Infrapatellar fat pad adipose tissue-derived macrophages display a predominant CD11c+CD206+ phenotype and express genotypes attributable to key features of OA pathogenesis.

Objectives: In knee osteoarthritis (OA), macrophages are the most predominant immune cells that infiltrate synovial tissues and infrapatellar fat pads (IPFPs). Both M1 and M2 macrophages have been described, but their role in OA has not been fully investigated. Therefore, we investigated macrophage subpopulations in IPFPs and synovial tissues of knee OA patients and their correlation with disease severity, examined their transcriptomics, and tested for factors that influenced their polarization. Methods: Synovial tissues and IPFPs were obtained from knee OA patients undergoing total knee arthroplasty. Macrophages isolated from these joint tissues were characterized via flow cytometry. Transcriptomic profiling of each macrophage subpopulations was performed using NanoString technology. Peripheral blood monocyte-derived macrophages (MDMs) were treated with synovial fluid and synovial tissue- and IPFP-conditioned media. Synovial fluid-treated MDMs were treated with platelet-rich plasma (PRP) and its effects on macrophage polarization were observed. Results: Our findings show that CD11c+CD206+ macrophages were predominant in IPFPs and synovial tissues compared to other macrophage subpopulations (CD11c+CD206-, CD11c-CD206+, and CD11c-CD206- macrophages) of knee OA patients. The abundance of macrophages in IPFPs reflected those in synovial tissues but did not correlate with disease severity as determined from Mankin scoring of cartilage destruction. Our transcriptomics data demonstrated highly expressed genes that were related to OA pathogenesis in CD11c+CD206+ macrophages than CD11c+CD206-, CD11c-CD206+, and CD11c-CD206- macrophages. In addition, MDMs treated with synovial fluid, synovial tissue-conditioned media, or IPFP-conditioned media resulted in different polarization profiles of MDMs. IPFP-conditioned media induced increases in CD86+CD206+ MDMs, whereas synovial tissue-conditioned media induced increases in CD86+CD206- MDMs. Synovial fluid treatment (at 1:8 dilution) induced a very subtle polarization in each macrophage subpopulation. PRP was able to shift macrophage subpopulations and partially reverse the profiles of synovial fluid-treated MDMs. Conclusion: Our study provides an insight on the phenotypes and genotypes of macrophages found in IPFPs and synovial tissues of knee OA patients. We also show that the microenvironment plays a role in driving macrophages to polarize differently and shifting macrophage profiles can be reversed by PRP.

Rapid decalcification of articular cartilage and subchondral bone using an ultrasonic cleaner with EDTA.

Articular cartilage and subchondral bones were used to be the samples for studying effects of drugs in the joint degenerative diseases such as osteoarthritis. Because of the deposition of mineral salts, articular cartilage and subchondral bones require decalcification process to soften the tissues. EDTA is a chelating agent that is commonly used to remove mineral salts, but this step is time-consuming and can take as long as 45 days. Commercial ultrasonic cleaner and microwave oven were reported to reduce the decalcification timing. The aim of this study is to determine and compare the decalcification of human articular cartilage and subchondral bone using EDTA together with ultrasonic cleaner or microwave oven. Hundred pieces of articular cartilage and subchondral bones obtained from osteoarthritis patients undergone total-knee-replacement were divided into 10 groups according to decalcification method (ultrasonic cleaner or microwave) and timing (2, 4, 6, 8, and 10 h). In each group, all cartilage and subchondral bone pieces were decalcified and sectioned, and subsequently stained with haematoxylin and eosin, Von Kossa, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, or caspase-3 immunohistochemistry. The optimal timing of decalcification of articular cartilage and subchondral bones using EDTA together with ultrasonic cleaner was at 8 and 10 h, while the timing using EDTA together with microwave oven was more than 10 h. Clear TUNEL and caspase-3 signals were obtained from samples decalcified using EDTA together with ultrasonic cleaner for 8 h. In summary, to our knowledge, this is the first study that compared EDTA decalcification between ultrasonic cleaner and microwave oven. Here, we report a new methodology for decalcification for articular cartilage and subchondral bones that reduces decalcification time from weeks to hours and is suitable for further pathological analyses.

Candida Administration Worsens Uremia-Induced Gut Leakage in Bilateral Nephrectomy Mice, an Impact of Gut Fungi and Organismal Molecules in Uremia.

