages were harvested and cultured in the presence of lipopolysaccharides (LPS) and hesperidin. MTT assay was performed to determine macrophage viability. The levels of TNF-α, IL-1β, IL-6 were determined by ELISA kits whereas NO levels were determined by Griess method. The results of the present study revealed a decrease in levels of TNF-α, IL-1β, IL-6 and NO in LPS treated groups. In conclusion, LPS activated the macrophage by promoting the production of immune mediators, whereas rutin, catechin, and hesperidin treatment showed anti-inflammatory activities by suppressing the cytokine production in rat macrophages.

M2 macrophage-derived exosomes promote the c-KIT phenotype of vascular smooth muscle cells during vascular tissue repair after intravascular stent implantation.

Rationale: For intravascular stent implantation to be successful, the processes of vascular tissue repair and therapy are considered to be critical. However, the mechanisms underlying the eventual fate of vascular smooth muscle cells (VSMCs) during vascular tissue repair remains elusive. In this study, we hypothesized that M2 macrophage-derived exosomes to mediate cell-to-cell crosstalk and induce dedifferentiation phenotypes in VSMCs.Methods: In vivo, 316L bare metal stents (BMS) were implanted from the left iliac artery into the abdominal aorta of 12-week-old male Sprague-Dawley (SD) rats for 7 and 28 days. Hematoxylin and eosin (HE) were used to stain the neointimal lesions. En-face immunofluorescence staining of smooth muscle 22 alpha (SM22α) and CD68 showed the rat aorta smooth muscle cells (RASMCs) and macrophages. Immunohistochemical staining of total galactose-specific lectin 3 (MAC-2) and total chitinase 3-like 3 (YM-1) showed the total macrophages and M2 macrophages. In vitro, exosomes derived from IL-4+IL-13-treated macrophages (M2Es) were isolated by ultracentrifugation and characterized based on their specific morphology. Ki-67 staining was conducted to assess the effects of the M2Es on the proliferation of RASMCs. An atomic force microscope (AFM) was used to detect the stiffness of the VSMCs. GW4869 was used to inhibit exosome release. RNA-seq was performed to determine the mRNA profiles of the RASMCs and M2Es-treated RASMCs. Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of the mRNAs. Western blotting was used to detect the candidate protein expression levels. T-5224 was used to inhibit the DNA binding activity of AP-1 in RASMCs.Results: M2Es promote c-KIT expression and softening of nearby VSMCs, hence accelerating the vascular tissue repair process. VSMCs co-cultured in vitro with M2 macrophages presented an increased capacity for de-differentiation and softening, which was exosome dependent. In addition, the isolated M2Es helped to promote VSMC dedifferentiation and softening. Furthermore, the M2Es enhanced vascular tissue repair potency by upregulation of VSMCs c-KIT expression via activation of the c-Jun/activator protein 1 (AP-1) signaling pathway.Conclusions: The findings of this study emphasize the prominent role of M2Es during VSMC dedifferentiation and vascular tissue repair via activation of the c-Jun/AP-1 signaling pathway, which has a profound impact on the therapeutic strategies of coronary stenting techniques.

Correction to: Protein and lipid homeostasis altered in rat macrophages after exposure to metallic oxide nanoparticles.

Unfortunately, the author names in the author group section were incorrectly captured in the published online paper.

IMPACT OF RONCOLEUKIN (RIL-2) ON THE LYMPHOCYTE-TRIGGERING ACTIVITY OF MACROPHAGES AFTER TRAUMATIC BRAIN INJURY IN VARIOUSLY AGED RATS

IL-1β is involved in both brain damage process and the mechanisms of brain regeneration processes. The goal of the present research is to measure the change in the production of lymphocyte-activating factor (LAF) after experimental traumatic injury (TBI) in variously aged rats and evaluate the possibility to correct LAF production with roncoleukin (rIL-2). LAF activity in the supernatants of peritoneal macrophages of injured rats was measured by its ability to have comitogenic effect on the proliferation of rat thymocytes stimulated by suboptimal lectin doses. LAF stimulation index was defined as ratio of stimulated to unstimulated levels of LTF. Results and discussion. The strongest suppression of microphages’ cytokine-producing function was found in older rats after TBI. rIL-2 injection significantly reversed the injuries caused by TBI even in older rats. These data confirm that exogenic IL-2 is able to activate the functional characteristics of innate immunity cells and that is has a normalizing effect on the immune cells of the older animals.

