Patient characterization
Patients with clinical cutaneous tuberculosis underwent a prospective study at the Integrated Chinese and Western Medicine Hospital in Nanjing, Jiangsu Province, China. The clinical manifestations were pale edema of the granuloma of the wound, purulent necrosis on the surface, unclear edge of the ulcer, dark red skin, white flocculent necrotic tissue like bean dregs in the abscess. Or the sinus opening can be seen and there are multiple branches extending in different directions. The wound is difficult to heal. During the course of the disease, the patient developed fatigue, fever, dizziness, night sweating, anorexia and weight loss.[4]
In this study, a total of 28 patients (E1-E28) who met the inclusion criteria were enrolled in the study. Ulcer tissue was obtained from all patients and HE staining (pathological diagnosis) was performed. Among them, 14 patients (E1-E14) were positive for HE staining and histopathologically positive (Fig. 1). They were treated with anti-tuberculosis drugs. Another 14 (E15-E28) control patients were non-specific infectious ulcers.
Expression profile of human local inflammatory granulation tissue
We performed a transcriptomic analysis of three experimental groups (E1-E3) and three control patients (E15-E17) using a whole human genomic oligonucleotide array. Based on this analysis, we identified 10,261 probes showing differential expression (adjusted p-value <0.05, fold-value ±1.2 (≈logFC±0.27), of which 2,594 genes had lower mRNA expression than the control group, 1,802 The gene expression was higher than that of the control group (Table 2). The relationship between gene expression profiles, the principal component analysis of the normalized gene expression data of the sample and the use of all unsupervised hierarchical clustering to examine the relationship between gene expression profiles. The largest variation in the data set (Fig. 2).
Table 2 Differential screening gene statistics in the study
|
|
mRNA(Regulated)
|
lncRNA(Regulated)
|
Multiple_Complex(Regulated)
|
circRNA(Regulated)
|
Up
|
Down
|
Total
|
Up
|
Down
|
Total
|
Up
|
Down
|
Total
|
Up
|
Down
|
Total
|
Case
|
Control
|
1802
|
2594
|
4396
|
2314
|
1282
|
3596
|
690
|
1204
|
1894
|
302
|
73
|
375
|
Upregulation and verification of chemokines in skin ulcer tissue
Chemokines are important components that direct immune cells to the site of infection and promote disease progression. Microarray results showed that mRNA expression in skin ulcer tissue was different from that in non-specific infectious ulcer local tissues, and tuberculosis-related genes were confirmed. This method provides expression data for 4,396 mRNAs showing up to 1802 genes upregulated in local inflammatory granulation tissue in cutaneous tuberculosis. The mRNA differential screening results showed that CXCL9, CXCL10, CXCL11 and CXCR3 were the most prominent upregulated chemokines. CXCL10 chemotactic leukocytes accumulate in the inflammation site, and CXCL10 binds to CXCR3 and exerts chemotaxis on T cells.(Fig.3)
GO and KEGG analyses
GO and KEGG analyses were performed to obtain detailed information on the biological functions and potential mechanisms of these differentially expressed mRNAs. A total of 4396 filtered mRNAs (2-fold change) were included in GO and KEGG analyses. GO analysis revealed that 1802 upregulated mRNAs in biological processes were involved in cellular responses to immune responses, adaptive immune response, interferon-gamma-mediated signaling pathway, inflammatory response, innate immune response, T cell costimulation regulation of immune response T cell activation and so on (Fig. 4) and in molecular function were analyzed tumor necrosis factor receptor binding, receptor activity, chemokine receptor activity (Fig. 5) and In cellular component were nucleosome, external side of plasma membrane, nuclear (Fig. 6). However, 2594 downregulated mRNAs were participants mitochondrial respiratory chain complex I assembly, muscle contraction, GO:0006120, tricarboxylic acid cycle (Fig. 7). KEGG analysis of the 1802 upregulated mRNAs Natural killer cell mediated cytotoxicity, Graft-versus-host disease, Chemokine signaling pathway, Antigen processing and presentation (Fig. 8).
Real-time quantitative RT-PCR verification sequencing results
The expression differences of CXCR3, CXCL9, CXCL10 and CXCL11 genes were detected by qRT-PCR. Compared with the control group, both CXCL9,CXCL10,CXCL11,CXCR3 were significantly up-regulated(Fig.9).