Anti-inflammatory Gene Expression Research Articles

Single-cell and spatial analyses reveal the association between gene expression of glutamine synthetase with the immunosuppressive phenotype of APOE+CTSZ+TAM in cancers.

An immunosuppressive state is regulated by various factors in the tumor microenvironment (TME), including, but not limited to, metabolic plasticity of immunosuppressive cells and cytokines secreted by these cells. We used single-cell RNA-sequencing (scRNA-seq) data and applied single-cell flux estimation analysis to characterize the link between metabolism and cellular function within the hypoxic TME of colorectal (CRC) and lung cancer. In terms of metabolic heterogeneity, we found myeloid cells potentially inclined to accumulate glutamine but tumor cells inclined to accumulate glutamate. In particular, we uncovered a tumor-associated macrophage (TAM) subpopulation, APOE+CTSZ+TAM, that was present in high proportions in tumor samples and exhibited immunosuppressive characteristics through upregulating the expression of anti-inflammatory genes. The proportion of APOE+CTSZ+TAM and regulatory T cells (Treg) were positively correlated across CRC scRNA-seq samples. APOE+CTSZ+TAM potentially interacted with Treg via CXCL16-CCR6 signals, as seen by ligand-receptor interactions analysis. Notably, glutamate-to-glutamine metabolic flux score and glutamine synthetase (GLUL) expression were uniquely higher in APOE+CTSZ+TAM, compared with other cell types within the TME. GLUL expression in macrophages was positively correlated with anti-inflammatory score and was higher in high-grade and invasive tumor samples. Moreover, spatial transcriptome and multiplex immunofluorescence staining of samples showed that APOE+CTSZ+TAM and Treg potentially colocalized in the tissue sections from CRC clinical samples. These results highlight the specific role and metabolic characteristic of the APOE+CTSZ+TAM subpopulation and provide a new perspective for macrophage subcluster-targeted therapeutic interventions or metabolic checkpoint-based cancer therapies.

Beneficial effect of TLR4 blockade by a specific aptamer antagonist after acute myocardial infarction

Experimental evidence indicates that the control of the inflammatory response after myocardial infarction is a key strategy to reduce cardiac injury. Cellular damage after blood flow restoration in the heart promotes sterile inflammation through the release of molecules that activate pattern recognition receptors, among which TLR4 is the most prominent. Transient regulation of TLR4 activity has been considered one of the potential therapeutic interventions with greater projection towards the clinic. In this regard, the characterization of an aptamer (4FT) that acts as a selective antagonist for human TLR4 has been investigated in isolated macrophages from different species and in a rat model of cardiac ischemia/reperfusion (I/R). The binding kinetics and biological responses of murine and human macrophages treated with 4FT show great affinity and significant inhibition of TLR4 signaling including the NF-κB pathway and the LPS-dependent increase in the plasma membrane currents (Kv currents). In the rat model of I/R, administration of 4FT following reoxygenation shows amelioration of cardiac injury function and markers, a process that is significantly enhanced when the second dose of 4FT is administered 24 h after reperfusion of the heart. Parameters such as cardiac injury biomarkers, infiltration of circulating inflammatory cells, and the expression of genes associated with the inflammatory onset are significantly reduced. In addition, the expression of anti-inflammatory genes, such as IL-10, and pro-resolution molecules, such as resolvin D1 are enhanced after 4FT administration. These results indicate that targeting TLR4 with 4FT offers new therapeutic opportunities to prevent cardiac dysfunction after infarction.

