Impaired mitochondria of Tregs decreases OXPHOS-derived ATP in primary immune thrombocytopenia with positive plasma pathogens detected by metagenomic sequencing

Patient characteristics and mNGS analysis

Seventy-five patients with newly diagnosed primary ITP with a median age of 53 years (range of 17–80 years) and a median platelet count of 11 × 109/L (range of 1–33 × 109/L) were enrolled to determine if they sustained with pathogen infection. Patients’ plasma was tested using mNGS. The clinical characteristics of the participants enrolled are shown in Table 1. As shown in Fig. 1A, the pathogen detection of plasma from ITP patients found 73.3% patients (55/75) were negative. Cytomegalovirus (CMV) were detected from 12% patients (9/75). Epstein-Barr virus (EBV) were detected from 5.3% patients (4/75). CMV and EBV were detected simultaneously in two patients. Aspergillusniger was detected from one patient and Candida parapsilosis was detected from two patients. Also, there were five kinds of bacteria were detected in the study, including 1 patient with B.fragilis, 2 patients with Helicobacter pylori, 1 patient with Richettsia, and 1 patient with Streptococcus sinensis and Neisseria flavescens. Candida parapsilosis and B.fragilis were detected simultaneously in 1 patient. Among the 7 patients accompany with bacterial or fungal infection, 71.4% (5/7) were NR after first-line treatment with corticosteroids.

Table 1 Clinical characteristics of study participantsFig. 1figure 1

Metagenomic next-generation sequencing (mNGS) of ITP patients’ plasma. A Pie charts show the distribution of detected pathogens in ITP patients. The stringently mapped reads number (SMRN) of CMV (B), EBV (C) and other detected pathogens (D). E The expression of anti-CMV IgG antibodies and anti-CMV IgM antibodies. ITP immune thrombocytopenia, CMV cytomegalovirus, EBV Epstein-Barr virus

Stringently mapped reads number of pathogens and related antibodies

The levels of detected pathogens and their related antibodies expression were then analyzed. The stringently mapped reads number (SMRN) was used to evaluate the levels of detected pathogens. The SMRNs of detected pathogens were shown in Fig. 1B, C and D. The SMRN of CMV was 3.11 ± 3.66 (Fig. 1B) and EBV was 4.75 ± 4.35 (Fig. 1C). The SMRN of bacterium and fungus was 21.56 ± 19.98 (Fig. 1D). All of the 9 patients with CMV shown anti-CMV IgG antibodies were positive which above 14 U/mL (108.61 ± 8.52 U/mL). However, anti-CMV IgM antibodies were all negative which below 18 U/mL (7.36 ± 0.71 U/mL) (Fig. 1E). In addition, 4 patients with EBV by mNGS shown anti-EBVCA IgM and IgG antibodies were negative (data not shown). In summary of the results of SMRNs, antibodies, and literature [26], infections of CMV and EBV in ITP patients in the study were not considered to have the ability causing the disease.

The change of lipid metabolic in ITP patients with positive antigens

Furthermore, LC–MS was used to study whether ITP patients with positive antigens could cause the change of metabolic. Positive plasma pathogens were detected in 7 ITP patients in the study. Twenty volunteers were enrolled as healthy controls. As shown in Fig. 2, the level of DAG (Fig. 2A) was increased significantly in positive pathogen-ITP patients compared to HC and negative pathogen-ITP patients (p = 0.023, and p = 0.042, respectively). The level of CER (Fig. 2B) was increased significantly in negative pathogen-ITP patients compared to HC (p = 0.029). However, the level of CER was not significantly different between positive pathogen-ITP patients and HC or negative pathogen-ITP patients. Also, the levels of CE (Fig. 2C), LPC (Fig. 2D), LPE (Fig. 2E), PC (Fig. 2F), PE (Fig. 2G), SM (Fig. 2H), and TAG (Fig. 2I) were not significantly different among 3 groups.

Fig. 2figure 2

Plasma lipid subclasses from comparing pathogen( +)-ITP, pathogen(-)-ITP patients, and healthy controls. A–I The plasma levels of DAG, CER, CE, LPC, LPE, PC, PE, SM, and TAG in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. Each bar graph represents mean ± SD. P values less than 0.05 were labeled in the figure. ITP immune thrombocytopenia, DAG diacylglycerol, CER ceramide, CE cholesteryl ester, LPC lysophosphatidylcholine, LPE lysophosphatidylethanolamine, PC phosphatidylcholine, PE phosphatidylethanolamine, SM sphingomyelin, TAG triacylglycerol

