Bifidobacterium animalis subsp. lactis LKM512 Alleviates Inflammatory Bowel Disease in Larval Zebrafish by Reshaping Microbiota.

Inflammatory bowel disease (IBD) is a worldwide issue, and the increased incidence has brought a heavy burden to patients and society. Gut microbiota is involved in the pathogenesis of IBD, and targeting the microbiota, such as probiotics, has emerged as a potential therapy for the treatment of IBD. Here, the effect of Bifidobacterium animalis ssp. lactis LKM512 (LKM512), an anti-aging probiotic, on dextran sulfate sodium salt (DSS)-induced IBD in larval zebrafish was determined. Supplementation of LKM512 promoted the survival rate of the larvae, together with increased locomotor activities and body length. In addition, LKM512 treatment enhanced mucus secretion and alleviated intestinal injury, and these results were associated with the upregulation of mucin-related and downregulation of inflammatory markers. Moreover, LKM512 increased the diversity of the microbiota and ameliorated the dysbiosis by increasing the abundance of Bacteroidetes and Firmicutes and reducing the abundance of Proteobacteria. Specifically, the abundance of beneficial bacteria, including the short-chain fatty-acids (SCFAs)-producing genera Lachnospiraceae_NK4A136_group, Muribaculaceae, and Alloprevotella, was increased by LKM512, while the abundance of harmful genera, such as Pseudomonas, Halomonas, and Escherichia-Shigella, was reduced by LKM512. Consistent with these findings, the microbial functions related to metabolism were partly reversed by LKM512, and importantly, fermentation of short-chain fatty acids-related functions were enhanced by LKM512. Therefore, LKM512 might be one potential probiotic for the prevention and treatment of IBD, and further studies that clarify the mechanism of LKM512 would promote the application of LKM512.

Enterorenal crosstalks in diabetic nephropathy and novel therapeutics targeting the gut microbiota.

The role of gut-kidney crosstalk in the progression of diabetic nephropathy (DN) is receiving increasing concern. On one hand, the decline in renal function increases circulating uremic toxins and affects the composition and function of gut microbiota. On the other hand, intestinal dysbiosis destroys the epithelial barrier, leading to increased exposure to endotoxins, thereby exacerbating kidney damage by inducing systemic inflammation. Dietary inventions, such as higher fiber intake, prebiotics, probiotics, postbiotics, fecal microbial transplantation (FMT), and engineering bacteria and phages, are potential microbiota-based therapies for DN. Furthermore, novel diabetic agents, such as glucagon-like peptide-1 (GLP-1) receptor agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, and sodium-dependent glucose transporter-2 (SGLT-2) inhibitors, may affect the progression of DN partly through gut microbiota. In the current review, we mainly summarize the evidence concerning the gut-kidney axis in the advancement of DN and discuss therapies targeting the gut microbiota, expecting to provide new insight into the clinical treatment of DN.

Anti-diabetic effects of astaxanthin on an STZ-induced diabetic model in rats.

Type 2 diabetes mellitus (T2DM), which is characterized by insulin resistance and relative insulin insufficiency, has become the most common chronic metabolic disease threatening global health. The preferred therapies for T2DM include lifestyle interventions and the use of anti-diabetic drugs. However, considering their adverse reactions, it is important to find a low-toxicity and effective functional food or drug for diabetes prevention and treatment. Astaxanthin is a potent antioxidant carotenoid found in marine organisms has been reported to prevent diet-induced insulin resistance and hepatic steatosis. To investigate the anti-diabetic effects of astaxanthin, male Wistar rats were fed a high-energy diet for 4 weeks, followed by a low dose streptozotocin (STZ) injection to induce the diabetes model, and the rats were then fed an astaxanthin-containing diet for another 3 weeks. Astaxanthin significantly decreased blood glucose and total cholesterol (TC) levels, and increased blood levels of high density lipoprotein cholesterol (HDL-C) in STZ-induced diabetic rats in a dose dependent manner. These results were associated with increased expression of insulin sensitivity related genes (adiponectin, adipoR1, and adipoR2) in vivo, thereby attenuating STZ-induced diabetes. In addition, we also compared the anti-diabetic effects of astaxanthin and monacolin K, which has been reported to downregulate hyperlipidemia and hyperglycemia. The results revealed that astaxanthin and monacolin K showed similar anti-diabetic effects in STZ-induced diabetic rats. Therefore, astaxanthin may be developed as an anti-diabetic agent in the future.

