Atg5 deficiency in basophils alleviates colon inflammation

We extracted the bone marrow from Atg5 knockout mice (Atg5 flox/flox Cre±, Atg5−/−), induced basophils using recombinant mouse IL-3, and further improved their purity using flow cytometry. Basophils were sorted using the markers FcεRIα+ CD117− CD11c− CD49b+ (Fig. 1A). After sorting, basophil purity, as confirmed by flow cytometry, reached 98.7% (Fig. 1B). Western blotting results indicated a significant decrease in Atg5 expression in the knockout group compared to that in the wild-type group (Fig. 1C). In addition, we found that the expression level of inflammatory cytokines (IL-4, IL-6, and IL-13) in the knockout group was significantly decreased compared to the wild-type group (Fig. 1D). We evaluated the effects of basophil depletion and adoptive transfer levels in MRL/lpr mice. The experimental design and timelines are shown in Fig. S1. The results showed that one-time basophil depletion led to a significant decrease in the percentage of basophils in the peripheral blood of MRL/lpr mice and remained low for over 10 days (Fig. S2). Meanwhile, one-time basophil transfer led to a significant increase in the percentage of basophils in the peripheral blood, which persisted for over 10 days (Fig. S3). To investigate the impact of basophil autophagy deficiency on lupus progression, MRL/lpr mice were divided into three groups—a control, an Atg5+/+ basophil adoptive transfer, and an Atg5−/− basophil adoptive transfer groups. Additionally, MRL/MpJ mice served as a separate control group for the MRL/lpr mice. Both the control group and the MRL/MpJ mice received tail vein injections of normal saline. The Atg5+/+ basophil adoptive transfer group underwent basophil depletion, followed by the transfer of Atg5+/+ basophils at 6, 8, 10, 12, and 14 weeks. Similarly, the Atg5−/− basophil adoptive transfer group underwent basophil depletion, followed by the transfer of Atg5−/− basophils at the same intervals.

Fig. 1

Atg5 deficiency in basophils improves colon inflammation in MRL/lpr mice. A Flow cytometry sorting of bone marrow-induced basophils. B Flow cytometry verification of basophil purity. C Western blotting analysis of Atg5 levels in basophils. D qPCR analysis of IL-4, IL-6, and IL-13 levels in basophils. E Morphological observation of colonic tissues in mice. F Statistical analysis of colonic tissues in mice. G Hematoxylin and Eosin (H&E) staining for assessing colonic inflammation in mice. H Statistical analysis of colonic inflammation in mice. *P < 0.05; ** P < 0.01; *** P < 0.001

All mice were euthanized at 16 weeks of age to assess disease progression. Colon length was found to be significantly longer in the Atg5−/− group than in the control group (Fig. 1E, F). Colonic pathological staining revealed significantly fewer inflammatory cells in the Atg5−/− group than in the control group, suggesting that Atg5−/− basophils ameliorate colonic inflammation in mice with lupus (Fig. 1G, H).

Atg5 deficiency in basophils reduces autoantibody levels, inflammatory cytokine expression, and improves metabolism

Autoantibodies and inflammatory cytokines are key metrics for assessing lupus severity [25]. Autoantibody detection revealed significantly lower levels of anti-nuclear and anti-dsDNA antibodies in the Atg5−/− basophil adoptive transfer group than that in the control and Atg5+/+ basophil adoptive transfer groups (Fig. 2A, B). Subsequent cytokine analysis showed that the TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-13, and IL-17 levels were lower in the Atg5−/− basophils adoptive transfer group compared to that in the control and Atg5+/+ basophil adoptive transfer groups (Fig. 2C–J). These findings suggest that Atg5 deficiency in basophils reduces in vivo autoantibody and inflammatory cytokine production.