МОЛЕКУЛЯРНО-КЛЕТОЧНЫЕ МАРКЕРЫ НЕЙРОВОСПАЛЕНИЯ И СОСТОЯНИЕ КОГНИТИВНЫХ ФУНКЦИЙ В ОТДАЛЕННЫЙ ПЕРИОД ПОСЛЕ ПРОЛОНГИРОВАННОГО γ-, n-ОБЛУЧЕНИЯ МЫШЕЙ В НИЗКИХ ДОЗАХ

The study aimed to investigate the effect of low-dose rate (2.13 mGy/h) prolonged γ,n-irradiation at 0.05 and 0.5 Gy on brain microglia, expression of pro- and anti-inflammatory cytokine genes, behavior and cognition of C57Bl/6 mice two months and one year after exposure. The decrease in the number of resting microglia and the increase in the proportion of activated microglia in brain cell preparations were observed after γ,n-irradiation at a dose of 0.5 Gy. Immunohistochemical analysis revealed the increased number of microglia cells and unaltered number of astrocytes in the hippocampus after γ,n-irradiation at both doses. Expression of TNFα and IL-1β pro-inflammatory cytokine genes in the hippocampus was increased after irradiation at both doses. Expression of TGFβ and IL-4 anti-inflammatory cytokine genes was decreased two months after irradiation only at a dose of 0.5 Gy, suggesting more intensive neuroinflammation. Analysis performed a year later revealed an age-related decrease in the number of resting microglia and increase in proportion of activated microglia compared to young animals, with more profound changes in male mice. The state of microglia in control and irradiated mice was similar one year after exposure. Neither two months nor one year after irradiation the impairments of motor activity and spatial memory of mice were detected; nevertheless, mice irradiated at a dose of 0.5 Gy demonstrated anxious behavior and decreased spatial learning in Morris water maze one year after exposure. In summary, we showed that γ, n-irradiation at doses of 0.05 and 0.5 Gy induced neuroinflammation two months after exposure. No difference from the control mice was observed one year after irradiation. Disturbances of behavior and spatial memory of irradiated mice were not detected.

Transcriptome responses of intestinal epithelial cells induced by membrane vesicles of Listeriamonocytogenes

Membrane vesicles (MVs) serve as an essential virulence factor in several pathogenic bacteria. The release of MVs by Listeria monocytogenes is only recently recognized; still, the enigmatic role of MVs in pathogenesis is yet to be established. We report the transcriptome response of Caco-2 cells upon exposure to MVs and the L. monocytogenes that leads to observe the up-regulation of autophagy-related genes in the early phase of exposure to MVs. Transcription of inflammatory cytokines is to the peak at the fourth hour of exposure. An array of differentially expressed genes was associated with actin cytoskeleton rearrangement, autophagy, cell cycle arrest, and induction of oxidative stress. At a later time point, transcriptional programs are generated upon interaction with MVs to evade innate immune signals, by modulating the expression of anti-inflammatory genes. KEGG pathway analysis is palpably confirming that MVs appear principally responsible for the induction of immune signaling pathways. Besides, MVs induced the expression of cell cycle regulatory genes, likely responsible for the ability to prolong host cell survival, thus protecting the replicative niche for L. monocytogenes. Notably, we identified several non-coding RNAs (ncRNAs), possibly involved in the regulation of early manipulation of the host gene expression, essential for the persistence of L. monocytogenes.

Glutathione Protects against Paraquat-Induced Oxidative Stress by Regulating Intestinal Barrier, Antioxidant Capacity, and CAR Signaling Pathway in Weaned Piglets.

Endogenous glutathione (GSH) effectively regulates redox homeostasis in the body. This study aimed to investigate the regulatory mechanism of different dietary levels of GSH supplementation on the intestinal barrier and antioxidant function in a paraquat-induced stress-weaned piglet model. Our results showed that dietary 0.06% GSH supplementation improved the growth performance of weaned piglets under normal and stressful conditions to some degree and decreased the diarrhea rate throughout. Exogenous GSH improved paraquat-induced changes in intestinal morphology, organelle, and permeability and reduced intestinal epithelial cell apoptosis. Moreover, GSH treatment alleviated intestinal oxidative stress damage by upregulating antioxidant (GPX4, CnZnSOD, GCLC, and GCLM) and anti-inflammatory (IL-10) gene expression and downregulating inflammatory cytokines (IFN-γ and IL-12) gene expression. Furthermore, GSH significantly reduced the expression levels of constitutive androstane receptor (CAR), RXRα, HSP90, PP2Ac, CYP2B22, and CYP3A29, and increased the expression levels of GSTA1 and GSTA2 in the jejunum and ileum of paraquat-induced piglets. We conclude that exogenous GSH protects against oxidative stress damage by regulating the intestinal barrier, antioxidant capacity, and CAR signaling pathway.