Neuroprotective effects of ProBeptigen/CMI-168 on aging-induced cognitive decline and neuroinflammation in mice: a comparison with essence of chicken.

Neuroinflammation and cognitive decline are the key pathological features in aging that bring detrimental impacts upon quality of life. However, there is no effective anti-aging pharmacological therapy thus far. Dietary supplements in particular essence of chicken (EC) has been found to be an effective remedy for alleviating mental stress and improving memory. In addition, a novel hydrolyzed chicken extract, ProBeptigen/CMI-168 (PB), showed beneficial effects on cognitive ability. However, the antiaging effect and possible mechanism of PB and EC are still unknown. Here, we investigated the antiaging effects of PB and EC on hippocampus-related cognitive decline and neuroinflammation in aged mice. PB and EC were administered for 16 weeks in 10-month-old mice. Both PB and EC treatments ameliorated age-related deterioration of learning and memory, and attenuated oxidative stress and inflammation in the hippocampus. These results were associated with decreased inflammatory cytokine levels and increased neurotransmitter levels in the hippocampus. The overall effects of improving aging-induced cognitive decline were more robust in PB-treated mice, while EC was effective in decreasing oxidative stress and inflammation. Moreover, alterations in the diversity and composition of the gut microbiota in aged mice were also regulated by both PB and EC, which induced distinguished features in the gut microbiota and their related functions. This study showed that PB exerts neuroprotective effects in aged mice, the mechanism of which might be different from that of EC. Therefore, PB has a potential as dietary supplement for ameliorating cognitive dysfunction and neuroinflammation in elderly individuals.

Pirfenidone prevents and reverses hepatic insulin resistance and steatohepatitis by polarizing M2 macrophages.

Nonalcoholic steatohepatitis (NASH) is associated with lipotoxic liver injury, leading to insulin resistance, inflammation, and fibrosis. Despite its increased global incidence, very few promising treatments for NASH are available. Pirfenidone is an antifibrotic agent used to treat pulmonary fibrosis; it suppresses the pulmonary influx of T cells and macrophages. Here, we investigated the effect of pirfenidone in a mouse model of lipotoxicity-induced NASH via a high-cholesterol and high-fat diet. After 12 weeks of feeding, pirfenidone administration attenuated excessive hepatic lipid accumulation and peroxidation by reducing the expression of genes related to lipogenesis and fatty acid synthesis and enhancing the expression of those related to fatty acid oxidation. Flow cytometry indicated that pirfenidone reduced the number of total hepatic macrophages, particularly CD11c+CD206-(M1)-type macrophages, increased the number of CD11c-CD206+(M2)-type macrophages, and subsequently reduced T-cell numbers, which helped improve insulin resistance and steatohepatitis. Moreover, pirfenidone downregulated the lipopolysaccharide (LPS)-induced mRNA expression of M1 marker genes and upregulated IL-4-induced M2 marker genes in a dose-dependent manner in RAW264.7 macrophages. Importantly, pirfenidone reversed insulin resistance, hepatic inflammation, and fibrosis in mice with pre-existing NASH. These findings suggest that pirfenidone is a potential candidate for the treatment of NASH.

Protective effects of astaxanthin on a combination of D-galactose and jet lag-induced aging model in mice.

Oxidative stress caused free radical and mitochondrial damage plays a critical role in the progression of aging and age-related damage at the cellular and tissue levels. Antioxidant supplementation has received growing attention and the effects of antioxidant on aging are increasingly assessed in both animal and human studies. However, additional and more promising treatments that contribute to the expansion of anti-aging therapies are needed. Astaxanthin, a super antioxidant carotenoid and free radical scavenger, inhibits lipid peroxidation more potently than vitamin E. In the present study, we investigated the preventative effects of astaxanthin on aging using an accelerated aging model: mice chronically treated with a combination of D-galactose and jet lag. After 6 weeks of treatment, astaxanthin administration tended to protect the liver weight loss in aged mice. It is probably by upregulating the mRNA expression of galactose-1-phosphate uridyltransferase, which contribute to the enhancement of D-galactose metabolism. Astaxanthin supplementation also improved muscle endurance of aged mice in a swimming test. These results were associated with reduced oxidative stress in serum and increased anti-oxidative enzymes activities and mRNA expression in vivo. Moreover, astaxanthin reversed the dysregulation of aging-related gene expression caused by the combination of D-galactose and jet lag in the liver and kidney of mice. In conclusion, astaxanthin prevents liver weight loss, ameliorates locomotive muscular function, exerts significant anti-aging effects by reducing oxidative stress and improving the expression of age-related genes in D-galactose and jet lag-induced aging model.