Fig. 2

Atg5 deficiency in basophils decreases autoantibody expression, reduces inflammatory cytokine levels, and improves metabolism in MRL/lpr mice. A, B Statistical analysis of plasma anti-nuclear antibodies and anti-dsDNA antibodies, respectively. C–J Statistical analysis of plasma cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)−1β, IL-2, IL-4, IL-6, IL-13, and IL-17. K–T Levels of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), α-hydroxybutyrate dehydrogenase (α-HBDH), cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), bile acids, and A/G ratio. *P < 0.05; ** P < 0.01

To explore the metabolic changes in SLE, we measured the plasma levels of enzymes, proteins, and lipids. The results indicated significantly lower plasma levels of ALT, AST, and LDH in the Atg5−/− basophil adoptive transfer group than in the control and the Atg5+/+ basophil adoptive transfer groups (Fig. 2K–M), reflecting an improved liver metabolism. Plasma CK levels showed a decreasing trend (Fig. 2N), indicating that the damage to the muscle tissue of the mice was alleviated. These findings suggest that basophil autophagy deficiency inhibits amino acid metabolism. α-HBDH has been reported to be closely associated with SLE and may serve as a predictive marker [26]. However, our results showed no significant changes in its levels (Fig. 2O). Our results indicated decreased plasma levels of cholesterol, LDL, and bile acids (Fig. 2P, R, S); however, HDL and the A/G ratio levels increased in the Atg5−/− basophil adoptive transfer group compared to those in the control and Atg5+/+ basophil adoptive transfer groups (Fig. 2Q, T). These findings suggest that an Atg5 deficiency in basophils improves metabolism in vivo.

Atg5 deficiency in basophils improves gut microbiota dysbiosis

We collected fecal samples from the control (Atg5+/+) and Atg5−/− basophil transplanted MRL/lpr mice for metagenomic sequencing to examine the impact of Atg5 deficiency in basophils on the gut microbiota composition in MRL/lpr mice. Principal component analysis revealed significant separation at the genus and species levels between the control and Atg5−/− groups (Fig. 3A–D).

Fig. 3

Metagenomic analysis of fecal samples from the control (Atg5+/+) and Atg5−/− basophil transplanted MRL/lpr mice. A, B Principal component analysis. C, D Taxonomic abundance plots. E–L Statistical analysis using Metastats at the genus level in control and Atg5−/− groups of mice. (M–V) Statistical analysis using Metastats at the species level in control and Atg5−/− groups of mice. *P < 0.05; ** P < 0.01

At the genus level, Ligilactobacillus and Faecalitalea were more abundant in Atg5−/− mice than that in control mice (Fig. 3E–L). At the species level, the abundance of Bacteroides_sp_CAG_927, Bacteroides_pyogenes, Bacteroides_ihuae, Phocaeicola_plebeius, Phocaeicola_salanitronis, Phocaeicola_barnesiae, Barnesiella_sp_CU968, Barnesiella_sp_An22, Duncaniella_sp_C9, and Duncaniella dubosii decreased in the Atg5−/− group compared to that in the control mice (Fig. 3M–V). The linear discriminant analysis confirmed similar trends. In the control group, Bacteroides_sp_CAG_927, Barnesiella_sp_CU968, and Duncaniella dubosii were significantly more abundant. Conversely, the abundance of Ligilactobacillus and Faecalitalea was significantly increased following basophil transplantation in Atg5-knockout mice (Fig. 4A, B). We analyzed the species-level changes in fecal metagenome sequencing results to identify potential microbial markers of SLE.

Fig. 4

Linear discriminant analysis Effect Size (LEfSe) analysis of fecal metagenomics in the control (Atg5+/+) and Atg5−/− basophil transplanted MRL/lpr mice. (A, B) Cladograms and bar plots generated via LEfSe identify significantly different microbiota taxa between groups (Linear discriminant analysis > 2.5, P < 0.05). The diameter of each node in the cladograms is proportional to the relative abundance of the taxonomic unit. Red indicates taxa enriched in the Atg5−/− group and green indicates taxa enriched in the control group

These results indicate that transplanting basophils with Atg5 deficiency can correct the gut microbiota imbalance in MRL/lpr mice. This procedure increased the abundance of beneficial bacteria such as Ligilactobacillus and Faecalitalea while significantly reducing the abundance of potentially pathogenic bacteria, including Phocaeicola_salanitronis, Bacteroides_sp_CAG_927, Barnesiella_sp_CU968, and Duncaniella dubosii.