Tryptophan metabolism and gut flora profile in different soybean protein induced enteritis of pearl gentian groupers.

The substitution of high-level soy meals for fish meal (FM) generally leads to fish enteritis, accompanied by significant variations in gut flora. Relevant studies have pointed out a close relationship between tryptophan metabolism mediated by gut flora and vertebrate inflammatory bowel disease. Present study examines the role of tryptophan metabolism and gut flora profile in fish enteritis caused by different soybean meals. The 960 groupers were randomly assigned into 4 groups (n = 4), which including: (1) FM (the control group, fed with 50% FM feed), (2) SBM40 (replacing 40% FM with soybean meal), (3) SPC40 (replacing 40% FM with soybean protein concentrate), and (4) FSBM40 (replacing 40% FM with fermented soybean meal). Under average temperature and natural light, the groupers were cultivated with feeds of iso-nitrogen and iso-lipid for 10 weeks. The results showed that soybean meal feeds at all experimental levels had negative effects on fish gut physiology and growth performance. Typical enteritis features and fluctuations of immune system occur, which can be observed in the enzyme activities of total superoxide dismutase and lysozyme and in the contents of immunoglobulin M, complement 3 and complement 4. 16SrDNA high-throughput sequencing indicated that it greatly influenced the gut flora with the abundance of maleficent bacteria, like Vibrio, amplified with increasing dietary soybean meals. According to the "3 + 2" full-length transcriptome sequencing, soy meals at the three experimental levels inhibited the key gene expressions of tryptophan metabolic pathway in fish gut, however, there are some differences in the types of key genes that are inhibited. The canonical correlation analysis showed that the changes in key gene expressions in tryptophan metabolic pathway had a positive correlation with the expressions of pro-inflammatory genes (P < 0.05) and negatively correlated with the expression of anti-inflammatory genes (P < 0.05). It is speculated from this study that tryptophan metabolism is closely related to fish soy meal-related enteritis, and the abnormal tryptophan metabolism caused by intestinal flora imbalance may play an important role. In the future research, we can further study the tolerance of fish to soy meals feed from two aspects of tryptophan metabolism and intestinal flora changes.

Adipocyte-Secreted IL-6 Sensitizes Macrophages to IL-4 Signaling.

Complex bidirectional cross talk between adipocytes and adipose tissue immune cells plays an important role in regulating adipose function, inflammation, and insulin responsiveness. Adipocytes secrete the pleiotropic cytokine IL-6 in response to both inflammatory and catabolic stimuli. Previous studies have suggested that IL-6 secretion from adipocytes in obesity may promote adipose tissue inflammation. Here, we investigated catabolic stimulation of adipocyte IL-6 secretion and its impact on adipose tissue immune cells. In obesity, catecholamine resistance reduces cAMP-driven adipocyte IL-6 secretion in response to catabolic signals. By restoring adipocyte catecholamine sensitivity in obese adipocytes, amlexanox stimulates adipocyte-specific IL-6 secretion. We report that in this context, adipocyte-secreted IL-6 activates local macrophage STAT3 to promote Il4ra expression, thereby sensitizing them to IL-4 signaling and promoting an anti-inflammatory gene expression pattern. Supporting a paracrine adipocyte to macrophage mechanism, these effects could be recapitulated using adipocyte conditioned media to pretreat bone marrow-derived macrophages prior to polarization with IL-4. The effects of IL-6 signaling in adipose tissue are complex and context specific. These results suggest that cAMP-driven IL-6 secretion from adipocytes sensitizes adipose tissue macrophages to IL-4 signaling.