Pilose antler polypeptides ameliorates hypoxic-ischemic encephalopathy by activated neurotrophic factors and SDF1/CXCR4 axis in rats.

Hypoxic-ischemic encephalopathy (HIE) is a complex condition which is associated with high mortality and morbidity. However, few promising treatments for HIE exist. In the present study, the central objective was to identify the therapeutic effect of pilose antler polypeptides (PAP) on HIE in rats. Sprague-Dawley (SD) rats (14 days old) were used and divided into three groups, including control group, hypoxic-ischemia (HI) group and PAP group. After 21 days of treatment, locomotor activity was improved in PAP-treated rats, brain atrophy was decreased and cerebral edema was mitigated to some extent. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis indicated that PAP administration decreased the expressions of inflammatory cytokines and apoptosis genes in hippocampus compared with HI group. Furthermore, the mRNA expressions of genes related to neurotrophic factors were significantly increased in the hippocampus. In addition, the expressions of oxidative stress markers were all down-regulated after PAP administration. Moreover, PAP up-regulated both the mRNA and protein levels of SDF1 and CXCR4, which may activate the SDF1/CXCR4 axis to moderate brain injury. These results suggest that PAP may be potentially used in the treatment of HIE.

An individual 12-h shift of the light-dark cycle alters the pancreatic and duodenal circadian rhythm and digestive function.

In mammals, behavioral and physiological rhythms are controlled by circadian clocks which are entrained by environmental light and food signals. However, how the environmental cues affect digestive tract's circadian clock remains poorly understood. Therefore, in order to elucidate the effect of light cue on the resetting of the peripheral clocks, we investigated the expressions of clock genes (Bmal1, Cry1, Rev-erbα, Per1, and Per2) and digestive function genes (Cck, Cck-1r, Sct, Sctr, and Ctrb1) in the pancreas and duodenum of rats after the light-dark (LD) cycle reversal for 7 days. We found that both the clock genes and digestive function genes exhibited a clear and similar daily rhythmicity in the pancreas and duodenum of rats. After reversal of the LD cycle for 7 days, the expressions of clock genes in pancreas, including Bmal1, Cry1, and Rev-erbα were affected; whereas the expression of Per1 gene failed to fit the cosine wave. However, in the duodenum the shifted genes were Bmal1, Rev-erbα, and Per2; in parallel, the Per1 gene expression also lost its circadian rhythm by reversal of the LD cycle. Therefore, the acrophases of the clock genes were shifted in a tissue- and gene-specific manner. Furthermore, the profiles of the digestive function genes, including Sctr and Ctrb1, were also affected by changes in LD cycle. These observations suggest that the mechanisms underlying the pancreatic and duodenal clocks are distinct, and there may be a potential linkage between the circadian clock system and the digestive system.

Reply to the Letter to the Editor by Li et al.: Bioinformatics Analysis in Mice with Diet-Induced Nonalcoholic Steatohepatitis Treated with Astaxanthin and Vitamin E.

n/a.

Hepatic Transcriptome Profiles of Mice with Diet-Induced Nonalcoholic Steatohepatitis Treated with Astaxanthin and Vitamin E.

Astaxanthin alleviates hepatic lipid accumulation and peroxidation, inflammation, and fibrosis in mice with high-cholesterol, high-cholate, and high-fat (CL) diet-induced nonalcoholic steatohepatitis (NASH) [...].