Atg5 deficiency in basophils affects gut microbiota function

To assess the effects of transplanting basophils with Atg5 deficiency on gut microbiota function, we annotated our fecal metagenomics sequencing data using the KEGG database. Pathway profiles were generated for microbial communities at each sampling site. A total of 308,190 annotated Unigenes were mapped to canonical KEGG reference pathways, distributed across six signaling cascades (Fig. 5A). These genes were categorized into several groups: “Carbohydrate metabolism” (28,140 members), “Amino acid metabolism” (18,939 members), “Energy metabolism” (12,049 members), “Endocrine system” (1,895 members), “Endocrine and metabolic disease” (1,046 members), “Immune system” (823 members), and “Immune disease” (181 members). We observed significant upregulation in the “Starch and sucrose metabolism (map00500),” “Fructose and mannose metabolism (map00051),” and “Glycerolipid metabolism (map00561)” pathways in the Atg5−/− group compared to that in the control group. Conversely, the pathways for “Alanine, aspartate, and glutamate metabolism (map00250),” “Oxidative phosphorylation (map00190),” and “arginine biosynthesis (map00220)” were significantly downregulated in the Atg5−/− group (Fig. 5B).

Fig. 5

Atg5 deficiency in basophils affects the gut microbiota function in MRL/lpr mice. A Predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classification of metagenomic Unigenes. The numbers on the bar chart represent the count of annotated Unigenes. B Predicted KEGG level 3 functional classification. C Network diagram of the correlation between different bacteria and KEGG pathways. Red lines indicate positive correlations, whereas blue lines indicate negative correlations (Spearman's rank correlation test). D–G Distribution of microbiota species and disease severity indexes in redundancy analysis (RDA). Arrow length indicates the magnitude of the correlation between disease severity indexes and sample distribution, with longer lines indicating greater correlation. The acute angle between arrow lines and axes indicated positive correlation, whereas the obtuse angle indicates negative correlation. H, I Spearman's rank correlation coefficient heatmap. H Correlation analysis between microbiota species abundance and specific metabolites. I Correlation analysis between specific indexes and lupus disease severity indexes (plasma levels of autoantibodies, pro-inflammatory cytokines, enzymes, proteins, lipids). R-values are represented by different colors, with red squares indicating a positive correlation, blue squares indicating a negative correlation, and darker shades indicating a stronger positive/negative correlation. Numbers in the heatmap cells represent P-values. *P < 0.05, **P < 0.01

In the Atg5−/− group, significantly enriched bacteria such as Ligilactobacillus and Faecalitalea positively correlated with the “Starch and sucrose metabolism (map00500),” “Fructose and mannose metabolism (map00051),” and “Glycerolipid metabolism (map00561)” pathways. These bacteria negatively correlated with the “Alanine, aspartate and glutamate metabolism (map00250),” “Oxidative phosphorylation (map00190),” and “arginine biosynthesis (map00220)” pathways. Conversely, in the control group, bacteria such as Bacteroides_sp_CAG_927, Bacteroides_pyogenes, Bacteroides_ihuae, and others showed positive correlations with “Alanine, aspartate, and glutamate metabolism (map00250),” “Oxidative phosphorylation (map00190),” and “arginine biosynthesis (map00220)” pathways. These bacteria also negatively correlated with “Starch and sucrose metabolism (map00500),” “Fructose and mannose metabolism (map00051),” and “Glycerolipid metabolism (map00561)” pathways (Fig. 5C).

To explore the role of gut microbiota in lupus pathogenesis, we conducted Spearman's correlation analyses between various gut microbiota profiles and lupus severity indices, including plasma levels of autoantibodies, inflammatory cytokines, and other metabolic markers. Elevated levels of Ligilactobacillus and Faecalitalea were negatively correlated with plasma autoantibodies (anti-dsDNA, anti-nuclear), inflammatory cytokines (TNF-α, IFN-γ, IL-1β), and metabolic indices (HDL, albumin, A/G ratio). Conversely, Bacteroides_sp_CAG_927, Bacteroides_pyogenes, and other bacteria showed positive correlations with plasma autoantibodies, inflammatory cytokines, and metabolic indices such as AST, ALT, CK, and LDL (Fig. 5D–I). Consequently, transplanting basophils with Atg5 deficiency may alleviate lupus via modulating gut microbiota populations, particularly those of Ligilactobacillus and Faecalitalea. These bacteria may ameliorate lupus symptoms by enhancing carbohydrate metabolism and suppressing amino acid metabolism, potentially reducing autoantibodies and inflammatory cytokines, thereby improving plasma metabolism.