Low-dose IL-2 reduces IL-21+ T cell frequency and induces anti-inflammatory gene expression in type 1 diabetes

Despite early clinical successes, the mechanisms of action of low-dose interleukin-2 (LD-IL-2) immunotherapy remain only partly understood. Here we examine the effects of interval administration of low-dose recombinant IL-2 (iLD-IL-2) in type 1 diabetes using high-resolution single-cell multiomics and flow cytometry on longitudinally-collected peripheral blood samples. Our results confirm that iLD-IL-2 selectively expands thymic-derived FOXP3+HELIOS+ regulatory T cells and CD56bright NK cells, and show that the treatment reduces the frequency of IL-21-producing CD4+ T cells and of two innate-like mucosal-associated invariant T and Vγ9Vδ2 CD8+ T cell subsets. The cellular changes induced by iLD-IL-2 associate with an anti-inflammatory gene expression signature, which remains detectable in all T and NK cell subsets analysed one month after treatment. These findings warrant investigations into the potential longer-term clinical benefits of iLD-IL-2 in immunotherapy.

The Role of Interferon Regulatory Factor 1 in Regulating Microglial Activation and Retinal Inflammation.

Microglia are resident immune cells in the central nervous system (CNS). Microglial activation plays a prominent role in neuroinflammation and CNS diseases. However, the underlying mechanisms of microglial activation are not well understood. Here, we report that the transcription factor interferon regulatory factor 1 (IRF1) plays critical roles in microglial activation and retinal inflammation by regulating pro- and anti-inflammatory gene expression. IRF1 expression was upregulated in activated retinal microglia compared to those at the steady state. IRF1 knockout (KO) in BV2 microglia cells (BV2ΔIRF1) created by CRISPR/Cas9 genome-editing technique causes decreased microglia proliferation, migration, and phagocytosis. IRF1-KO decreased pro-inflammatory M1 marker gene expression induced by lipopolysaccharides (LPS), such as IL-6, COX-2, and CCL5, but increased anti-inflammatory M2 marker gene expression by IL-4/13, such as Arg-1, CD206, and TGF-β. Compared to the wild-type cells, microglial-conditioned media (MCM) of activated BV2ΔIRF1 cell cultures reduced toxicity or death to several retinal cells, including mouse cone photoreceptor-like 661 W cells, rat retinal neuron precursor R28 cells, and human ARPE-19 cells. IRF1 knockdown by siRNA alleviated microglial activation and retinal inflammation induced by LPS in mice. Together, the findings suggest that IRF1 plays a vital role in regulating microglial activation and retinal inflammation and, therefore, may be targeted for treating inflammatory and degenerative retinal diseases.

Effects of cysteine addition to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus).

As the precursor of taurine, cysteine serves physiological functions, such as anti-oxidative stress and immune improvement. Investigation of cysteine and its derivatives has made positive progress in avian and mammalian species, yet the study and application of cysteine in aquatic animals are relatively rare. Therefore, we evaluated the effects of supplementing a low-fishmeal diet with various levels of cysteine on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae of the juvenile golden pompano (Trachinotus ovatus). According to our study, exogenous supplementation with 0.6-1.2% cysteine greatly increased the final body weight (FBW) and specific growth rate (SGR) of golden pompano compared to the control group. Under the present conditions, the optimum dietary cysteine supplementation level for golden pompano was 0.91% based on the polynomial regression analysis of SGR. Meanwhile, we found that the Nrf2/Keap1/HO-1 signaling pathway was notably upregulated with the increase of exogenous cysteine, which increased antioxidant enzyme activity in serum and gene expression in the intestine and reduced the level of reactive oxygen species (ROS) in the serum of golden pompano. In addition, morphological analysis of the midgut demonstrated that exogenous cysteine improved muscle thickness and villi length, which suggested that the physical barrier of the intestine was greatly strengthened by cysteine. Moreover, cysteine increased the diversity and relative abundance of the intestinal flora of golden pompano. Cysteine suppressed intestinal NF-κB/IKK/IκB signaling and pro-inflammatory cytokine mRNA levels. Conversely, intestinal anti-inflammatory cytokine gene expression and serum immune parameters were upregulated with the supplementary volume of cysteine and improved intestine immunity. Further, exogenous cysteine supplementation greatly reduced the mortality rate of golden pompano challenged with S. agalactiae. In general, our findings provide more valuable information and new insights into the rational use of cysteine in the culture of healthy aquatic animals.