Targeting and imaging of monocyte-derived macrophages in rat's injured artery following local delivery of liposomal quantum dots

Quantum dots offer superior optical features and hold a great potential as an imaging tool in comparison to ‘conventional’ fluorescent dyes. However, in vivo application in inflammatory-associated disorders is limited due to potential toxicity following systemic administration. Vascular inflammation contributes to cardiovascular diseases such as restenosis (re-narrowing of the artery following angioplasty), and poor prognosis is associated with the increased number of monocytes-derived macrophages (MDMs) in the arterial wall. Local administration of a suitable delivery system targeting MDMs could provide effective fluorescent imaging while minimizing systemic exposure and toxicity. We report here on the physicochemical characteristics and the structural stability of MDMs-targeted liposomal QDs (LipQDs), cellular uptake and cytotoxicity, the systemic biodistribution of LipQDs following local intra-luminal administration of LipQDs in carotid-injured rats vs. systemic administration, and imaging of QDs in the arterial tissue. The local treatment with LipQDs was found to be a suitable approach for targeting QDs to MDMs in the injured artery. In contrast to free QDs, the LipQDs formulation exhibited unique properties including structural and fluorescent stability, increased accumulation and retention for up to 24 h, and targeting properties enabling imaging of MDMs. MDMs imaging by targeted nanoparticles (NPs) could potentially serve for the detection of MDMs density in the injured artery for diagnostic purposes.

Functional polarization of macrophages of rats with progesterone-induced obesity treated with melanin from the Antarctic yeast Nadsoniella nigra

Progesterone-induced obesity develops in women who use this drug for contraception and the menopause treatment, though its mechanisms remain poorly understood. We studied functional M1 and M2 polarizations of the abdominal cavity macrophages of rats with progesterone induced obesity during 28 days of administration. The effect of melanin from the Antarctic yeast Nadsoniella nigra (Chaetothyriales, Herpotrichiellaceae, Nadsoniella Issatsch, 1914) was investigated. The NO level was determined by the accumulation of nitrites, ROS level was estimated by the NBT-test, arginase activity was assayed by the reaction of L-arginine hydrolysis. The body weights of rats administrated progesterone increased by 27% and continued to increase one month after withdrawal of progesterone (55% higher than control). Melanin prevents the weight gain when administered during one month after progesterone withdrawal. The NO production by peritoneal macrophages of obese animals intensified by 31% indicating their polarization towards pro-inflammatory M1 type. Production of ROS did not change. A 14% increase in arginase activity was observed, indicating the inhibition of M2 (anti-inflammatory) polarization. In the progesterone withdrawal group all these rates significantly decreased, indicating a reduction in the functional activity of peritoneal macrophages’. Melanin decreased the NO and ROS production by 60% and 18% respectively in comparison with the progesterone group and unexpectedly reduced arginase activity. Our data provide evidence of the spread of inflammation in response to progesterone-induced obesity. Peritoneal macrophages are involved in the inflammation in obesity, undergoing polarization towards the pro-inflammatory phenotype. The long-term consequences of such inflammation include the continuation of weight gain and likely the development of systemic inflammation associated with the exhaustion of the functional capacity of peritoneal cavity macrophages. Melanin has an anti-obesity effect and exhibits anti-inflammatory properties preventing progesterone-induced weight gain and macrophage M1 polarization. This requires detailed elucidation and can be valuable in designing countermeasures to prevent obesity outcomes.

RM-CSF efficiently replaces L929 in generating mouse and rat bone marrow-derived macrophages for in vitro functional studies of immunity to intracellular bacteria

Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as a prototype. We compared rM-CSF with L929 supernatant in terms of their effects on mouse and rat macrophage growth, maturation patterns, surface marker expression, and the expression of selected genes. Further, we compared macrophage infectivity and bacterial replication using LVS. Finally, we compared the in vitro function of BMDMs co-cultured with splenocytes from vaccinated animals in terms of their control of intramacrophage bacterial replication, as well as production of cytokines and nitric oxide. We demonstrated that rM-CSF produced BMDMs with similar, or minimal, phenotypic and gene expression outcomes compared to those generated with media containing L929 supernatant. Most importantly, functional outcomes were similar. Taken together, our data support the use of the rM-CSF in cell culture media as an alternative to L929-supplemented media for functional bioassays that use C57BL/6J mouse or Fischer 344 rat BMDMs to study intracellular infections. This comparison therefore facilitates future protocol standardization.