The changes of CD4 T cells in ITP patients with positive antigens

As the immune system may be abnormally regulated by positive antigens, T cell subsets and function were evaluated for the changes of T cells in positive pathogen-ITP patients. Representative dot plots of Th1, Th2, Tregs, Th17, CD4 + TNFα + , and CD4 + TGFβ + cells in the PBMCs of HC, positive pathogen-ITP patient, and negative pathogen-ITP patient were shown in Fig. 3A, B, C, D, E and F. The percentages of Th1 cells (Fig. 3G) and CD4+ TNFα+ cells (Fig. 3K) were decreased significantly in positive pathogen-ITP and negative pathogen-ITP patients, as compared to HC (p < 0.05). The percentages of Th2 cells (Fig. 3H) and Th17 cells (Fig. 3J) were increased significantly in positive pathogen-ITP patients and negative pathogen-ITP patients, as compared to HC (p < 0.05). The percentages of Tregs were increased significantly in positive pathogen-ITP patients, comparing to negative pathogen-ITP patients and HC (p = 0.033 and 0.003, respectively) (Fig. 3I). The ratios of Treg/Th17 (Fig. 3M) and Th1/Th2 (Fig. 3N) were decreased significantly in negative pathogen-ITP patients than HC (p = 0.004, and p < 0.001, respectively). The ratio of Th1/Th2 was decreased significantly in positive pathogen-ITP patients as compared to that of HC (p < 0.001). However, there were no significant differences in the percentages of Th1, Th2, Th17, CD4+ TNFα+ , CD4+ TGFβ+ cells (Fig. 3L), Treg/Th17 ratio, and Th1/Th2 ratio between positive pathogen-ITP and negative pathogen-ITP patients.

Fig. 3figure 3

Flow cytometry analysis of Th1, Th2, Treg, Th17, CD4+ TNFα+ , CD4+ TGFβ+ cells in pathogen( +)-ITP, pathogen(-)-ITP patients, and healthy controls. A–F Representative dot plots of Th1, Th2, Tregs, Th17, CD4+ TNFα+ , and CD4+ TGFβ+ cells in the PBMCs of pathogen( +)-ITP, pathogen(−)-ITP patient, and healthy control. G–N The percentages of Th1, Th2, Tregs, Th17, CD4+ TNFα+ , and CD4+ TGFβ+ cells and the ratio of Treg/ Th17 and Th1/Th2 in the PBMCs of pathogen( +)-ITP, pathogen(-)-ITP patients, and healthy controls. Each bar graph represents mean ± SD. P values less than 0.05 were labeled in the figure. ITP, immune thrombocytopenia

The changes of CD8 T cells in ITP patients with positive antigens

As shown in Fig. 4, representative dot plots of CD8+ Ki67+ , CD8+ GnB+ , CD8+ TNFα+ , CD8+ IFNγ+ , and CD8+ IL17+ cells in the PBMCs of HC, positive pathogen-ITP patient, and negative pathogen-ITP patient were shown in Fig. 4A, B, C, D and E. The percentages of CD8+ Ki67+ (Fig. 4F) and CD8+ IL17+ (Fig. 4J) cells were increased significantly in positive pathogen-ITP and negative pathogen-ITP patients than HC (p < 0.05). However, the percentages of CD8+ TNFα+ (Fig. 4G) and CD8 + IFNγ + (Fig. 4I) cells were decreased significantly in positive pathogen-ITP and negative pathogen-ITP patients than HC (p < 0.05). CD8+ GnB+ T cells (Fig. 4H) were increased significantly in negative pathogen-ITP than HC (p = 0.04). There were no significant differences in the percentages of CD8+ Ki67+ , CD8+ TNFα+ , CD8+ GnB+ , CD8+ IFNγ+ , and CD8+ IL17+ T cells between positive pathogen-ITP and negative pathogen-ITP patients.

Fig. 4figure 4

Flow cytometry analysis of CD8+ Ki67+ , CD8+ TNFα+ , CD8+ Granzyme B+ , CD8+ IFNγ+ , and CD8+ IL17+ cells in pathogen(+)-ITP, pathogen(−)-ITP patients, and healthy controls. A–E Representative dot plots of CD8+ Ki67+ , CD8+ Granzyme B+ , CD8+ TNFα+ , CD8 + IFNγ+ , and CD8+ IL17+ cells in the PBMCs of pathogen( +)-ITP, pathogen(-)-ITP patient, and healthy control. F–J The percentages of CD8+ Ki67+ , CD8+ TNFα+ , CD8+ Granzyme B+ , CD8+ IFNγ+ , and CD8+ IL17+ cells in the PBMCs of pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. Each bar graph represents mean ± SD. P values less than 0.05 were labeled in the figure. ITP immune thrombocytopenia

Changes of mitochondria membrane potential in ITP patients with positive antigens

