Patient Characteristics and mNGS Analysis
At first, pathogen detection by mNGS of plasma from ITP patients was performed in our study to assess whether patient with pathogen infection. For this part, 75 patients with newly diagnosed primary ITP were enrolled. The ITP patients shown 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). Clinical characteristics of the participants enrolled in the study are shown in Table 1. As shown in Figure 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 our study, including one patient with B.fragilis, two patients with Helicobacter pylori, one patient with Richettsia, and one patient with Streptococcus sinensis and Neisseria flavescens. Candida parapsilosis and B.fragilis were detected simultaneously in one patient. Among the seven patients accompany with bacterial or fungus infection, 71.4% (5/7) were NR after first-line treatment with corticosteroids.
Table 1
Clinical characteristics of study participants.
Characteristics
|
ITP patients
|
Healthy Controls
|
(n=75)
|
(n=20)
|
Median age (range)
|
53 (17-80)
|
45 (26-70)
|
Gender
|
|
|
Female
|
47
|
13
|
Male
|
28
|
7
|
Platelet count (×109/L), median (range)
|
11 (1-33)
|
243 (140-320)
|
Bleeding score at initial diagnosis,
mean (range)
|
2.25 (1-5)
|
-
|
Response to corticosteroids
|
|
|
CR+PR
|
49
|
-
|
NR
|
26
|
-
|
ITP, immune thrombocytopenia; CR, complete response; R, response; NR, no response.
Stringently Mapped Reads Number of Pathogens and Related Antibodies
Next, we analyzed the levels of detected pathogens and their related antibodies expression. The Stringently mapped reads number (SMRN) was used to evaluate the levels of detected pathogens. The SMRNs of detected pathogens were shown in Figure 1B, C, and D. The SMRN of CMV was 3.11 ± 3.66 (Figure 1B) and EBV was 4.75 ± 4.35 (Figure 1C). The SMRN of bacterium and fungus was 21.56 ± 19.98 (Figure 1D). All of the nine 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) (Figure 1E). In addition, four patients with EBV by mNGS shown anti-EBVCA IgM and IgG antibodies were negative (data no shown). Taken together the results of SMRN, antibodies, and previously reported(22), CMV and EBV in ITP patients in the study were considered to have not the ability to cause diseases.
The Change of Lipid Metabolic in ITP Patients with Positive Antigens
In the next step, we used LC-MS to study whether ITP patients with positive antigens could cause the change of metabolic. Positive plasma pathogens were detected in seven ITP patients in the study. Twenty healthy controls were also enrolled for this part. As shown in Figure 2, the level of DAG (Figure 2A) was increased significantly in positive pathogen-ITP patients compared to healthy controls and negative pathogen-ITP patients (p=0.023 and 0.042, respectively). The level of CER (Figure 2B) was increased significantly in negative pathogen-ITP patients compared to healthy controls (p=0.029). However, there was no significant difference in the level of CER between positive pathogen-ITP patients and healthy controls or negative pathogen-ITP patients. Also, there were no significant differences in the levels of CE (Figure 2C), LPC (Figure 2D), LPE (Figure 2E), PC (Figure 2F), PE (Figure 2G), SM (Figure 2H), and TAG (Figure 2I) among the three groups.
The Changes of CD4 T Cells in ITP Patients with Positive Antigens
As the immune system may be abnormally regulated by positive antigens, further research on T cell subsets and function were performed to evaluate 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 control, positive pathogen-ITP patient, and negative pathogen-ITP patient were shown in Figure 3A, B, C, D, E, and F. The percentages of Th1 cells (Figure 3G) and CD4+TNFα+ cells (Figure 3K) were decreased significantly in positive pathogen-ITP and negative pathogen-ITP patients compared to healthy controls (p<0.05). The percentages of Th2 cells (Figure 3H) and Th17 cells (Figure 3J) were increased significantly in positive pathogen-ITP patients and negative pathogen-ITP patients compared to healthy controls (p<0.05). The percentages of Tregs were increased significantly in positive pathogen-ITP patients compared to negative pathogen-ITP patients and healthy controls (p=0.033 and 0.003, respectively) (Figure 3I). The ratios of Treg/Th17 (Figure 3M) and Th1/Th2 (Figure 3N) were decreased significantly in negative pathogen-ITP patients than healthy controls (p=0.004 and <0.001, respectively). The ratio of Th1/Th2 was decreased significantly in positive pathogen-ITP patients than healthy controls (p<0.001). However, there were no significant differences in the percentages of Th1, Th2, Th17, CD4+TNFα+, CD4+TGFβ+ cells (Figure 3L), Treg/Th17 ratio, and Th1/Th2 ratio between positive pathogen-ITP and negative pathogen-ITP patients.