Atg5 deficiency in basophil transplantation improves plasma metabolic abnormalities

We employed widely targeted metabolomics to analyze the plasma metabolome of MRL/lpr mice and investigated the association between gut microbiota and plasma metabolites. OPLS-DA revealed a distinct difference between the control and Atg5−/− groups (Fig. 6A). A volcano plot revealed 66 upregulated and 33 downregulated metabolites in the Atg5−/− group (Fig. 6B). Our metabolomic analysis revealed increased levels of GLA, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and OPPC, whereas the levels of arginine, methylmalonic acid, and 2-hydroxyglutaric acid decreased in the Atg5−/− group compared to those in the control group (Fig. 6C–H).

Fig. 6

Atg5 deficiency in basophil transplantation improves plasma metabolic abnormalities in MRL/lpr mice. A Orthogonal partial least squares-discriminant analysis (OPLS-DA) score chart for the control (green) and Atg5−/− (red) mice groups. B Volcano plot of differential metabolites. Selection criteria for differential metabolites are variable importance in projection (VIP) > 1 and P-value < 0.05. C–H Violin plots of specific metabolites in correlation analysis. The box within the violin plot center represents the interquartile range. The thin black lines extending from it represent the 95% confidence interval. The central black horizontal line corresponds to the median, whereas the external shapes depict the data distribution density. I–M RDA between microbiota species abundance and disease severity indexes. Distribution of samples and lupus disease severity indexes. The figure shows the microbiota species distribution and disease severity indexes in RDA. Arrow length indicates the magnitude of the correlation between disease severity indexes and sample distribution, with longer lines indicating greater correlation. The acute angle between arrow lines and axes indicated a positive correlation, whereas the obtuse angle indicates a negative correlation. (N–P) Spearman's rank correlation coefficient heatmap. N Correlation analysis between microbiota species abundance and specific metabolites. O, P Correlation analysis between specific metabolites and lupus disease severity indexes. R-values are represented by different colors, with red squares indicating a positive correlation, blue squares indicating a negative correlation, and darker shades indicating a stronger positive/negative correlation. Numbers in the heatmap cells represent P-values. *P < 0.05, **P < 0.01

We conducted a correlation analysis to explore the functional relationship between the gut microbiota and differential plasma metabolites. The results of the redundancy analysis and Spearman's correlation analyses between these metabolites and lupus severity indices revealed that in the Atg5−/− group, enriched species, such as Ligilactobacillus and Faecalitalea, showed positive correlations with beneficial metabolites (GLA, DLPC, and OPPC) and negative correlations with harmful metabolites (arginine, methylmalonic acid, 2-hydroxyglutaric acid). Results of the relationship between these metabolites and autoantibodies, inflammatory cytokines, and biochemical indices indicated that GLA, DLPC, and OPPC negatively correlated with plasma autoantibodies (anti-dsDNA, anti-nuclear) and inflammatory cytokines (TNF-α, IFN-γ, and IL-1β). GLA, DLPC, and OPPC were negatively correlated with enzymes (AST, ALT, LDH, CK, and α-HBDH) and lipids (globulin, cholesterol, LDL, bile acid), but positively with HDL, albumin, and the A/G ratio (Fig. 6I–P). Therefore, transplanting basophils with Atg5 deficiency could improve the lupus condition by modulating lipid and amino acid metabolism through alterations in gut microbiota populations, particularly those of Ligilactobacillus and Faecalitalea. These bacteria may ameliorate lupus by promoting beneficial metabolites (GLA, DLPC, and OPPC) and reducing harmful metabolites (arginine, methylmalonic acid, and 2-hydroxyglutaric acid), thus lowering autoantibody and inflammatory cytokine levels and improving biochemical metabolism (Fig. 6Q).