Cytokines in Lung Transplantation.

Lung transplantation has developed significantly in recent years, but post-transplant care and patients' survival still need to be improved. Moreover, organ shortage urges novel modalities to improve the quality of unsuitable lungs. Cytokines, the chemical mediators of the immune system, might be used for diagnostic and therapeutic purposes in lung transplantation. Cytokine monitoring pre- and post-transplant could be applied to the prevention and early diagnosis of injurious inflammatory events including primary graft dysfunction, acute cellular rejection, bronchiolitis obliterans syndrome, restrictive allograft syndrome, and infections. In addition, preoperative cytokine removal, specific inhibition of proinflammatory cytokines, and enhancement of anti-inflammatory cytokines gene expression could be considered therapeutic options to improve lung allograft survival. Therefore, it is essential to describe the cytokines alteration during inflammatory events to gain a better insight into their role in developing the abovementioned complications. Herein, cytokine fluctuations in lung tissue, bronchoalveolar fluid, peripheral blood, and exhaled breath condensate in different phases of lung transplantation have been reviewed; besides, cytokine gene polymorphisms with clinical significance have been summarized.

Heterogeneity of T cells and macrophages in chlorine-induced acute lung injury in mice using single-cell RNA sequencing

Objective Chlorine (Cl2), as an asphyxiant toxicant, induced poisoning incidents and acute lung injury (ALI) occur frequently. The specific pathogenesis of Cl2-induced ALI remains unclear. Immune cells play an important role in the process of lung damage. We used single-cell RNA sequencing (scRNA-seq) technology to explore T cells and macrophages molecular mechanism. Methods Female BALB/c mice were exposed to 400 ppm Cl2 for 15 min. scRNA-seq technology was used to observe the heterogeneity of T cells and macrophages. Hematoxylin–eosin (H&E) staining was used to evaluate the degree of lung injury. Immunofluorescence was used to verify the highly expressed genes of our interest. Results A total of 5316 to 7742 cells were classified into eight different cell types. Several new highly expressed anti-inflammatory and pro-inflammatory genes were found in T cells and macrophages, which were further verified in vitro. Through the pseudotime analysis of macrophages, it was found that the expression of pro-inflammatory and anti-inflammatory genes showed opposite trends in the development of Cl2-induced ALI. This study also mapped T cells-macrophage communication and identified the development of several important receptor-ligand complexes in Cl2-induced ALI. Conclusions These findings are worthy of further exploration and provide new resources and directions for the study of Cl2-induced ALI in mice, especially in immune and inflammation mechanisms.

Role of low-intensity pulsed ultrasound in regulating macrophage polarization to accelerate tendon-bone interface repair.

Low-intensity pulsed ultrasound (LIPUS) has been proven to accelerate the healing of the tendon-bone interface (TBI), and macrophages are considered to play an important regulatory role. This study was designed to explore the polarization of macrophages during treatment of TBI injury with LIPUS. In a rat model of rotator cuff tear, LIPUS or mock sonication (controls) was administered from 1 week postoperatively. The supraspinatus-supraspinatus tendon-humerus complexes were harvested for further evaluation at different time points for measures such as new bone formation, TBI maturity, ultimate failure load and stiffness, and types of macrophages. In vitro, bone marrow-derived macrophageswere cultured, and polarization was identified after stimulation with or without LIPUS (the LIPUS or control groups, respectively). Two weeks posttreatment, the LIPUS group showed higher bone volume/total volume ratios and better TBI maturity scores. Six weeks posttreatment, the failure load of the LIPUS group was significantly higher than that of the control group. LIPUS also accelerated initial inflammatory macrophage accumulation and facilitated anti-inflammatory macrophage polarization (M2) in the late period. In the in vitro macrophage polarization model, the LIPUS group showed a higher proportion of M2 macrophages and mRNA expression of anti-inflammatory genes than the control group, while there was no significant difference in the proinflammatory macrophages between the two groups. Our observations revealed that macrophage polarization may be a potential mechanism of LIPUS treatment for TBI repair.