The impact of gut fungi and (1→3)-ß-d-glucan (BG), a major fungal cell wall component, on uremia was explored by Candida albicans oral administration in bilateral nephrectomy (BiNx) mice because of the prominence of C. albicans in the human intestine but not in mice. As such, BiNx with Candida administration (BiNx-Candida) enhanced intestinal injury (colon cytokines and apoptosis), gut leakage (fluorescein isothiocyanate [FITC]-dextran assay, endotoxemia, serum BG, and bacteremia), systemic inflammation, and liver injury at 48 h postsurgery compared with non-Candida BiNx mice. Interestingly, uremia-induced enterocyte apoptosis was severe enough for gut translocation of viable bacteria, as indicated by culture positivity for bacteria in blood, mesenteric lymph nodes (MLNs), and other organs, which was more severe in BiNx-Candida than in non-Candida BiNx mice. Candida induced alterations in the gut microbiota of BiNx mice as indicated by (i) the higher fungal burdens in the feces of BiNx-Candida mice than in sham-Candida mice by culture methods and (ii) increased Bacteroides with decreased Firmicutes and reduced bacterial diversity in the feces of BiNx-Candida mice compared with non-Candida BiNx mice by fecal microbiome analysis. In addition, lipopolysaccharide plus BG (LPS+BG), compared with each molecule alone, induced high supernatant cytokine levels, which were enhanced by uremic mouse serum in both hepatocytes (HepG2 cells) and macrophages (RAW264.7 cells). Moreover, LPS+BG, but not each molecule alone, reduced the glycolysis capacity and mitochondrial function in HepG2 cells as determined by extracellular flux analysis. Additionally, a probiotic, Lactobacillus rhamnosus L34 (L34), attenuated disease severity only in BiNx-Candida mice but not in non-Candida BiNx mice, as indicated by liver injury and serum cytokines through the attenuation of gut leakage, the fecal abundance of fungi, and fecal bacterial diversity but not fecal Gram-negative bacteria. In conclusion, Candida enhanced BiNx severity through the worsening of gut leakage and microbiota alterations that resulted in bacteremia, endotoxemia, and glucanemia.IMPORTANCE The impact of fungi in the intestine on acute uremia was demonstrated by the oral administration of Candida albicans in mice with the removal of both kidneys. Because fungi in the mouse intestine are less abundant than in humans, a Candida-administered mouse model has more resemblance to patient conditions. Accordingly, acute uremia, without Candida, induced intestinal mucosal injury, which resulted in the translocation of endotoxin, a major molecule of gut bacteria, from the intestine into blood circulation. In acute uremia with Candida, intestinal injury was more severe due to fungi and the alteration in intestinal bacteria (increased Bacteroides with decreased Firmicutes), leading to the gut translocation of both endotoxin from gut bacteria and (1→3)-ß-d-glucan from Candida, which synergistically enhanced systemic inflammation in acute uremia. Both pathogen-associated molecules were delivered to the liver and induced hepatocyte inflammatory responses with a reduced energy production capacity, resulting in acute uremia-induced liver injury. In addition, Lactobacillus rhamnosus attenuated intestinal injury through reduced gut Candida and improved intestinal bacterial conditions.

Anti-inflammatory and anti-oxidant effects of aloe vera in rats with non-alcoholic steatohepatitis.

BACKGROUND: Aloe vera exerts several biological activities, such as, anti-inflammatory, antioxidant, and antimicrobial effects. It was recently shown to reduce insulin resistance and triglyceride level. We hypothesized that aloe vera would have beneficial effects in alleviating non-alcoholic steatohepatitis (NASH) in rats. AIM: To examine the therapeutic effects of aloe vera in NASH rats. METHODS: All rats were randomly divided into 3 groups (n = 6 in each group). Rats in the control group were fed ad libitum with a standard diet for 8 wk. Rats in the NASH group were fed ad libitum with a high-fat high-fructose diet (HFHFD) for 8 wk. Rats in the aloe vera group were fed ad libitum with a HFHFD and aloe vera in dimethylsulfoxide (50 mg/kg) by gavage daily for 8 wk. Liver samples were collected at the end of the treatment period. RESULTS: Hepatic malondialdehyde (MDA) levels increased significantly in the NASH group as compared with the control group (377 ± 77 nmol/mg vs 129 ± 51 nmol/mg protein, respectively, P < 0.001). Glutathione (GSH) levels were significantly lower in the NASH group than the control group (9 ± 2 nmol/mg vs 24 ± 8 nmol/mg protein, respectively, P = 0.001). The expression of interleukin-18 (IL-18), nuclear factor-kappa ß, and caspase-3 increased, while peroxisome proliferator-activated receptor gamma decreased in the NASH group compared with the controls. Following aloe vera administration, MDA levels decreased (199 ± 35 nmol/mg protein) and GSH increased (18 ± 4 nmol/mg protein) markedly. Steatosis, hepatocyte ballooning, lobular inflammation and increased hepatocyte apoptosis were observed in the NASH group. Aloe vera treatment attenuated these changes in liver histology. CONCLUSION: Aloe vera attenuated oxidative stress, hepatic inflammation and hepatocyte apoptosis, thus improving liver pathology in rats with NASH.

