Table showing morphometric data from microscopy.

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and show no evidence of immune sensitization to Mycobacterium tuberculosis (Mtb) as defined by persistently negative tuberculin skin tests (TST) and interferon gamma release assays (IGRA). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophil Mtb infection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected with Mtb H37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. Since there was no significant differential transcriptional response at 1h between infected PMNHITTIN and PMNHIT, we focused on the 6h timepoint. When compared to uninfected PMN, PMNHITTIN displayed 3106 significantly upregulated and 3548 significantly downregulated differentially expressed genes (DEGs) (absolute cutoff of a log2FC of 0.2, FDR HIT demonstrated 3816 significantly upregulated and 3794 significantly downregulated DEGs following 6h Mtb infection. Contrasting the log2FC 6h infection response to Mtb from PMNHITTIN against PMNHIT, 2285 genes showed significant differential response between the two groups. Overall PMNHITTIN had a lower fold change response to Mtb infection compared to PMNHIT. According to pathway enrichment, Apoptosis and NETosis were differentially regulated between HITTIN and HIT PMN responses after 6h Mtb infection. To corroborate the blunted NETosis transcriptional response measured among HITTIN, fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTIN compared to PMNHIT, showing that PMNHITTIN have a distinct response to Mtb.

艾滋病病毒感染者（Persons living with HIV, PLWH）罹患结核病（tuberculosis, TB）的风险显著升高。在长期反复暴露于结核分枝杆菌（Mycobacterium tuberculosis, Mtb）后，部分PLWH始终未发展为结核病，且结核菌素皮肤试验（tuberculin skin tests, TST）与γ干扰素释放试验（interferon gamma release assays, IGRA）持续呈阴性，未表现出针对Mtb的免疫致敏迹象。该群体被明确界定为HIV阳性且持续呈结核病、结核菌素及IGRA阴性表型（HITTIN）。为探究HITTIN表型个体特有的潜在先天免疫机制，本研究将其中性粒细胞针对Mtb的感染应答与另一组无结核病病史，但结核菌素试验与IGRA持续呈阳性的PLWH（下称HIT组）进行对比。研究人员分离得到17例HITTIN个体的中性粒细胞（PMNHITTIN）与11例HIT个体的中性粒细胞（PMNHIT），分别用Mtb H37Rv菌株感染1小时与6小时。提取细胞总RNA并开展RNA测序（RNAseq）分析。由于感染后1小时，PMNHITTIN与PMNHIT两组间未出现显著的转录组差异应答，故本研究将分析重点聚焦于6小时时间点。与未感染的中性粒细胞相比，PMNHITTIN组共有3106个显著上调、3548个显著下调的差异表达基因（differentially expressed genes, DEGs），筛选标准为log2倍变化（log2FC）绝对值≥0.2及错误发现率（FDR）阈值。HIT组在6小时Mtb感染后，则分别检出3816个显著上调与3794个显著下调的DEGs。对比两组中性粒细胞在6小时Mtb感染后的log2FC应答特征，共鉴定出2285个基因在两组间存在显著应答差异。整体而言，PMNHITTIN对Mtb感染的转录应答幅度显著低于PMNHIT组。通路富集分析结果显示，感染6小时后，细胞凋亡与中性粒细胞胞外陷阱形成（NETosis）在HITTIN组与HIT组的中性粒细胞应答中存在差异化调控。为验证HITTIN组中观察到的NETosis转录应答减弱现象，研究人员通过荧光显微镜观察发现，相较于PMNHIT，PMNHITTIN的中性粒细胞胞外陷阱形成量与细胞丢失率均相对更低，证实PMNHITTIN对Mtb感染具有独特的应答特征。