Micronutrient Antioxidants and Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the most important chronic liver diseases worldwide and has garnered increasing attention in recent decades. NAFLD is characterized by a wide range of liver changes, from simple steatosis to nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. The blurred pathogenesis of NAFLD is very complicated and involves lipid accumulation, insulin resistance, inflammation, and fibrogenesis. NAFLD is closely associated with complications such as obesity, diabetes, steatohepatitis, and liver fibrosis. During the progression of NAFLD, reactive oxygen species (ROS) are activated and induce oxidative stress. Recent attempts at establishing effective NAFLD therapy have identified potential micronutrient antioxidants that may reduce the accumulation of ROS and finally ameliorate the disease. In this review, we present the molecular mechanisms involved in the pathogenesis of NAFLD and introduce some dietary antioxidants that may be used to prevent or cure NAFLD, such as vitamin D, E, and astaxanthin.

Spermidine activates adipose tissue thermogenesis through autophagy and fibroblast growth factor 21.

Spermidine exerts protective roles in obesity, while the mechanism of spermidine in adipose tissue thermogenesis remains unclear. The present study first investigated the effect of spermidine on cold-stimulation and ß3-adrenoceptor agonist-induced thermogenesis in lean and high-fat diet-induced obese mice. Next, the role of spermidine on glucose and lipid metabolism in different types of adipose tissue was determined. Here, we found that spermidine supplementation did not affect cold-stimulated thermogenesis in lean mice, while significantly promoting the activation of adipose tissue thermogenesis under cold stimulation and ß3-adrenergic receptor agonist treatment in obese mice. Spermidine treatment markedly enhanced glucose and lipid metabolism in adipose tissues, and these results were associated with the activated autophagy pathway. Moreover, spermidine up-regulated fibroblast growth factor 21 (FGF21) signaling and its downstream pathway, including PI3K/AKT and AMPK pathways in vivo and in vitro. Knockdown of Fgf21 or inhibition of PI3K/AKT and AMPK pathways in brown adipocytes abolished the thermogenesis-promoting effect of spermidine, suggesting that the effect of spermidine on adipose tissue thermogenesis might be regulated by FGF21 signaling via the PI3K/AKT and AMPK pathways. The present study provides new insight into the mechanism of spermidine on obesity and its metabolic complications, thereby laying a theoretical basis for the clinical application of spermidine.

DPP-4 inhibition by linagliptin ameliorates age-related mild cognitive impairment by regulating microglia polarization in mice.

Extensive preclinical evidence demonstrates a causative link between insulin signaling dysfunction and the pathogenesis of Alzheimer's disease (AD), and diabetic drugs may represent a promising approach to fighting AD. However, it remains to be determined which antidiabetic drugs are more effective in preventing cognitive impairment. Thus, the present study investigated the effect of dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin on cognitive impairment in middle-aged mice by comparing it with the effect of metformin. We found that DPP-4 activity increased in the hippocampus of middle-aged mice, and DPP-4 was mainly expressed by microglia rather than astrocytes and oligodendrocytes. DPP-4 directly regulated M1/M2 microglia polarization following LPS or IL-4 stimulation, while DPP-4 inhibitor, linagliptin, suppressed M1-polarized activation and induced M2-polarized activation. Both linagliptin and metformin enhanced cognitive ability, increased hippocampal synaptic plasticity and neurogenesis, and decreased age-related oxidative stress and inflammation by regulating microglia polarization in the hippocampus of middle-aged mice. The combination of linagliptin and metformin showed a maximum protective effect compared to the individual drugs alone. Loss of macrophage inflammatory protein-1α (MIP-1α), a DPP-4 substrate, abrogated the cognitive protection and anti-inflammation effects of linagliptin. Therefore, the current investigation exhibits a potential utility for DPP-4 inhibition in attenuating microglia-mediated inflammation and preventing mild cognitive impairment (MCI) in middle-aged mice, and the effect was partly mediated by MIP-1α.

Multigenerational Adaptation Can Enhance the Pathogen Resistance of Plants via Changes in Rhizosphere Microbial Community Assembly.