Effects of exogenous taurine supplementation on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae in juvenile golden pompano (Trachinotus ovatus) fed with a low-fishmeal diet.

Taurine has various biological functions in fish, playing an essential role in growth, resistance to oxidative stress, and intestine immunity. Here, we evaluated the effects of exogenous taurine added to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus). Our study showed that exogenous taurine supplementation of 1.2% (T3 group) greatly enhanced the weight gain rate and specific growth rate (SGR) of juvenile golden pompano, significantly upregulating growth-related factor expression in the brain and liver, as well as the levels of growth-related parameters in the serum. Polynomial regression analysis using SGR estimated the optimal dietary taurine level for golden pompano at 1.18%. Moderate exogenous taurine also increased the muscular thickness and villus length within the intestine, maintained intestinal physical barrier stability, activated the Nrf2/Keap-1/HO-1 signaling pathway, increased intestinal antioxidant enzyme gene expression and antioxidant enzyme activity in the serum, and upregulated immunoglobulin and complement levels in parallel with declining reactive oxygen species (ROS) levels in the serum. Antioxidant factor expression was also upregulated in the intestine. Furthermore, supplementation suppressed NF-κB signaling and intestinal pro-inflammatory cytokine gene expression, increased anti-inflammatory cytokine gene expression, and improved intestine immunity. Finally, taurine supplementation improved the survival rate of golden pompano challenged with S. agalactiae. Overall, our findings provide additional information and support for the rational use of taurine in healthy aquatic animal farming.

Immunomodulatory effect of IL-1RA in LPS-activated macrophage/dental pulp stem cells co-culture.

Lipopolysaccharides (LPS)-activated human dental pulp stem cells (hDPSCs) and macrophage co-cultures showed downregulated TNF-α secretion that is modulated by hDPSCs through IDO axis, whereas the secretory levels of IL-1β remained unchanged. Therefore, sustained production of IL-1β could contribute to progressive dental pulp inflammation. However, the role of interleukin-1 receptor antagonist (IL-1RA) in downregulating the secretion of IL-1β and TNF-α in LPS-activated M0/M1/M2 macrophage and hDPSCs co-culture has not been studied yet. Therefore, the aim of the present study was to determine the immunomodulatory role of blocking IL-1 receptors in DPSCs macrophage co-culture activated with LPS. Human monocytic cell line THP-1 was polarized to M0, M1 and M2 macrophages and co-cultured with hDPSCs. The viability of the co-cultured cells was assessed by apoptosis assay. Co-cultures were activated with LPS followed by the assessment of gene expression and protein levels of IL-1β and TNF-α with and without IL-1RA blocking via qRT-PCR and cytokine flex assay by flow cytometry. Data from three separate experiments were analysed using one-way anova followed by Tukey's post hoc test and a p-value of <.05 was considered statistically significant. THP-1-derived M0, M1 and M2 macrophages co-cultured with hDPSCs showed spindle and round-shaped cells, with >90% viability when assessed by apoptosis assay. Inflammatory TNF-α and IL-1β profiles in stimulated co-cultures showed upregulated IL-1β, whereas TNF-α was downregulated (p < .05). Anti-inflammatory gene expression levels of IL-10 and TGF-β were downregulated (p < .05). Blocking with IL-1RA resulted in a remarkable decrease in IL-1β at the gene expression and protein production levels whilst TNF-α levels remained low (p < .05). Levels of anti-inflammatory cytokine IL-10 showed no significant difference. Blocking the IL-1 receptor in hDPSCs and macrophage (M0, M1, M2) co-cultures activated with LPS resulted in downregulation of inflammatory cytokines IL-1β and TNF-α. These findings highlight the immunomodulatory effect of IL-1RA in inflammatory conditions of dental pulp infections.

Application of Bone Marrow-Derived Macrophages Combined with Bone Mesenchymal Stem Cells in Dual-Channel Three-Dimensional Bioprinting Scaffolds for Early Immune Regulation and Osteogenic Induction in Rat Calvarial Defects.