Development of Dried Emulsion/Mannitol Composite Microparticles through a Unique Spray Nozzle for Efficient Delivery of Hydrophilic Anti-tuberculosis Drug against Alveolar Macrophages.

As alveolar macrophages are attractive targets for the treatment of tuberculosis, effective methods for delivery to alveolar macrophages are under development. We investigated a pulmonary formulation for the efficient delivery of high water-soluble drugs at high concentration targeting alveolar macrophages. In this study, a surfactant-coated high water-soluble drug complex (SDC, a hydrophobic dried emulsion), which can preferably target alveolar macrophages and be expected to deliver drug at a high concentration, was prepared in the first process. OCT313, a high water-soluble sugar derivative with anti-tuberculosis activity was used. Then, a unique two-solution, mixing-type nozzle was used to prepare the SDC nanoparticles in mannitol (MAN) microparticles (SDC/MAN microparticles) because it was difficult to disperse the SDC nanoparticles in aqueous solution. The single micron size of OCT313-SDC/MAN microparticles contained OCT313-SDC nanoparticles (mean particle size of OCT313-SDC nanoparticles, 277.9 nm; drug contents, 1.31 ± 0.041 wt%). We found that the treatment of SDC/MAN microparticles exhibited significantly higher drug accumulation in macrophage cells (Raw264.7 cells, 7.5-fold, at 4 h after treatment) in vitro and in alveolar macrophages in rats (9.1-fold, at 4 h after treatment) in vivo than that of drug alone. These results suggest that the SDC/MAN microparticle formulation prepared by spray drying through a two-solution mixing-type nozzle provides efficient delivery of a water-soluble drug targeting alveolar macrophages and may be useful for tuberculosis treatment.

Palmitic acid induces guanylin gene expression through the Toll-like receptor 4/nuclear factor-κB pathway in rat macrophages.

Recently, we showed that double-transgenic rats overexpressing guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), specifically in macrophages demonstrate an antiobesity phenotype and low-expression levels of proinflammatory cytokines in the mesenteric fat even when fed a high-fat diet. Here, we examined the levels and mechanism of Gn and GC-C transcription following saturated fatty acid and lipopolysaccharide (LPS), an activator of Toll-like receptor 4 (TLR4), exposure by using the NR8383 macrophage cell line. In addition, the levels of guanylin and cGMP were increased by addition of either palmitic acid or LPS. Next, we investigated the interaction of the gene transcription and nuclear factor-κB (NF-κB) by using an NF-κB inhibitor and chromatin immunoprecipitation assay. We showed that palmitic acid induced Gn gene expression via TLR4 and NF-κB. Moreover, we demonstrated that NF-κB binding to the Gn promoter was responsible for the induction of gene transcription by palmitic acid or LPS. Our results indicate that saturated fatty acids such as palmitic acid activate Gn gene expression via the NF-κB pathway, raising the possibility that the activated Gn-GC-C system may contribute to the inhibition of high-fat diet-induced proinflammatory cytokines in macrophages.

Nrf2 Suppression Delays Diabetic Wound Healing Through Sustained Oxidative Stress and Inflammation.

Impaired wound healing is one of the major complications of diabetes, involving prolonged inflammation, delayed re-epithelialization, and consistent oxidative stress. The detailed mechanism remains unclear, and there is currently no effective treatment for diabetic wound healing. In this study, we aim to investigate the potential role and effect of nuclear factor erythroid-2–related factor-2 (Nrf2) activation on diabetic wound healing. In vitro experiments in rat macrophages showed that hyperglycemia treatment suppresses Nrf2 activation, resulting in oxidative stress with decreased expression of antioxidant genes, including NAD(P)H:quinone oxidoreductase 1 and heme oxygenase 1, together with increased secretion of proinflammatory cytokines, including interleukin 1β (IL1β), IL6, and monocyte chemoattractant protein-1. Both Nrf2 overexpression and Nrf2 activator dimethyl fumarate (DMF) treatment significantly ameliorated oxidative stress and inflammation. On the other hand, both Nrf2 knockdown or Nrf2 inhibitor ML385 mimicked the effect of diabetes. Further in vivo experiments in rats showed that DMF treatment significantly accelerated wound healing in streptozocin-induced diabetic rats with increased expression of antioxidant enzymes and decreased secretion of proinflammatory cytokines, while Nrf2 inhibitor ML385 mimicked the effect of diabetes. We conclude that Nrf2 activation accelerates impaired wound healing by ameliorating diabetes-mediated oxidative stress and inflammation. This provides a new clinical treatment strategy for diabetic wound healing using Nrf2 activator DMF.