Gut leakage enhances sepsis susceptibility in iron-overloaded ß-thalassemia mice through macrophage hyperinflammatory responses.

Iron overload induces intestinal-permeability defect (gut leakage), and gut translocation of organismal molecules might enhance systemic inflammation and sepsis severity in patients with thalassemia (Thal). Hence, iron administration in Hbbth3/+ mice, heterozygous ß-globin-deficient Thal mice, was explored. Oral iron administration induced more severe secondary hemochromatosis and gut leakage in Thal mice compared with wild-type (WT) mice. Gut leakage was determined by 1) FITC-dextran assay, 2) spontaneous serum elevation of endotoxin (LPS) and (1→3)-ß-d-glucan (BG), molecular structures of gut-organisms, and 3) reduction of tight-junction molecules with increased enterocyte apoptosis (activated caspase-3) by immunofluorescent staining. Iron overload also enhanced serum cytokines and increased Bacteroides spp. (gram-negative bacteria) in feces as analyzed by microbiome analysis. LPS injection in iron-overloaded Thal mice produced higher mortality and prominent cytokine responses. Additionally, stimulation with LPS plus iron in macrophage from Thal mice induced higher cytokines production with lower ß-globin gene expression compared with WT. Furthermore, possible gut leakage as determined by elevated LPS or BG (>60 pg/mL) in serum without systemic infection was demonstrated in 18 out of 41 patients with ß-thalassemia major. Finally, enhanced LPS-induced cytokine responses of mononuclear cells from these patients compared with cells from healthy volunteers were demonstrated. In conclusion, oral iron administration in Thal mice induced more severe gut leakage and increased fecal gram-negative bacteria, resulting in higher levels of endotoxemia and serum inflammatory cytokines compared with WT. Preexisting hyperinflammatory cytokines in iron-overloaded Thal enhanced susceptibility toward infection.NEW & NOTEWORTHY Although the impact of iron accumulation in several organs of patients with thalassemia is well known, the adverse effect of iron accumulation in gut is not frequently mentioned. Here, we demonstrated iron-induced gut-permeability defect, impact of organismal molecules from gut translocation of, and macrophage functional defect upon the increased sepsis susceptibility in thalassemia mice.

Plasma miR-370-3P as a Biomarker of Sepsis-Associated Encephalopathy, the Transcriptomic Profiling Analysis of Microrna-Arrays From Mouse Brains.

The diagnosis of sepsis-associated encephalopathy (SAE), an alteration of conscious from sepsis, is difficult due to the similarity to altered states of conscious that occur from other causes. Transcriptomic analyses between mouse brains at 24 h after cecal ligation and puncture (CLP) (SAE brain as evaluated by SHIRPA score) and at 120 h post-CLP (survivor) were performed to discover the SAE biomarker. Then, candidate microRNAs were validated in mouse and patient samples.As such, increased miR-370-3p in SAE mouse-brains (compared with recovery phase) was demonstrated by transcriptomic miR-profiling and was highly expressed in brain (but not other organs) of 24 h post-CLP mice. Plasma miR-370-3p also increased in CLP but was non-detectable in bilateral-nephrectomy (BiNx, a representative model of acute uremic encephalopathy) despite blood brain barrier permeability defect (determined by plasma s100ß and Evan blue dye assay) in both conditions. In parallel, high plasma miR-370-3p was demonstrated in patients with SAE (but not sepsis alone or uremia) suggesting the specificity toward SAE. The association among TNF-α, miR-370-3p and brain apoptosis was demonstrated by high serum TNF-α and increased brain apoptosis in SAE mice, TNF-α (but not other cytokines) activated miR-370-3p expression in PC-12 neuron cell, and increased cell apoptosis in miR-370-3p transfected PC-12 after incubation with TNF-α.In conclusion, miR-370-3p increased in brain and plasma of SAE mice but not uremic encephalopathy. Perhaps, TNF-α enhances cell susceptibility toward brain apoptosis in SAE, in part, through miR-370-3p induction in neuron. Our pilot results in patients with SAE supported the possibility that plasma miR-370-3p is an interesting SAE biomarker candidate. Further studies are warranted.

Increased susceptibility against Cryptococcus neoformans of lupus mouse models (pristane-induction and FcGRIIb deficiency) is associated with activated macrophage, regardless of genetic background.

