Cervical Cutaneous Tuberculosis Shows the Inflammatory Infection Environment of Local Necrotic Granulation Tissue through the Expression Profile of Chemokine

Background Tuberculosis is an important infectious disease that jeopardizes human health. Research on extrapulmonary tuberculosis is still rare, especially cutaneous tuberculosis, a more refractory extrapulmonary tuberculosis. This study is to explore the inflammatory and immunobiomarkers of patients with cutaneous tuberculosis and to analyze the potential functions and pathways involved in the pathogenesis of M. tuberculosis (Mtb) infection, in order to support cutaneous tuberculosis diagnosis and treatment.Methods Analysis of mRNA identifies biomarkers in cutaneous tuberculosis and non-specific infectious ulcer tissue. Analyze the correlation between gene ontology (GO) and the Kyoto Gene and Genomic Encyclopedia (KEGG) biological pathways, immunity, inflammation and chemokines. Chemotaxis factors such as CXCL9, CXCL10, CXCL11 and CXCR3 were compared by quantitative PCR analysis.Results The mRNA expression levels of CXCL9, CXCL10, CXCL11 and CXCR3 in local necrotic granulation tissue of cutaneous tuberculosis were significantly increased compared with those of normal tissues. GO analysis indicated that activation of various immune responses and inflammatory responses may be associated with up-regulation of chemokine genes. Gene Ontology (GO) and Kyoto Biogenomics and Genomics Encyclopedia (KEGG) biological pathway analysis revealed the relationship between immune, inflammatory and chemokine pathways.Conclusion GO analysis and KEGG analysis of differentially expressed mRNA revealed potential functions and pathways associated with the onset of Mtb infection.

tuberculosis is the most common, that is, tuberculosis such as lymphatic tuberculosis, bone and joint tuberculosis, breast tuberculosis, etc. directly spread to adjacent skin tissues, or through ulcerated skin through blood, and lymphatic tissue is damaged. Cutaneous tuberculosis occur in the neck, underarms and perianal. Because the skin surface is weak, the nerves and blood vessels are densely distributed, and the skin and mucous membranes affect the healing ability of the ulcer; the wounds are not healed for a long time, or recurrent after pseudo-healing, which seriously affects the health of the patient.
In recent years, with the vigorous development of biology, people have become more and more clear about the pathogenesis of tuberculosis, but the recent research is more concentrated on tuberculosis research [1]. At present, research on cutaneous tuberculosis is rare. Although there have been many reports highlighting risk factors and cytokine expression in various forms of EPTB, extrapulmonary tuberculosis [2,3], knowledge of changes in pathogen-associated tissue expression in hosts is very inadequate.So far, only a few reports have studied chemokines and chemokine receptors in human tissues, which are responsive in TB patients [4].
Understanding an important aspect of host-pathogen interaction is to identify host tissue responses to infection and to identify causal factors associated with disease performance.
Chemokines are superfamilies of small molecular weight proteins (8 to 10-kd) with 20% to 70% homology in the amino acid sequence, which are involved in cell activation and proliferation and leukocyte recruitment to sites of inflammation such as ligands. They make an important impact. The receptor (CXCL10 / CXCR3) is involved in the selective migration of lymphocytes to the intestinal mucosa [5]. They are subdivided into families based on the relative positions of cysteine residues.
There are at least four chemokine families, but only two are widely characterized.

Tissue samples
In this study, we included 14 patients (E1-E14) who were diagnosed with cutaneous tuberculosis at the Integrated Chinese and Western Medicine Hospital in Nanjing, Jiangsu Province, China. None of these patients had anti-tuberculosis treatment and no active tuberculosis, mental illness, impaired immune function (symptomatic HIV infection, confirmed or suspected HIV infection, leukemia, lymphoma or systemic malignancy), immunosuppressive therapy (corticosteroids, alkylating agents, antimetabolites, radiation therapy). In addition, 14 non-specific infectious ulcer patients (E15-E24) matched with cutaneous tuberculosis patients, such as age and gender, were included for comparative study. (Table 1) Among them, 6 patients with E1-E3 and E8-E10 were selected for local lesion tissue transcription analysis of CXCR3, CXCL9, CXCL0, CXCL11 mRNA expression. Finally, the differential expression levels of CXCR3, CXCL9, CXCL0 and CXCL11 chemokines in the local lesions of these 28 patients were verified by quantitative PCR. Table 1 summarizes the demographic and clinical characteristics of all patients with cutaneous tuberculosis and non-specific infectious ulcers. Informed consent was obtained from all patients or legal guardians. This study was approved by the hospital ethics committee.

Patient characterization
Patients with clinical cutaneous tuberculosis underwent a prospective study at the Integrated Chinese 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.

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).

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.

Discussion
Tuberculosis is one of the top ten causes of infectious diseases that endanger human health and cause human death. People with AIDS often die from tuberculosis infection. China is a country with a high burden of tuberculosis.
Lymphatic tuberculosis (LNTB) is the most common form of extrapulmonary tuberculosis, leading to lymphadenopathy cutaneous tuberculosis. Cutaneous tuberculosis is often referred to as tuberculous ulcer in China.
However, the lack of a clear pathogenesis hinders diagnosis and treatment. Reported CXCL9, CXCL10 mRNA expression levels associate with cutaneous tuberculosis. Literature reported, IFN-g signaling, via T-bet, also promotes CXCR3 expression on a subset of Th1-specific regulatory T cells [6]. The chemokine receptor CXCR3 is a G protein-coupled receptor found predominantly on T cells that is activated by three ligands as follows: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Three ligands, CXCL11 is the most potent inducer of CXCR3 internalization and is the physiologic inducer of CXCR3 internalization after T cell contact with activated endothelial cells [7]. C-X-C motif chemokine 10 (CXCL10) is also known as IP-10. The biological function of CXCL10 is to induce chemotaxis, cell growth, angiogenesis, and apoptosis by binding to its surface chemokine receptor CXCR3, and CXCL10 is recognized as an inflammatory chemokine. Moreover, CXCL10 plays an important role in various pathological states. CXCL10 had been reported to act as a marker for the diagnosis of tuberculosis [8,9].The production of CXCL9 is specifically induced by IFN-γ [10],and IFN-γ plays an important role in the pathogenesis of tuberculosis-related diseases. [11] Specific blockage of CXCR3 (a CXCL10 receptor) significantly decreased the severity of chronic pulmonary inflammation by decreasing the recruitment of inflammatory cells. [12] Similarly, mRNA CXCL10 expression was up-regulated in tuberculous ulcer tissues, namely Log FC.
Given their pleiotropic nature, CXCR3 ligands play their part in a myriad of diseases. Involvement of all CXCR3 ligands has been observed in various angiogenesis-related pathologies as well as immunological disorders, the latter being mostly related to autoimmunity or infection.
In this experiment, a microarray of whole genome mRNA expression profiles of local inflammatory granulation tissue of cutaneous tuberculosis patients was compared with local inflammatory granulation tissue of non-specific infectious ulcer patients.
However, our detection found that the mRNA expression levels of CXCR3, CXCL9, CXCL10, CXCL11 were       Twenty pathways of transcriptomic analysis of cutaneous tuberculosis inflammatory granulation tissue patients compared to non-specific infectious ulcer patient controls.

Figure 9
Real-time quantitative RT-PCR verification sequencing results

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