Effect of Hydrogen Sulfide on the Mitogen-Activated Protein Kinase Signaling Pathway in Cultured Skin Macrophages of Burned Rats

BackgroundThis study aims to investigate the effect of hydrogen sulfide on the mitogen-activated protein kinases signaling pathway in in vitro cultured skin macrophages of burned rats. Materials and methodsThirteen healthy Sprague–Dawley rats were divided into five groups: normal control group, burned control group, sodium hydrogen sulfide group, glibenclamide group, and sodium hydrogen sulfide + glibenclamide group. The burned rats were made into a deep II° 5% total body surface area flame burn injury model. The skin basement macrophages were separated from the skin of normal rats and the wound skin of burned rats and cultured. At 1, 6, and 12 h after intervention, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 protein levels were detected by Western blot, and ERK, p38, and JNK messenger RNA (mRNA) levels were detected by reverse transcription polymerase chain reaction. ResultsDifferences in ERK, p38, and JNK mRNA and protein levels between the normal control group and burned control group were statistically significant (P < 0.05). At the same time point, the ERK, p38, and JNK mRNA and protein levels in the NaSH group were different from those in other groups, and the differences were statistically significant (P < 0.05). ConclusionsHydrogen sulfide has a regulatory effect on ERK, JNK, and p38 in the mitogen-activated protein kinases signaling pathway in macrophages of burned rats.

Role of heme oxygenase-1 on lipopolysaccharide-induced activation of NLRP3 inflammasomes in alveolar macrophages of rats

Objective To evaluate the role of heme oxygenase-1 (HO-1) on lipopolysaccharide (LPS)-induced activation of NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes in alveolar macrophages of rats. Methods NR8383 cells of rat alveolar macrophages cultured in vitro were seeded in 96-well plates at a density of 4×104 cells/ml and divided into 4 groups (n=48 each) using a random number table method: control group (group C), group LPS (group L), LPS+ HO-1 siRNA group (group L+ HO-1 siRNA), and LPS+ Con siRNA group (group L+ Con siRNA). The cells were cultured in normal culture atmosphere in group C. NR8383 cells were stimulated with 10 μg/ml LPS in L, L+ HO-1 siRNA and L+ Con siRNA groups.Cells were transfected with HO-1 siRNA at 48 h before stimulation with LPS in group L+ HO-1 siRNA and with Con siRNA at 48 h before stimulation with LPS in group L+ Con siRNA.The cells were collected and incubated for 24 h after stimulation with LPS for measurement of the cell viability, expression of HO-1 and NLRP3 mRNA (by real-time polymerase chain reaction), expression of HO-1, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 (by Western blot), and concentrations of interleukin-1beta (IL-1β) and interleukin-18 (IL-18) in the supernatant (by enzyme-linked immunosorbent assay). Results Compared with group C, the cell viability was significantly decreased, the expression of HO-1 and NLRP3 protein and mRNA, ASC and caspase-1 was up-regulated, and the IL-1β and IL-18 concentrations were increased in L, L+ HO-1 siRNA and L+ Con siRNA groups (P 0.05). Conclusion HO-1 is involved in LPS-induced activation of NLRP3 inflammasomes in alveolar macrophages of rats. Key words: Heme oxygenase-1; Lipopolysaccharides; NLR family, pyrin domain-containing 3 protein

Analysis of the impact of CSF-1 administration in adult rats using a novel Csf1r-mApple reporter gene.