The host immune response to biomaterials is critical for determining scaffold fate and bone regeneration outcomes. Three-dimensional (3D) bioprinted scaffolds encapsulated with living cells can improve the inflammatory microenvironment and further accelerate bone repair. Here, we screened and adopted 8% methacrylamidated gelatin (GelMA)/1% methacrylamidated hyaluronic acid (HAMA) as the encapsulation system for rat bone marrow-derived macrophages (BMMs) and 3% Alginate/0.5 mg/mL graphene oxide (GO) as the encapsulation system for rat bone mesenchymal stem cells (BMSCs), thus forming a dual-channel bioprinting scaffold. The 8% GelMA/1% HAMA/3% Alginate/0.5 mg/mL GO (8/1/3/0.5) group could form a scaffold with a stable structure, good mechanical properties, and satisfied biocompatibility. When exploring the crosstalk between BMMs and BMSCs in vitro, we found that BMSCs could promote the polarization of BMMs to M2 type at the early stage, reduce the pro-inflammatory gene expression, and increase anti-inflammatory gene expression; conversely, BMMs can promote the osteogenic differentiation of BMSCs. In addition, in the model of rat calvarial defects, the dual-channel scaffold encapsulated with BMMs and BMSCs was more effective than the single-cell scaffold and the acellular scaffold. The paracrine of BMMs and BMSCs in the biodegradable dual-channel scaffold effectively promoted the M2-type polarization of macrophages in the microenvironment of early bone defects, avoided excessive inflammatory responses, and further promoted bone repair. In conclusion, our findings suggested that using 3D bioprinting to simultaneously encapsulate two primary cells of BMMs and BMSCs in a dual-channel system may be an effective way to promote bone repair from the perspective of early immune regulation and late induction of osteogenesis.

Analysis and identification of key anti-inflammatory molecules in Eerdun Wurile and exploration of their mechanism of action in microglia

Ethnomedicine Eerdun Wurile (EW) can significantly promote poststroke neuro-recovery through modulation of microglia polarization. Fraction 4–6 (F4-6) isolated from EW via serial fractionation inhibits the expression of pro-inflammatory genes in LPS stimulated microglia. However, the key active molecules of F4-6 have not been identified. Herein, we identified alantolactone (Ala) and dehydrodiisoeugenol (Deh) as the active anti-inflammatory components of F4-6 by UPLC-qTof MS analysis. We confirmed that, F4-6, Ala, Deh and mixture of Ala and Deh (Mix) downregulate the expression of several pro-inflammatory genes including Ccl2, Cox2 and Il6 in LPS-treated microglia in a similar pattern. At the same time upregulate the expression of anti-inflammatory genes including Hmox1, Tgfβ, Igf1 and Creb1. Moreover, the conditioned culture media obtained from F4-6 treated microglia significantly enhanced proliferation of N2a cells, and promoted neurite outgrowth possibly through upregulation of Nefh and Dlg4. Mechanistically, F4-6 strongly downregulated the expression of NF-κB p65, while also inhibiting the nuclear translocation of p65, leading to the suppression of transcription of pro-inflammatory genes initiated by NF-κB. Collectively, our data identified and quantified the key chemicals of EW and provide insights into the optimization of the herbal composition for neuroprotection.

Substance P Reduces Infarct Size and Mortality After Ischemic Stroke, Possibly Through the M2 Polarization of Microglia/Macrophages and Neuroprotection in the Ischemic Rat Brain.