The severity of cryptococcosis in lupus from varying genetic-backgrounds might be different due to the heterogeneity of lupus-pathogenesis. This study explored cryptococcosis in lupus mouse models of pristane-induction (normal genetic-background) and FcGRIIb deficiency (genetic defect). Because the severity of lupus nephritis, as determined by proteinuria and serum creatinine, between pristane and FcGRIIb-/- mice were similar at 6-month-old, Cryptococcus neoformans was intravenously administered in 6-month-old mice and were age-matched with wild-type. Indeed, the cryptococcosis disease severity, as evaluated by mortality rate, internal-organ fungal burdens and serum cytokines, between pristane and FcGRIIb-/- mice was not different. However, the severity of cryptococcosis in wild-type was less severe than the lupus mice. On the other hand, phagocytosis activity of peritoneal macrophages from lupus mice (pristane and FcGRIIb-/-) was more predominant than the wild-type without the difference in macrophage killing-activity among these groups. In addition, the number of active T helper cells (Th-cell) in the spleen, including Th-cells with intracellular IFN-γ, from lupus mice (pristane and FcGRIIb-/-) was higher than wildtype. Moreover, these active Th-cells were even higher after 2 weeks of cryptococcal infection. These data support enhanced macrophage activation through prominent Th-cells in both lupus models. In conclusion, an increased susceptibility of cryptococcosis in both lupus models was independent to genetic background. This might due to Th-cell enhanced macrophage phagocytosis with the interference of macrophage killing activity from Cryptococcal immune-evasion properties.

Helicobacter pylori Infection Increased Anti-dsDNA and Enhanced Lupus Severity in Symptomatic FcγRIIb-Deficient Lupus Mice.

The defect on Fc gamma receptor IIb (FcγRIIb), the only inhibitory FcγR, has been identified as one of the genetic factors increasing susceptibility to lupus. The prevalence of Helicobacter pylori (HP) and FcγRIIb dysfunction-polymorphisms are high among Asians, and their co-existence is possible. Unfortunately, the influence of HP against lupus progression in patients with lupus is still controversial. In this study, the interactions between these conditions were tested with HP infection in 24-week-old FcγRIIb-/- mice (symptomatic lupus). HP induced failure to thrive, increased stomach bacterial burdens and stomach injury (histology and cytokines) in both wild-type and FcγRIIb-/- mice. While the severity of HP infection, as determined by these parameters, was not different between both strains, antibodies production (anti-HP, anti-dsDNA and serum gammaglobulin) were higher in FcγRIIb-/- mice compared to wild-type. Accordingly, HP infection also accelerated the severity of lupus as determined by proteinuria, serum creatinine, serum cytokines, renal histology, and renal immune complex deposition. Although HP increased serum cytokines in both wild-type and FcγRIIb-/- mice, the levels were higher in FcγRIIb-/- mice. As such, HP also increased spleen weight and induced several splenic immune cells responsible for antibody productions (activated B cell, plasma cell and follicular helper T cell) in FcγRIIb-/- mice, but not in wild-type. These data describe the different systemic responses against localized HP infection from diverse host genetic background. In conclusion, the mutual interactions between HP and lupus manifestations of FcγRIIb-/-mice were demonstrated in this study. With the prominent immune responses from the loss of inhibitory signaling in FcγRIIb-/- mice, HP infection in these mice induced intense chronic inflammation, increased antibody production, and enhanced lupus severity. Thus, the increased systemic inflammatory responses due to localized HP inducing gastritis in some patients with lupus may enhance lupus progression. More studies are needed.

Vasculoprotective effects of combined endothelial progenitor cells and mesenchymal stem cells in diabetic wound care: their potential role in decreasing wound-oxidative stress.

To investigate whether the combined endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) could enhance angiogenesis and wound healing in diabetic mice. Balb/c nude mice were divided into five groups, including a control group, diabetic group (DM), DM injected with 1 × 10(6) cells MSCs, DM injected with 1 × 10(6) cells EPCs, and DM injected with combined 0.5 × 10(6) cells MSCs and 0.5 × 10(6) cells EPCs. After seven weeks, the mice were anesthetized, and bilateral full-thickness excision skin wounds were made on the dorsorostral back. The percentage of wound closure in DM group decreased significantly than in control and all other treated groups on day 7 and day 14 (P < 0.005). On day 14, the percentage of capillary vascularity in combine-treated group was significantly higher than in DM (P < 0.005). In the present study, we have demonstrated that the combined EPCs and MSCs can increase vascular endothelial growth factor (VEGF) level and angiogenesis which resulted in reduced neutrophil infiltration, decreased malondialdehyde (MDA) levels, and enhanced wound healing in diabetic mice model.