Macrophages are present in large numbers in every tissue in the body where they play critical roles in development and homeostasis. They exhibit remarkable phenotypic and functional diversity, underpinning their adaptation to specialized roles in each tissue niche. CSF1, signaling through the CSF1 receptor, which is restricted to monocyte-macrophage lineage cells in adults, is a critical growth factor controlling macrophage proliferation, differentiation, and many aspects of mature macrophage function. We have generated a macrophage reporter rat, utilizing a construct containing elements of the mouse Csf1r promoter and the highly conserved Fms intronic regulatory element to drive mApple fluorescent protein expression. Csf1r-mApple was robustly expressed in monocyte-macrophage lineage cells in rat bone marrow (BM), peripheral blood, and tissues, with detectable expression in granulocytes and B cells and no evidence of expression in hematopoietic precursors or non-hematopoietic cells. Here, we use the Csf1r-mApple transgene to highlight and dissect the abundance and heterogeneity of rat tissue macrophage populations, and to demonstrate parallel increases in blood monocytes and multiple tissue macrophage populations, including BM, liver, spleen, and lung, in response to CSF1 treatment in vivo. The Csf1r-mApple rat is a novel tool enabling analysis of rat macrophages in situ by direct imaging and providing an additional phenotypic marker to facilitate exploration of rat tissue macrophage phenotypic and functional heterogeneity.

Protein and lipid homeostasis altered in rat macrophages after exposure to metallic oxide nanoparticles

Metal oxide nanoparticles (NPs), such as ZnO, ZnFe2O4, and Fe2O3, are widely used in industry. However, little is known about the cellular pathways involved in their potential toxicity. Here, we particularly investigated the key molecular pathways that are switched on after exposure to sub-toxic doses of ZnO, ZnFe2O4, and Fe2O3 in the in vitro rat alveolar macrophages (NR8383). As in our model, the calculated IC50 were respectively 16, 68, and more than 200 μg/mL for ZnO, ZnFe2O4, and Fe2O3; global gene and protein expression profiles were only analyzed after exposure to ZnO and ZnFe2O4 NPs. Using a rat genome microarray technology, we found that 985 and 1209 genes were significantly differentially expressed in NR8383 upon 4 h exposure to ¼ IC50 of ZnO and ZnFe2O4 NPs, respectively. It is noteworthy that metallothioneins were overexpressed genes following exposure to both NPs. Moreover, Ingenuity Pathway Analysis revealed that the top canonical pathway disturbed in NR8383 exposed to ZnO and ZnFe2O4 NPs was eIF2 signaling involved in protein homeostasis. Quantitative mass spectrometry approach performed from both NR8383 cell extracts and culture supernatant indicated that 348 and 795 proteins were differentially expressed upon 24 h exposure to ¼ IC50 of ZnO and ZnFe2O4 NPs, respectively. Bioinformatics analysis revealed that the top canonical pathways disturbed in NR8383 were involved in protein homeostasis and cholesterol biosynthesis for both exposure conditions. While VEGF signaling was specific to ZnO exposure, iron homeostasis signaling pathway was specific to ZnFe2O4 NPs. Overall, the study provides resource of transcriptional and proteomic markers of response to ZnO and ZnFe2O4 NP-induced toxicity through combined transcriptomics, proteomics, and bioinformatics approaches.

Haloperidol Reduces the Activity of Complement and Induces the Anti-Inflammatory Transformation of Peritoneal Macrophages in Rats.

It is known that psychotropic substances affect the immune system. Unfortunately, chronic antipsychotic administration causes side toxicological effects, associated with oxidative stress. The mechanisms of these effects are still unclear. We investigated the impact of sub-chronic administration of haloperidol (Hal) on parameters of innate immunity and related systems in healthy rats and compared them with Hal content. Hal administration (0.5mg/kg, 3weeks) resulted in two-fold decrease of the activity of the complement system and hemostasis. Hal content correlated with the activity of the complement (r = -0.71), phagocytic activity of peritoneal macrophages (r = 0.78), leukocyte elastase (r = -0.71) and glutathione-S-transferase activity (r = -0.67). Hal fully blocked in vitro PMA-induced iNOS expression in macrophages and changed their morphology to "anti-inflammatory" phenotype. The comparison of in vivo and in vitro data showed that Hal has a direct effect on phagocytic component of innate immunity and an indirect effect on leukocyte elastase and antioxidant enzymes. The results obtained in the present study indicated that Hal significantly affects homeostasis and causes a number of complex biological transformations. Graphical Abstract.