Considering the role of CD4 T cell subsets in ITP and the difference of Tregs in positive pathogen-ITP and negative pathogen ITP patients, researches on mitochondrial of CD4 T cell subsets were performed to investigate the changes of mitochondrial function in positive pathogen-ITP patients. Representative dot plots of ROS and JC-1 in control, positive pathogen-ITP patient, and negative pathogen-ITP patient were shown in Fig. 5A and B. The MFI of ROS in Th1 (Fig. 5C), Th2 (Fig. 5D), and Th17 (Fig. 5E) cells were increased in positive pathogen-ITP and negative pathogen-ITP patients compared to healthy controls but without significantly. The ROS of Tregs (Fig. 5F) was decreased in positive pathogen-ITP compared to negative pathogen-ITP patients and HC but without significantly. The result of mitochondria membrane potential shown that mitochondria membrane potential was decreased significantly in Th2 (Fig. 5H), Th17 (Fig. 5I), and Tregs (Fig. 5J) in positive pathogen-ITP patients compared to HC (p = 0.004, 0.003, and 0.013, respectively). Mitochondria membrane potential were decreased significantly in Th17 and Tregs in negative pathogen-ITP patients compared to HC (p = 0.001 and 0.035, respectively). There was no significant difference in mitochondria membrane potential between positive pathogen-ITP and negative pathogen-ITP patients.

Fig. 5figure 5

Flow cytometry analysis of Th1, Th2, Th17, and Treg mitochondrial ROS and mitochondria membrane potential in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. A, B Representative plots of ROS and JC-1 in pathogen( +)-ITP, pathogen(−)-ITP patient, and healthy control. C–F The MFI of ROS of Th1, Th2, Th17, and Tregs in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. G–J The mitochondria membrane potential of Th1, Th2, Th17, and Tregs in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. JC-1 forms aggregates emit red fluorescence while loss of membrane potential becomes monomers emit green fluorescence. The greater the accumulation of green fluorescence, the lesser the mitochondria membrane potential. Each bar graph represents mean ± SD. P values less than 0.05 were labeled in the figure. ITP immune thrombocytopenia; ROS, reactive oxygen species

Suppressing OXPHOS in Tregs in ITP patients with positive antigens

Since mitochondria membrane potential were remarkably changed in positive pathogen-ITP patients and negative pathogen-ITP patients, the changes of mitochondria metabolism were tested. The glycolysis-derived ATP were decreased in Th1 cells in positive pathogen-ITP patients and negative pathogen-ITP patients compared to that of HC (p = 0.001 and 0.011, respectively) (Fig. 6A). The mitochondrial-derived ATP were also decreased in Th1 cells in positive pathogen-ITP patients than that of HC (p = 0.008) (Fig. 6B). The decrease of glycolysis-derived ATP in Th17 cells was shown in negative pathogen-ITP patients compared to HC (p = 0.027) (Fig. 6E). There were no significant differences in glycolysis-derived ATP in Th2 and Tregs among groups (Fig. 6C, G). Also, there were no significant differences in mitochondrial-derived ATP in Th2 and Th17 cells among groups (Fig. 6D, F). Compared to HC, the mitochondrial-derived ATP in Tregs in positive pathogen-ITP patients and negative pathogen-ITP patients were decreased (p < 0.001 and p = 0.015, respectively) (Fig. 6H).

Fig. 6figure 6

ATP production rate in Th1, Th2, Th17, and Treg in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. AH The production rate of glycolysis-derived ATP and mitochondrial-derived ATP of Th1, Th2, Th17, and Tregs in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. Each bar graph represents mean ± SD. P values less than 0.05 were labeled in the figure. ITP, immune thrombocytopenia

The overall metabolism flux was decreased in Th1 and Tregs in positive pathogen-ITP patients compared to that of HC (p < 0.001 and p = 0.004, respectively) (Fig. 7A, D). The overall metabolism flux in Th1 cells of negative pathogen-ITP patients was decreased than that of HC (p = 0.013) (Fig. 7A). There were no significant differences in the overall metabolism flux in Th2 and Th17 cells among groups (Fig. 7B, C). The data showed that a smaller proportion of ATP was generated by OXPHOS in positive pathogen-ITP patients compared to negative pathogen-ITP patients and HC (15.95% versus 18.63% and 25.5%, respectively), and a higher proportion of ATP was generated by glycolysis compared to negative pathogen-ITP patients and HC (84.05% versus 81.37% and 74.5%, respectively) (Fig. 7D). The ATP rate index of mitochondrial-ATP production rate versus glycolysis-ATP production rate of Tregs was decreased significantly in positive pathogen-ITP and negative pathogen-ITP patients compared to HC (p < 0.001) (Fig. 7H). The ATP rate index of Tregs was decreased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients (p = 0.036). There were no significant differences in ATP rate index of Th1, Th2, and Th17 cells among groups (Fig. 7E, F, G).

Fig. 7figure 7

Seahorse XF real-time ATP rate analysis of Th1, Th2, Th17, and Treg in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. A–D An overall metabolism flux of Th1, Th2, Th17, and Tregs in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. (E–H) The ATP rate index of Th1, Th2, Th17, and Tregs in pathogen( +)-ITP, pathogen(−)-ITP patients, and healthy controls. Each bar graph represents mean ± SD. P values less than 0.05 were labeled in the figure. ITP, immune thrombocytopenia

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