Nurzat Yeltai, Zhu Zhu, Zhang Yixin, Xu Heng
Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Exp Dermatol. 2023 Feb;32(2):186-197. doi: 10.1111/exd.14700. Epub 2022 Nov 17.
Keloids are pathological scars that invade normal surrounding tissue without self-limitation, causing pain, itching, cosmetic disfigurement, etc. Knowledge of the molecular mechanisms underlying keloids remains unclear; thus, there are no available biomarkers for its diagnosis, resulting in a diagnostic accuracy of only 81%, which may be resolved by seeking an effective biomarker. Given that keloids possess pathogenic features similar to those of autoimmune skin disease, this study aimed to utilise the single-cell V(D)J sequencing method to identify a potential biomarker and clarify the underlying biological mechanisms. Single-cell V(D)J sequencing was used to detect T cell receptor (TCR) diversity between keloid patients and healthy donors using peripheral blood samples, the results of which were further validated using reverse transcription-polymerase chain reaction (RT-PCR). Flow cytometry was used to analyse the mucosal-associated invariant T (MAIT) cell percentage, cytokine production, and activation marker expression levels in peripheral blood samples of keloid patients and normal donors. An immunofluorescence test was used to quantitatively analyse the distribution of MAIT cells in scar and healthy donor skin tissues. Single-cell V(D)J sequencing analysis showed that the usage frequency of the TRAJ33-one invariant chain of the TCR of MAIT cells was decreased in keloid patients. This result was validated by RT-PCR, which showed that significantly lower TCR Vα7.2-Jα33 was expressed in keloid patients compared with that in healthy donors and hypertrophic scar patients (p < 0.05). Flow cytometry and immunofluorescence tests further verified that MAIT cells decreased significantly both in the peripheral blood sample and lesions of keloid patients compared with those of healthy controls (p < 0.05). MAIT cells from keloid patients secreted less interferon (IFN)-γ than those from the healthy controls and hypertrophic scar group (p < 0.001). The percentage of PLZF+ MAIT cells was lowest in the peripheral blood samples of keloid patients (p < 0.05). The percentage of IL-18+ MAIT cells was lower in the peripheral blood samples of keloid patients compared with that in healthy donors (p < 0.05). These findings indicate that MAIT cells could be associated with keloids and may serve as potential biomarkers or therapeutic targets in the diagnosis of keloids.
瘢痕疙瘩是一种病理性瘢痕，会侵袭周围正常组织且无自我限制，可导致疼痛、瘙痒、外观毁损等。瘢痕疙瘩潜在的分子机制尚不清楚；因此，目前尚无用于其诊断的生物标志物，导致诊断准确率仅为81%，这可能需要通过寻找有效的生物标志物来解决。鉴于瘢痕疙瘩具有与自身免疫性皮肤病相似的致病特征，本研究旨在利用单细胞V(D)J测序方法来鉴定一种潜在的生物标志物，并阐明其潜在的生物学机制。采用单细胞V(D)J测序，利用外周血样本检测瘢痕疙瘩患者与健康供体之间的T细胞受体(TCR)多样性，其结果通过逆转录-聚合酶链反应(RT-PCR)进一步验证。采用流式细胞术分析瘢痕疙瘩患者和正常供体外周血样本中黏膜相关恒定T(MAIT)细胞百分比、细胞因子产生情况及活化标志物表达水平。采用免疫荧光试验定量分析MAIT细胞在瘢痕疙瘩和健康供体皮肤组织中的分布。单细胞V(D)J测序分析显示，瘢痕疙瘩患者中MAIT细胞TCR的TRAJ33-恒定链使用频率降低。RT-PCR验证了该结果，结果显示瘢痕疙瘩患者中TCR Vα7.2-Jα33的表达明显低于健康供体和增生性瘢痕患者(p<0.05)。流式细胞术和免疫荧光试验进一步证实，与健康对照相比，瘢痕疙瘩患者外周血样本和病变部位的MAIT细胞均显著减少(p<0.05)。瘢痕疙瘩患者的MAIT细胞分泌的干扰素(IFN)-γ少于健康对照和增生性瘢痕组(p<0.001)。瘢痕疙瘩患者外周血样本中PLZF+MAIT细胞百分比最低(p<0.05)。与健康供体相比，瘢痕疙瘩患者外周血样本中IL-18+MAIT细胞百分比更低(p<0.05)。这些发现表明，MAIT细胞可能与瘢痕疙瘩有关，并且可能作为瘢痕疙瘩诊断中的潜在生物标志物或治疗靶点。