Transplantation of L. murinus and F. rodentium improves metabolism through the increased production of GLA and OPPC

Based on fecal metagenomic sequencing of the control (Atg5+/+) and Atg5−/− basophil transplanted MRL/lpr mice, we conducted FMT targeting the most enriched taxa in Atg5−/− basophil groups, L. murinus and F. rodentium, and the control group's P. salivarius. Plasma autoantibody levels in MRL/lpr mice showed significantly lower anti-nuclear and anti-dsDNA antibodies in the F. rodentium FMT and L. murinus FMT groups compared to that in the controls and P. salivarius FMT, with the combined group exhibiting further reduction (Fig. 7A, B). This suggests that F. rodentium and L. murinus caused a reduction in autoantibody expression. Inflammatory cytokines TNF-α and IFN-γ were significantly lower in the L. murinus FMT, F. rodentium FMT, and combined groups versus controls and P. salivarius FMT, with no differences among the treatment groups (Fig. 7C, D). IL-4 and IL-17 levels were also reduced in the treatment groups compared to that in the controls, without significant variations between the groups (Fig. 7E, F). Intestinal pathology indicated less inflammatory cell deposition in the F. rodentium FMT and L. murinus FMT groups than that in controls and P. salivarius FMT, with the combined group showing further reduction (Fig. 7I, S4). These findings indicate that P. salivarius exacerbates intestinal inflammation in MRL/lpr mice, whereas F. rodentium and L. murinus ameliorate it, with a combined approach offering superior therapeutic benefits.

Fig. 7

Transplantation of Ligilactobacillus. murinus and Faecalibaculum. rodentium improves metabolism through the increased production of GLA and OPPC. A, B Statistical analysis of plasma anti-nuclear and anti-dsDNA antibodies in mice. C–F Statistical analysis of plasma cytokines, including TNF-α, IFN-γ, IL-4, and IL-17, in mice. G–H Statistical analysis of plasma arginine and γ-linolenic acid in mice. I H&E staining analysis of colon pathology in mice. J–Q Statistical analysis of plasma ALT, AST, LDH, CK, cholesterol, HDL, LDL, and A/G ratio in mice. R H&E staining analysis of liver pathology in mice. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Notably, in our analysis of amino acid and lipid profiles in MRL/lpr mice, we noted significantly lower arginine levels in the F. rodentium FMT and L. murinus FMT groups relative to the control and P. salanitronis FMT groups. The L. murinus + F. rodentium FMT group exhibited even lower arginine levels (Fig. 7G). In contrast, GLA levels were markedly higher in the F. rodentium FMT and L. murinus FMT groups, with the L. murinus + F. rodentium FMT group showing a further increase (Fig. 7H). These findings align with our plasma metabolomic data, suggesting that Atg5 deficiency in basophils enriches L. murinus and F. rodentium, promoting GLA production through lipid metabolism and improving overall metabolism, whereas basophil enrichment of P. salanitronis and other microbes through amino acid metabolism leads to arginine production and disease exacerbation. We then administered the most enriched metabolites from the control group (arginine) and the Atg5−/− basophil group (GLA and OPPC) to MRL/lpr mice to assess their impact on disease progression. The GLA and OPPC treatments significantly reduced AST, ALT, and LDH levels compared to the control and arginine groups, with the combined treatment showing further reductions in AST and ALT (Fig. 7J–L). CK levels remained unchanged across all groups (Fig. 7M). These results indicate that arginine increases transaminases in MRL/lpr mice, while GLA and OPPC ameliorate this increase, with the combined treatment showing superior therapeutic effects. Protein and lipid analyses revealed that GLA and OPPC interventions decreased cholesterol levels in MRL/lpr mice, probably by elevating HDL and reducing LDL (Fig. 7N–P). The A/G ratio improved with GLA and OPPC treatments, with no significant differences observed between combined and individual treatments (Fig. 7Q). Pathological assessments of livers from MRL/lpr mice treated with GLA and OPPC showed significant improvements in hepatocyte degeneration and necrosis compared to the control and arginine groups, with the combined treatment group showing further improvements (Fig. 7R, S5). In conclusion, Atg5 deficiency in basophils improves liver function and metabolism by enriching L. murinus and F. rodentium to produce GLA and OPPC.