Immunomodulatory Effects of Colistin on Macrophages in Rats by Activating the p38/MAPK Pathway

Objectives: Colistin has been identified in a Caenorhabditis elegans chemical screening as an immunostimulatory agent that activates the conserved p38/PMK-1 pathway and provides protection against pathogens. Here we aimed to extend those findings to a mammalian model and evaluate the immunomodulatory effects of colistin on rat macrophages. Methods: Macrophages were isolated from Sprague-Dawley (SD) rat. The effects of colistin on the cytokine secretion, phagocytic activity and protein expression were determined by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blotting analysis, respectively. The relative microRNA expression was determined by microarray, and Kyoto Encylopedia of Genes and Genomes (KEGG) was used to identify potential signaling pathways. Results: Our data showed that 5, 10, and 20 µg/ml colistin significantly increased the secretion of TNF-α, while 20 and 5 µg/ml colistin significantly increased the levels of IL-1β and IL-6, respectively. Flow cytometry results showed that the relative mean fluorescence intensity and percentage of phagocytosis in colistin treatment groups were significantly higher compared with the control group, while the increased phagocytosis phenomenon can be blocked by p38 inhibitor. The phagocytic ability of macrophages against Staphylococcus aureus was significantly increased after colistin treatment. Microarray and KEGG pathway analyses revealed that mitogen-activated protein kinase (MAPK), mammalian target of rapamycin (mTOR), chemokine, and B cell receptor were the main pathways involved in the colistin stimulation process. Western blotting analysis demonstrated that the phosphorylated p38 protein level of colistin treatment groups was increased in a dose dependent manner. Conclusions: Present study is the first to demonstrate that colistin had immunomodulatory effects on macrophages in mammals, and the p38/MAPK pathway was involved in such colistin-induced immunomodulatory effect.

Antibiotic resistance and virulence genes in nisin-resistant Enterococcus faecalis isolated from raw buffalo milk modulate the innate functions of rat macrophages.

To elucidate the antibiotic resistance and virulence genes of nisin-resistant Enterococcus faecalis isolated from raw buffalo milk and to study the effect of nisin-sensitive and -resistant E. faecalis on the innate immunity of rats. Slanetz-Bartley agar plates containing nisin were used to isolate nisin-resistant E. faecalis. The virulence factors were ascertained using quantitative real-time polymerase chain reaction. Cell viability, phagocytosis, intracellular survival and enzyme assays were performed to investigate the interaction of E. faecalis with rat macrophages. Nisin-resistant E. faecalis was less prone to phagocytosis and survived longer inside the macrophages, due to reduced production of reactive oxygen species and nitric oxide. The viability and activation of macrophages was also reduced in the presence of resistant E. faecalis, as observed by enhanced lactate dehydrogenase production and reduced β-galactosidase. Nisin-resistant E. faecalis and its virulence factors were reported in raw buffalo milk. This study shows that nisin-resistant variants exhibited cross resistance to antibiotics and suppressed the innate immune responses of rats by directly affecting macrophage activity. This study elucidated the contamination of raw buffalo milk by nisin-resistant E. faecalis, which may pose food safety risk.

The effects of silica dust on the expression of MyD88 and TRAF6 mRNA of lung macrophages in rats

Objective: To investigate the effects of silica dust on the expression of Myeloid differentiation factor 88 (MyD88) mRNA and tumor necrosis factor receptor-associated factor (TRAF6) mRNA of lung macrophages in rats. Methods: Selecting 40 SPF-class Wistar rats with average weight (200±20) g randomly divided into control group and 30 d, 60 d, 120 d experimental groups with 10 rats in each group according to body weight. The experimental groups rats were injected with 1 ml of SiO(2) (100 mg/ml) suspension through the trachea into lung only once, then they were respectively killed after 30, 60, 120 days. The control group rats were injected with 1 ml of saline into lung, and killed after 120 days. The lungs of the rats were taken for pathological observation. Lung macrophages were extracted and counted, and their activity was detected by MTT. RT-qPCR was used to assess the relative contents of MyD88 mRNA and TRAF6 mRNA. Results: Silica dust inhalation led to infiltration of lung tissue cells, thickening the alveolar wall and destruction of alveolar structure. The longer the exposure to dust, the more obvious the results were. The number of macrophages in all experimental groups and activity in the 30 d, 60 d groups were significantly higher than that in the control group (P<0.05) . Among them, 30 d group had the largest number and the highest activity. Compared with the control group, the expression of MyD88 mRNA and TRAF6 mRNA of lung macrophages in rats increased in the experimental groups (P<0.05) , especially in the 60 d group. Conclusion: Silica dust inhalation can increase the expression of MyD88 and TRAF6 in macrophages, suggesting that silica dust can induce silicosis fibrosis by activating TLR/NF-κB signal pathway.