The Changes of CD8 T Cells in ITP Patients with Positive Antigens
As shown in Figure 4, representative dot plots of CD8+Ki67+, CD8+Granzyme B+, CD8+TNFα+, CD8+IFNγ+, and CD8+IL17+ cells in the PBMCs of control, positive pathogen-ITP patient, and negative pathogen-ITP patient were shown in Figure 4A, B, C, D, and E. The percentages of CD8+Ki67+ (Figure 4F) and CD8+IL17+ (Figure 4J) cells were increased significantly in positive pathogen-ITP and negative pathogen-ITP patients than healthy controls (p<0.05). However, the percentages of CD8+TNFα+ (Figure 4G) and CD8+IFNγ+ (Figure 4I) cells were decreased significantly in positive pathogen-ITP and negative pathogen-ITP patients than healthy controls (p<0.05). There were no significant differences in the percentages of CD8+Ki67+, CD8+TNFα+, CD8+Granzyme B+ (Figure 4H), CD8+IFNγ+, and CD8+IL17+ cells between positive pathogen-ITP and negative pathogen-ITP patients.
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, further research 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 Figure 5A and B. The MFI of ROS in Th1 (Figure 5C), Th2 (Figure 5D), and Th17 (Figure 5E) cells were increased in positive pathogen-ITP and negative pathogen-ITP patients compared to healthy controls but without significantly. The ROS of Tregs (Figure 5F) was decreased in positive pathogen-ITP compared to negative pathogen-ITP patients and healthy controls but without significantly. The result of mitochondria membrane potential shown that mitochondria membrane potential was decreased significantly in Th2 (Figure 5H), Th17 (Figure 5I), and Tregs (Figure 5J) in positive pathogen-ITP patients compared to healthy controls (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 healthy controls (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.
Suppressing OXPHOS in Tregs in ITP Patients with Positive Antigens
Since mitochondria membrane potential were shown changed in positive pathogen-ITP patients and negative pathogen-ITP patients, the changes of mitochondria metabolism were performed in the study. There is a decrease of glycolysis-derived ATP in Th1 cells in positive pathogen-ITP patients and negative pathogen-ITP patients compared to HC (p=0.001 and 0.011, respectively) (Figure 6A). There also is a decrease of mitochondrial-derived ATP in Th1 cells in positive pathogen-ITP patients than those in healthy controls (p=0.008) (Figure 6B). The decrease of glycolysis-derived ATP in Th17 cells was shown in negative pathogen-ITP patients compared to healthy controls (p=0.027) (Figure 6E). There were no significant differences in glycolysis-derived ATP in Th2 and Tregs among groups (Figure 6C, G). Also, there were no significant differences in mitochondrial-derived ATP in Th2 and Th17 cells among groups (Figure 6D, F). Compared to HC, there is a decrease of mitochondrial-derived ATP in Tregs in positive pathogen-ITP patients and negative pathogen-ITP patients (p<0.001 and p=0.015, respectively) (Figure 6H).
The data shown a decrease of an overall metabolism flux in Th1 and Tregs in positive pathogen-ITP patients compared to HC (p<0.001 and p=0.004, respectively) (Figure 7A, D). There is a decrease of an overall metabolism flux in Th1 cells in negative pathogen-ITP patients than those in HC (p=0.013) (Figure 7A). There were no significant differences in the overall metabolism flux in Th2 and Th17 cells among groups (Figure 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 healthy controls (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 healthy controls (84.05% versus 81.37% and 74.5%, respectively) (Figure 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 healthy controls (p<0.001) (Figure 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 the ATP rate index of Th1, Th2, and Th17 cells among groups (Figure 7E, F, G).