Plants withstand pathogen attacks by recruiting beneficial bacteria to the rhizosphere and passing their legacy on to the next generation. However, the underlying mechanisms involved in this process remain unclear. In our study, we combined microbiomic and transcriptomic analyses to reveal how the rhizosphere microbiome assembled through multiple generations and defense-related genes expressed in Arabidopsis thaliana under pathogen attack stress. Our results showed that continuous exposure to the pathogen Pseudomonas syringae pv tomato DC3000 led to improved growth and increased disease resistance in a third generation of rps2 mutant Arabidopsis thaliana. It could be attributed to the enrichment of specific rhizosphere bacteria, such as Bacillus and Bacteroides. Pathways associated with plant immunity and growth in A. thaliana, such as MAPK signaling pathways, phytohormone signal transduction, ABC transporter proteins, and flavonoid biosynthesis, were activated under the influence of rhizosphere bacterial communities. Our findings provide a scientific basis for explaining the relationship between beneficial microbes and defense-related gene expression. Understanding microbial communities and the mechanisms involved in plant responses to disease can contribute to better plant management and reduction of pesticide use.

Time-dependent effect of REV-ERBα agonist SR9009 on nonalcoholic steatohepatitis and gut microbiota in mice.

The circadian clock is involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), and the target pathways of many NASH candidate drugs are controlled by the circadian clock. However, the application of chronopharmacology in NASH is little considered currently. Here, the time-dependent effect of REV-ERBα agonist SR9009 on diet-induced NASH and microbiota was investigated. C57BL/6J mice were fed a high-cholesterol and high-fat diet (CL) for 12 weeks to induce NASH and then treated with SR9009 either at Zeitgeber time 0 (ZT0) or ZT12 for another 6 weeks. Pharmacological activation of REV-ERBα by SR9009 alleviated hepatic steatosis, insulin resistance, liver inflammation, and fibrosis in CL diet-induced NASH mice. These effects were accompanied by improved gut barrier function and altered microbial composition and function in NASH mice, and the effect tended to be stronger when SR9009 was injected at ZT0. Moreover, SR9009 treatment at different time points resulted in a marked difference in the composition of the microbiota, with a stronger effect on the enrichment of beneficial bacteria and the diminishment of harmful bacteria when SR9009 was administrated at ZT0. Therefore, the time-dependent effect of REV-ERBα agonist on NASH was partly associated with the microbiota, highlighting the potential role of microbiota in the chronopharmacology of NASH and the possibility of discovering new therapeutic strategies for NASH.

Corrigendum: Depiction of immune heterogeneity of peripheral blood from patients with type II diabetic nephropathy based on mass cytometry.

[This corrects the article DOI: 10.3389/fendo.2022.1018608.].

Bifidobacterium and Lactobacillus improve inflammatory bowel disease in zebrafish of different ages by regulating the intestinal mucosal barrier and microbiota.

AIMS: Inflammatory bowel disease (IBD) patients are accompanied by impaired intestinal barrier integrity and gut microbiota dysbiosis. Strategies targeting the gut microbiota are potential therapies for preventing and ameliorating IBD. MAIN METHODS: The potential roles of two probiotic stains, Bifidobacterium longum BL986 (BL986) and Lactobacillus casei LC122 (LC122), on intestinal mucosal barrier function and microbiota in IBD zebrafish of different ages were investigated. KEY FINDINGS: BL986 and LC122 treatment promoted the development and increased the microbiota diversity in larval zebrafish. Both probiotic treatment ameliorated mortality, promoted intestinal mucus secretion, and reduced the expression of inflammatory markers, thereby improving intestinal mucosal barrier function in dextran sulfate sodium salt (DSS)-induced ulcerative colitis (UC) and 2,4,6-trinitro-benzenesulfonicacid (TNBS)-induced Crohn's disease (CD) models in zebrafish. Moreover, the composition and function of microbiota were altered in IBD zebrafish, and probiotics treatment displayed prominent microbiota features. BL986 was more potent in the DSS-induced UC model, and increased the abundance of Faecalibaculum and butyric acid levels. LC122 exerted better protection against TNBS-induced CD, and increased the abundance of Enhydrobacter and acetic acid levels. Furthermore, the effect of probiotics was stronger in larval and aged zebrafish. CONCLUSION: The impact of probiotics on IBD might differ from the subtypes of IBD and the age of the zebrafish, suggesting the types of disease and age should be taken into full consideration during the practical usage of probiotics.

Enhanced effect of daytime restricted feeding on the circadian rhythm of streptozotocin-induced type 2 diabetic rats.