Substance-P (SP) is an 11 amino acid neuropeptide that is known to stimulate the peripheral mobilization of bone marrow mesenchymal stem cells and M2 polarization in monocytes/macrophages in a variety of acute and chronic tissue injuries. To examine the role of SP in protection and recovery from acute ischemic brain injury, experimental ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAo) in rats for 1h with subsequent reperfusion. Two injections of SP, immediately and one day post-tMCAo, resulted in approximately threefold lower mortality and 40% less infarct volume than those of saline-treated rats at seven days post-tMCAo. At 4.5h, SP markedly increased CD11b/c+CD163+/CD 206+ cells in the blood, which were concomitantly decreased in the bone marrow, suggesting that SP preferentially mobilized M2-polarized monocytes. After two days, SP increased the expression of neuroprotective and anti-inflammatory genes in the ischemic brain and induced neuronal survival in the brain penumbra. Additionally, SP markedly increased CD68+CD163+ and CD68+CD206+ M2 microglia/macrophages in the ischemic brain during seven days post-tMCAo. Furthermore, SP preserved the blood‒brain barrier in the ischemic brain, which was confirmed by the abundant levels of SMI71+ brain endothelial cells that colocalized with α-SMA+ pericytes. The beneficial effects of SP on functional recovery and tissue preservation were maintained for six weeks. Collectively, SP treatment in the early phase of ischemic stroke markedly suppressed the destructive inflammatory response and improved the microenvironment for tissue protection and repair.

The single-cell landscape of kidney immune cells reveals transcriptional heterogeneity in early diabetic kidney disease

The pathogenesis of diabetic kidney disease (DKD) involves multifactorial processes that converge to initiate and advance the disease. Although DKD is not typically classified as an inflammatory glomerular disease, mounting evidence supports the involvement of kidney inflammation as a key contributor in DKD pathogenesis, particularly through macrophages. However, detailed identification and corresponding phenotypic changes of macrophages in DKD remain poorly understood. To capture the gene expression changes in specific macrophage cell subsets in early DKD, we performed single-cell transcriptomic analysis of CD45-enriched kidney immune cells from type 1 diabetic OVE26 mice at two time points during the disease development. We also undertook a focused analysis of mononuclear phagocytes (macrophages and dendritic cells). Our results show increased resident and infiltrating macrophage subsets in the kidneys of mice with diabetes over time, with heightened expression of pro-inflammatory or anti-inflammatory genes in a subset-specific manner. Further analysis of macrophage polarization states in each subset in the kidneys showed changes consistent with the continuum of activation and differentiation states, with gene expression tending to shift toward undifferentiated phenotypes but with increased M1-like inflammatory phenotypes over time. By deconvolution analysis of RNAseq samples and by immunostaining of biopsies from patients with DKD, we further confirmed a differential expression of select genes in specific macrophage subsets essentially recapitulating the studies in mice. Thus, our study provides a comprehensive analysis of macrophage transcriptomic profiles in early DKD that underscores the dynamic macrophage phenotypes in disease progression.

Effectiveness of new selenium-enriched mutated probiotics in reducing inflammatory effects of piroxicam medication in liver and kidney.

Piroxicam is used to treat the pain, swelling, and stiffness associated with osteoarthritis and rheumatoid arthritis, but it has many side effects, such as hypertension, elevation of liver enzymes, and hepatitis. This study used selenium-enriched probiotics to reduce the side effects of piroxicam on the liver and kidney tissues and functions. Forty-eight male albino mice were randomly assigned to control, piroxicam (P), piroxicam plus selenium-enriched Lactobacillus plantarum PSe40/60/1 (P + SP), piroxicam plus selenium-enriched Bifidobacterium longum BSe50/20/1 (P + SB), selenium-enriched L. plantarum PSe40/60/1 (SP), and selenium-enriched B. longum BSe50/20/1 (SB) groups. In this study, the function of the liver and kidney was biochemically determined; the histopathology of the liver and kidney tissues was microscopically examined and the expression of inflammatory and anti-inflammatory genes in liver and kidney tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Liver and kidney functions were significantly reduced in the piroxicam group compared with control. Liver and kidney tissues were damaged in the piroxicam group while they appeared more or less normal in the SB group. The expression of inflammatory genes was significantly up-regulated in the liver and kidney tissues of the piroxicam group compared to the control group. The expression of anti-inflammatory genes was significantly down-regulated in the liver and kidney of the piroxicam group and up-regulated in the liver and kidney of the SB group compared to the control group. Therefore, these mutated strains of probiotics were useful in reducing the side effects of the piroxicam drug on the liver and kidney.