There is increasing awareness of the link between impaired circadian clocks and multiple metabolic diseases. However, the impairment of the circadian clock by type 2 diabetes has not been fully elucidated. To understand whether and how the function of circadian clock is impaired under the diabetic condition, we examined not only the expression of circadian genes in the heart and pineal gland but also the behavioral rhythm of type 2 diabetic and control rats in both the nighttime restricted feeding (NRF) and daytime restricted feeding (DRF) conditions. In the NRF condition, the circadian expression of clock genes in the heart and pineal gland was conserved in the diabetic rats, being similar to that in the control rats. DRF shifted the circadian phases of peripheral clock genes more efficiently in the diabetic rats than those in the control rats. Moreover, the activity rhythm of rats in the diabetic group was completely shifted from the dark phase to the light phase after 5 days of DRF treatment, whereas the activity rhythm of rats in the control group was still under the control of the suprachiasmatic nucleus (SCN) after the same DRF treatment. Furthermore, the serum glucose rhythm of type 2 diabetic rats was also shifted and controlled by the external feeding schedule, ignoring the SCN rhythm. Therefore, DRF shows stronger effect on the reentrainment of circadian rhythm in the type 2 diabetic rats, suggesting that the circadian system in diabetes is unstable and more easily shifted by feeding stimuli.

Bifidobacterium animalis subsp. lactis lkm512 Attenuates Obesity-Associated Inflammation and Insulin Resistance Through the Modification of Gut Microbiota in High-Fat Diet-Induced Obese Mice.

SCOPE: The impacts of longevity-promoting probiotic Bifidobacterium animalis subsp. lactis LKM512 (LKM512) on metabolic disease remain unclear. Here, the authors aim to explore the potential of LKM512 on the host physiological function and gut microbiota in high-fat diet-induced obese mice. METHODS AND RESULTS: LKM512 are orally administrated for 12 weeks, and the effects of LKM 512 on systemic inflammation and insulin resistance, as well as gut microbiota, are investigated in high-fat (HF) diet-induced obese mice. LKM512 supplementation ameliorates hepatic lipid accumulation, attenuates hepatic and adipose tissue inflammation, and improves intestinal barrier function. These results are associated with improved insulin sensitivity and metabolic endotoxemia. Furthermore, the colonization of LKM512 induces an increase in polyamine metabolism and production, together with significant alternations in the composition and function of gut microbiota in obese mice, which are correlated with these improved metabolic phenotypes in the host. CONCLUSION: The probiotic strain LKM512 may become a promising strategy to improve obesity and related metabolic disorders.

Depiction of immune heterogeneity of peripheral blood from patients with type II diabetic nephropathy based on mass cytometry.

Diabetic nephropathy (DN) is the most prominent cause of chronic kidney disease and end-stage renal failure. However, the pathophysiology of DN, especially the risk factors for early onset remains elusive. Increasing evidence has revealed the role of the innate immune system in developing DN, but relatively little is known about early immunological change that proceeds from overt DN. Herein, this work aims to investigate the immune-driven pathogenesis of DN using mass cytometry (CyTOF). The peripheral blood mononuclear lymphocytes (PBMC) from 6 patients with early-stage nephropathy and 7 type II diabetes patients without nephropathy were employed in the CyTOF test. A panel that contains 38 lineage markers was designed to monitor immune protein levels in PBMC. The unsupervised clustering analysis was performed to profile the proportion of individual cells. t-Distributed Stochastic Neighbor Embedding (t-SNE) was used to visualize the differences in DN patients' immune phenotypes. Comprehensive immune profiling revealed substantial immune system alterations in the early onset of DN, including the significant decline of B cells and the marked increase of monocytes. The level of CXCR3 was dramatically reduced in the different immune cellular subsets. The CyTOF data classified the fine-grained differential immune cell subsets in the early stage of DN. Innovatively, we identified several significant changed T cells, B cell, and monocyte subgroups in the early-stage DN associated with several potential biomarkers for developing DN, such as CTLA-4, CXCR3, PD-1, CD39, CCR4, and HLA-DR. Correlation analysis further demonstrated the robust relationship between above immune cell biomarkers and clinical parameters in the DN patients. Therefore, we provided a convincible view of understanding the immune-driven early pathogenesis of DN. Our findings exhibited that patients with DN are more susceptible to immune system disorders. The classification of fine-grained immune cell subsets in this present research might provide novel targets for the immunotherapy of DN.